Ultrabithorax
Polycomb mediates repression of Ubx (McCall, 1994), while Trithorax acts positively to maintain Ubx expression (Breen, 1993).
Silencing also occurs in imaginal disc cells. Fragments which mediate silencing in anterior regions of imaginal discs contain embryonic silencers and HB target sites. bxd is not under hb control itself, but the silencing activity of BXD depends on combination with
fragments containing HB protein binding sites. Since silencing by BXD also requires Polycomb function, this suggests that BXD contains target sites for PC or for PC-like proteins (Christen, 1994).
Trithorax complex genes, absent, small, or homeotic discs 1 and 2 interact with homeotic genes. The consequences of ash1 and ash2 mutations on the expression of homeotic selector genes in imaginal discs demonstrate that both ash1 and ash2 are trans-regulatory elements of homeotic selector gene regulation. Hypomorphic ash1 mutations cause variegated expression of Antennapedia, Sex combs reduced, Ultrabithorax, and engrailed. (LaJeunesse, 1995).
The ash2 transcript detected in null mutant
larvae is interpreted as a maternally derived product. This suggests
that the zygotic requirement for ash2 begins during the
third larval instar and is consistent with phenotypes of
imaginal discs from mutant larvae. For example, in haltere discs from mid third instar mutant larvae there is no expression of an Antennapedia
reporter gene and nearly uniform accumulation of Ultrabithorax protein
as in normal haltere discs, whereas
in haltere discs from late third instar mutant larvae
there is ectopic expression of an Antennapedia reporter
gene and patchy accumulation of Ultrabithorax protein. Finding the first detectable 53kDa Ash2 protein in late third instar larvae
suggests that whatever activity is responsible for producing this smaller Ash2 product is not present before the
end of the third larval instar. The fact that the final
Ash2 protein is significantly different in larvae and
pupae suggests that it has different functions in larvae
and pupae. This may represent a novel mechanism for
a single gene product to have multiple functions (Adamson, 1996).
A regulatory element in the Ubx gene responds to Pc-G and trx-G genes. Transposons, genetically engineered pieces of DNA carrying the regulatory element to new ectopic sites, create new binding sites for Pc-G products in the new sites in which they integrate. The transposons carry Pc-G response elements (PRE), DNA regions responsive to the repressive affect of Pc-G genes. PREs and Pc-G proteins establish a repressive complex that keeps itself and other distal enhancers repressed in cells where they were first active and then repressed, and maintains this repressed state over many cell divisions. PRE functions to silence these remote enhancers or to maintain expression regulated by trx-group products. Hunchback mediates repression at the PRE. The trx-G products stimulate the expression of separate and distinct enhancers, active in imaginal discs (Chan, C-S. 1994).
Newer evidence maintains that silencing of late Ubx enhancers by PRE is Hunchback-independent. If a PRE is combined with late enhancers, including a Wingless responsive CNS element or imaginal disc enhancers, repression of a reporter gene can be established everywhere in the embryo, irrespective of the presence or absence of Hunchback protein. If, however, these late enhancers are combined with a Ubx early enhancer, one that is silenced early, as well as with a PRE, then repression is established only where the reporter gene was inactive at early stages. These results imply that the Polycomb complex is not dependent on Hunchback, and suggest that the pattern of silencing reflects rather the level of activity of the gene at the time the Polycomb repressive protein complex is formed (Poux, 1996).
moira (mor) is a member of the trithorax group of homeotic gene regulators in Drosophila. moira is required for the function of multiple homeotic
genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal
tissues. Heterozygous mor mutations suppress the following Polycomb-induced phenotypes: The requirement for moira function is at the level of transcription. The ability of moira mutations to supppress Antp homeotic phenotypes is dependent on the promoter.
moira is also required for transcription of the engrailed segmentation gene in the
imaginal wing disc. Because homozygous mor clones have phenotypes similar to those seen in clones of cells that have lost en function, en transcription was examined in clones of cells in the posterior wing. In the absence of transcriptional activation by mor, the pattern of en is altered. Greatly reduced en expression is found in wing clones. The abnormalities caused by the loss of moira function in germ
cells suggest that at least one other target gene requires moira for normal oogenesis (Brizuela, 1997).
Arresting cell division using the string mutation or blocking DNA
replication with aphidicolin failed to prevent ectopic expression of the homeotic gene Ultrabithorax
in Polycomb mutants. Thus, even in the absence of DNA replication, Pc is required to maintain spatially
restricted patterns of homeotic gene expression (Gould, 1990).
The wave of differentiation that traverses the Drosophila eye disc requires rapid transitions in gene expression that are controlled by a number of signaling molecules also required in other developmental processes. A mosaic genetic screen has been used to systematically identify autosomal genes required for the normal pattern of photoreceptor differentiation, independent of their requirements for viability. In addition to genes known to be important for eye development and to known and novel components of the Hedgehog, Decapentaplegic, Wingless, Epidermal growth factor receptor, and Notch signaling pathways, several members of the Polycomb and trithorax classes of genes, encoding general transcriptional regulators, were identified. Mutations in these genes disrupt the transitions between zones along the anterior-posterior axis of the eye disc that express different combinations of transcription factors. Different trithorax group genes have very different mutant phenotypes, indicating that target genes differ in their requirements for chromatin remodeling, histone modification, and coactivation factors (Janody, 2004).
Very similar phenotypes were observed in clones mutant for Pc or E(z), which encode components of two distinct complexes implicated in transcriptional repression. Although likely null alleles for both genes were used, the phenotype of E(z) clones appeared slightly stronger, with a greater likelihood of derepressing hth in posterior regions of the eye disc. The E(z) protein has been shown to act as a histone methyltransferase for H3 K27 within a complex that also includes Extra sex combs (Esc), Suppressor of zeste 12 [Su(z)12], and the histone-binding protein NURF-55. esc appears to act only early in embryonic development, while E(z) and Su(z)12 are continuously required to repress inappropriate homeotic gene expression in wing imaginal discs. The core PRC1 complex contains Pc, as well as Ph, Psc, and dRing1, and can prevent SWI/SNF complexes from binding to a chromatin template. Pc, Psc, and ph are all required to prevent homeotic gene misexpression in wing discs; however, Psc and ph act redundantly with closely related adjacent genes. The two complexes are thought to be linked through binding of the Pc chromodomain to K27-methylated H3. The stronger phenotype of E(z) mutations in the eye disc might suggest that methylation of H3 K27 can recruit other proteins in addition to Pc (Janody, 2004).
In the eye disc, loss of E(z) or Pc leads to misexpression of the homeotic gene Ubx, but this does not seem to account for the entire phenotype. Although Ubx is sufficient to turn on tsh ectopically, misexpression of hth and tsh can occur in E(z) or Pc clones in which Ubx is not misexpressed. This suggests that hth and tsh are either direct targets of Pc/E(z)-mediated repression or targets of a downstream gene other than Ubx, possibly one of the homeotic genes not examined. Tsh misexpression would be sufficient to explain the suppression of photoreceptor differentiation in clones close to the morphogenetic furrow, since it is able to maintain expression of Hth and Ey and, in combination with them, to repress eya. Misexpression of Tsh can also account for overgrowth of Pc or E(z) mutant cells at the posterior margin of the eye disc (Janody, 2004).
Loss of maternal brahma function blocks
oogenesis; individuals homozygous for extreme brm alleles die as late embryos with no obvious pattern
defects (Brizuella, 1994). Since it has not been possible to generate embryos lacking both
maternal and zygotic brm function, the exact role of brm in embryonic development is not clear.
Information concerning the role of brm after embryogenesis has been derived primarily from the
analysis of hypomorphic brm alleles. Individuals trans-heterozygous for certain combinations of brm
alleles survive to adulthood and exhibit developmental abnormalities similar to those arising from
reduced expression of Antp-C and Bx-C genes, including the transformation of first legs to second legs
and the fifth abdominal segment to a more anterior identity (Brizuella, 1994). Because the
effect of complete loss of brm function had not been examined, it was unclear whether brm is also involved in other processes. To clarify the role of brm in Drosophila development, mosaic analysis has been used to determine the null phenotype of brm mutations. As an
alternative approach, site-directed mutagenesis was used to generate dominant-negative brm mutations
and investigate the functions of evolutionarily conserved domains within the Brm protein (Elfring, 1998).
A dominant-negative brm mutation (DNbrm) was generated by replacing a conserved lysine in the ATP-binding site of the Brm protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the high molecular weight (2 million Daltons) Brm complex. Expression of the dominant-negative Brm protein causes
peripheral nervous system defects, homeotic transformations, and decreased viability. Individuals bearing
one or two copies of the dominant-negative Brm are viable, but frequently exhibit partial transformations
of haltere to wing, as evidenced by an increase in haltere size and the appearance of ectopic bristles on
the capitellum. Approximately one third of dominant-negative Brm adults exhibit this
transformation, which is presumably caused by the decreased expression of the Ultrabithorax
gene. Increasing the ratio of dominant-negative Brm to wild-type Brm to 2:1 is lethal. Thus,
the dominant-negative brm mutation behaves as an antimorphic allele of brm. Expression of the dominant-negative Brm protein in patterns identical to the segmentation genes hairy or engrailed has no effect on embryonic viability or segmentation.
The lack of an embryonic phenotype resulting from embryonic expression of the dominant-negative Brm protein may be caused by the high maternal expression of wild-type Brm protein, which is sufficient to allow
embryogenesis to proceed to near completion in the absence of zygotic brm function. Expression of the dominant-negative protein in imaginal tissues after embryogenesis leads to greatly reduced viability.
Individuals reared at 20° display partial transformation of first leg
to second leg, as evidenced by a reduction in the number of sex comb teeth on the first leg. This phenotype is also seen in adults trans-heterozygous for hypomorphic brm alleles and is presumably caused by decreased expression of the Sex combs
reduced (Scr) gene. Adults reared at 20° also display twinning of mechanosensory bristles, a phenotype similar to that observed in clones of brm2 tissue.
Expression of the dominant negative protein also has dramatic effects on the
size and morphology of the wing; mutant wings are reduced in size, and the L5 and the posterior
cross-vein (PCV) are usually absent. Defects in the campaniform sensilla, a
class of sensory organs important for flight, are also observed with high frequency. These defects fall into four classes:
missing sensilla, duplication or triplication of sensilla, transformation of sensilla into bristles, and the
appearance of ectopic sensilla. Ectopic sensilla and bristles are observed most
frequently on the L3 vein. Three sensilla (L3-1, L3-2, and L3-3) and no bristles are normally found on
this vein. By contrast, approximately one-half of mutant wings display one or two additional sensilla
on L3. Ectopic bristles are observed on this vein in approximately one-fifth of mutant wings (Elfring, 1998).
Transvection is the phenomenon by which the expression of a gene can be controlled by its homologous counterpart in trans,
presumably due to pairing of alleles in diploid interphase cells. Transvection or trans-sensing phenomena have been reported
for several loci in Drosophila, the most thoroughly studied of which is the Bithorax-Complex (BX-C). It is not known how
early trans-sensing occurs nor the extent or duration of the underlying physical interactions. The physical
proximity of homologous genes of the BX-C during Drosophila embryogenesis has been investigated by applying fluorescent in situ
hybridization techniques together with high-resolution confocal light microscopy and digital image processing. The association
of homologous alleles of the BX-C starts in nuclear division cycle 13, reaches a plateau of 70% in postgastrulating embryos,
and is not perturbed by the transcriptional state of the genes throughout embryogenesis.
After gastrulation, the pairing frequency of the Ubx and Abd-B
loci increased to 60-70%. Pairing frequencies never reach 100%,
indicating that the homologous associations are in equilibrium with a dissociated state. The effects of
translocations and a zeste protein null mutation, both of which strongly diminish transvection phenotypes, have been investigated as to the extent of
diploid homolog pairing. Although translocating one allele of the BX-C from the right arm of chromosome 3 to the left arm
of chromosome 3 or to the X chromosome abolishes trans-regulation of the Ultrabithorax gene, pairing of homologous alleles
surprisingly is reduced to only 20-30%. A zeste protein null mutation neither delays the onset of pairing nor leads to
unpairing of the homologous alleles (Gemkow, 1998).
The data support a model for trans-sensing based on physical
proximity and association of the homologous alleles. The
translocation mutants that were investigated demonstrate that
pairing can occur between segments of non-homologous
chromosomes, albeit with a reduced frequency compared to
that for the sites on homologous chromosomes. It is
concluded that the efficacy of the trans-sensing effect is
dependent on the sensitivity of the phenotype to the
transcriptional level of the gene in question and to the stability
or affinity of the specific paired locus.
What model might be consistent with these data and those of
others? In particular, what mechanism is responsible for the
stable pairing of chromosomes? The information that loci on
extreme positions of the 3R chromosome (near the centromere,
central to the chromosome arm and close to the telomere) show
the same frequency and onset of pairing argues against a zipper
mechanism by which a centromeric association leads to a linear
pairing along the chromosome to the telomere. The equal
pairing frequencies observed for the translocation to the X
chromosome and the opposite arm of chromosome 3 also
argue against the centromere having a dominant role in the
pairing mechanism and furthermore require flexibility in the
chromosomal arms with respect to each other. In addition, the
pairing properties of chromosome 4 argue against an earlier
association of centromeric heterochromatin than euchromatic
loci or a dominant role of heterochromatin-binding proteins in
the recognition process. The heterochromatin-binding protein,
HP1, does not appear in embryos until nuclear cycle 10,
increasing dramatically in cycle 14.
Although HP1 has been implicated as a protein causing
heterochromatin aggregation, the data do not support a model
by which heterochromatin regions would preferentially interact
and drive homologue recognition and pairing. The HP1-
binding loci remain independent in diploid embryonic nuclei
even as late as stage 12. Distinct centromeres can be discerned
in many stage 14 diploid nuclei in which chromosome 4 lies
outside the regions occupied by the other centromeres (Gemkow, 1998).
No
preferential disposition of the unpaired BX-C is seen in the lateral
dimension of the nucleus in cycle 14 embryos. In cycle 14
embryos, the largest interallelic distance measured for probes
from the BX-C is close to the diameter of the nucleus but is not
as large as the axial dimension of the nucleus. This is most easily
explained by the fact that, following the rapid early division
cycles, the chromosomes are all still oriented with their
centromeres at the apical surface, although no
chromocenter exists. Decondensation of the chromosomes in
this orientation predetermines a preferred axial disposition of the
locus. That this interpretation is probably correct is supported by results for chromosome 4, which always lies on the apical
surface in blastoderm embryos. These observations are compatible with a multipoint
recognition of sequences dispersed along the chromosome,
resulting in globally stable interactions despite relatively
unstable (short lifetime) individual associations. The
combination of numerous such associations would lead to a
sudden increase in the overall stability of the paired
chromosome, i.e. to a highly cooperative pairing along the
whole chromosome once a threshold number of interactions
was established. If the pairing is driven by associations of the
chromosomes through protein-protein interactions then each
individual paired site will have a finite binding constant and be
in equilibrium with its unpaired state. Thus, if any
individual locus (in this case, the genes of the BX-C) is probed there will
be a probability (or frequency) of dissociation (in this case
about 30%-35%). The fact that the paired state
predominates and that the distance between unpaired loci
becomes greatly reduced at later times during embryogenesis
is consistent with this model. The
paired alleles on the chromosome may be likened to the buttons on a shirt or blouse. Any one button may become unfastened yet the
whole garment will not open. In addition, the probability of
the unpaired region to pair again will be much higher than in
the original recognition step since the local concentration, i.e.
the total volume available to the homologous loci is reduced
by some orders of magnitude due to the associations of sites
flanking the test locus (Gemkow, 1998).
Several conclusions can be derived from these data: (1) the
homology search occurs with approximately the same kinetics
for the BX-C as for chromosome 4, as well as other sites on
chromosome 3 as soon as the cell cycle is lengthened beyond
successive S and M phases, (2) the frequency of pairing in
postmitotic embryos reflects the affinity of the recognition
elements scaled by the size of the chromosomal target locus,
and (3) pairing or association of the chromosomes may be
mediated through protein-protein interactions.
To address this latter point, a protein was investigated that
influences transvection of some genes (in particular white,
yellow, Ubx and decapentaplegic), shows self-association, and binds at several hundred loci on
polytene chromosomes: the protein Zeste. It has been argued that the self-association tendency
of this protein promotes associations of Zeste-binding sites
in both cis and trans. In addition, there are a number of clustered
Zeste-binding sites in the upstream control region for the
Ubx gene, the
locus that was investigated in these experiments. The effect of a zeste null
mutation on pairing at the Ubx locus was of interest, since this
mutant changes the Ubx phenotypes in transvection-sensitive
experiments. A higher variation of the pairing
frequencies was observed but a similar mean was found for both the onset and extent of
pairing, when compared to wild type; that is, mutant embryos were
detected with pairing frequencies as low as 50%. Local decondensation of the fluorescent signals was detected in some of
the chromosomes at the BX-C in zeste mutants. It is
possible that the Polycomb group protein complexes
themselves, which assemble at the PREs in the BX-C, are a stronger determinant of the local pairing
affinity at this locus. Such a possibility is supported by the
observation of the trans interactions of PREs.
The present hypothesis is that many specific protein-protein
interactions are responsible for recognition and pairing along
the chromosomes and that they comprise proteins that have
other functions such as enhancers or repressors. There is clear indication from data on mitotic recombination, that the length of time
between mitoses sets the lower limit on the frequency of
pairing. According to the model presented here,
sites with strong protein-protein interactions would act as
nucleating, sites but stable homolog associations along the
length of the chromatids would only occur by multipoint
recognition and interaction (Gemkow, 1998).
Drosophila Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF,
CHRAC, and ACF. To clarify the biological functions of Iswi, null and dominant-negative Iswi mutations were generated and characterized. Iswi
mutations affect both cell viability and gene expression during Drosophila development. Iswi mutations also cause striking alterations in the structure of the male X
chromosome. The Iswi protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for Iswi in transcriptional
repression. These findings reveal novel functions for the Iswi ATPase and underscore its importance in chromatin remodeling in vivo (Deuring, 2000).
To determine when Iswi is required during development, the lethal phase and phenotype of Iswi null mutants were examined. Individuals heterozygous for ISWI1 or ISWI2 are viable and phenotypically normal. ISWI1/Df(2R)vg-C individuals die during late larval or early pupal development and display no obvious homeotic transformations or other pattern defects. Similar results were obtained for both ISWI2/Df(2R)vg-C and ISWI1/ISWI2 individuals (Deuring, 2000).
In vitro studies have suggested that Iswi plays an important role in transcription by facilitating the interaction of transcription factors with chromatin. One of the best candidates for a transcription factor that requires Iswi for its activity is the GAGA factor. GAGA factor binds to GA-rich sequences near the promoters of a wide variety of Drosophila genes and is thought to activate transcription by altering local chromatin structure. As the ATPase subunit of NURF, Iswi assists the GAGA factor to remodel chromatin in vitro, suggesting that the two proteins may act in concert to modulate chromatin structure in vivo as well. To examine possible interactions between Iswi and GAGA factor in vivo, the phenotypes of mutations in the two genes have been compared. GAGA factor is encoded by Trithorax-like (Trl), a member of the trithorax group of homeotic gene activators. Trl mutations enhance mutations in trithorax and cause homeotic transformations resulting from the decreased transcription of homeotic genes. Trl mutations also enhance position effect variegation, suggesting that GAGA factor antagonizes the assembly or function of heterochromatin. Unlike Trl mutations, Iswi mutations fail to enhance or suppress position effect variegation. No dominant interactions could be detected between mutations in Iswi and other genes, including Trl, other trithorax group genes (trithorax and brm), and Polycomb, a repressor of homeotic genes that is thought to act at the level of chromatin structure. These data suggest that Iswi and GAGA factor play distinct roles in chromatin remodeling in vivo (Deuring, 2000).
To investigate the role of Iswi in transcriptional activation in vivo, the effect of Iswi mutations on the expression of two targets of the GAGA factor were examined: the segmentation gene engrailed (en) and the homeotic gene Ultrabithorax (Ubx). The expression of En protein is reduced dramatically in imaginal discs of ISWI1/ISWI2 mutant larvae. Similar results are observed for Ubx. These data suggest that Iswi is essential for the expression of both en and Ubx in imaginal discs, although the possibility that this interaction is indirect cannot be ruled out (Deuring, 2000).
To directly observe interactions between Iswi and chromatin in vivo, the distribution of Iswi protein on salivary gland polytene chromosomes in third instar larvae was examined by immunofluorescence microscopy. Consistent with a fairly general role in transcription or other processes, Iswi protein is present at a large number of euchromatic sites in the polytene chromosomes. The same pattern was observed using whole sera and affinity-purified antibodies. The chromosomal distribution of Iswi protein is not appreciably altered following heat shock (Deuring, 2000).
Iswi protein is also associated with a subset of heterochromatin, as evidenced by punctate staining at the chromocenter. It is difficult to analyze the distribution of heterochromatic proteins on salivary gland chromosomes, since heterochromatic sequences are underreplicated in polytene tissues. To more accurately map the regions of heterochromatin with which Iswi interacts, the distribution of Iswi protein on mitotic chromosomes from larval neuroblasts was examined. On mitotic chromosomes, Iswi protein is abundantly present on the euchromatic arms of all chromosomes and is concentrated in regions of heterochromatin enriched with middle-repetitive sequences. For example, on the heterochromatic Y chromosome, Iswi is concentrated in the h11–13 region, which is composed almost entirely of middle repetitive DNA families. By contrast, little Iswi protein is detected in regions containing predominantly satellite DNA. The distributions of Iswi and GAGA factor on polytene and mitotic chromosomes were determined by double-label immunofluorescence microscopy. Both GAGA factor and Iswi are associated with hundreds of sites in the euchromatin of polytene chromosomes, but the distributions of the two proteins do not overlap extensively. Even greater differences in the distributions of the two proteins were observed in mitotic chromosomes where the GAGA factor, but not Iswi, is associated with GAGA-satellite sequences. The lack of extensive colocalization does not rule out an interaction between Iswi and GAGA at specific loci, but it does suggest that Iswi and GAGA are not obligatory partners (Deuring, 2000).
The decrease in en and Ubx expression in Iswi mutant larvae is consistent with reports that Iswi is involved in transcriptional activation in vitro. Consequently, it was not anticipated that the distributions of Iswi and RNA Pol II on salivary gland polytene chromosomes would be mutually exclusive. The preferential association of Iswi with transcriptionally inactive regions suggests that Iswi may create changes in chromatin structure that are not conducive to RNA Pol II transcription in vivo. Although there is no direct evidence that Iswi represses transcription, such a function would be consistent with the proposal that Iswi acts antagonistically toward histone acetyltransferases to compact chromatin structure. Based on these observations, further investigation of the role of Iswi in transcriptional repression is clearly warranted (Deuring, 2000).
How can the distributions of Iswi and RNA Pol II on polytene chromosomes be reconciled with the effect of Iswi mutations on gene expression in imaginal discs and the ability of Iswi complexes to activate transcription in vitro? One possibility is that Iswi has roles in both transcriptional repression and activation. NURF, ACF, and CHRAC were purified from Drosophila embryo extracts, and nothing is known about the nature or relative abundance of Iswi complexes in larvae. Perhaps only one Iswi complex is associated with transcriptionally inactive chromatin in the larval salivary gland, while others are either less abundant or transiently interact with chromatin to activate transcription. It is also possible that the interaction of Iswi with en and Ubx is indirect. For instance, the decreased expression of the two genes may be a secondary consequence of reduced cell viability in Iswi mutant larvae (Deuring, 2000).
The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, mutations were isolated in the gene encoding the large NURF subunit, nurf301. NURF is shown to be required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is shown to be required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form (Badenhorst, 2002).
ISWI, the catalytic subunit of NURF, is required for expression of the homeotic gene engrailed (en). However, ISWI is also a component of two other chromatin remodeling complexes, ACF and
CHRAC. To resolve which ISWI-containing complex is required for homeotic gene expression, expression of Ultrabithorax (Ubx) and engrailed (en) were examined in nurf301 mutant animals. When both copies of nurf301 are mutated, in homozygous mutant
nurf3011 larvae, expression of the Ubx protein becomes undetectable. The normal expression of Ubx in the haltere and third leg discs of wild-type third instar larvae is absent in nurf301 mutant animals. Expression of the homeotic gene en requires nurf301. The normal expression of En in the posterior compartment of imaginal discs is abolished in nurf3012 mutants. Semiquantitative RT-PCR analysis confirms that Ubx and en transcript levels are reduced in nurf301 mutant animals. These results confirm that the defects in homeotic transcription seen in iswi mutants are caused by abrogated NURF function (Badenhorst, 2002).
A positive regulator of the Bithorax-Complex, E(bx), has
been localized genetically to 61A, the same cytological interval as nurf301. However, unlike numerous regulators of the
BX-C, E(bx) had not been cloned. Since NURF is required for
expression of Ubx, whether nurf301
corresponds to E(bx) was tested. Both alleles of E(bx) were no
longer extant, so whether the mutations that were isolated in
nurf301 recapitulated the published morphological properties
of E(bx) mutants was tested (Badenhorst, 2002).
nurf301 mutants, like E(bx), increase
the severity of bithorax (bx) mutant phenotypes.
bx is a DNA regulatory element required for correct expression
of Ubx in regions that give rise to the third (T3), but not
second thoracic segment (T2) of the adult fly. This expression distinguishes T3 from T2 identity. Loss or reduction of Ubx levels in bx mutant animals (Ubx6.28/bx34e and
Ubx6.28/bx8 mutant combinations causes a homeotic transformation of the third thoracic segment to the anterior second thoracic segment. Thus, the third thoracic segment, which is normally vestigial and naked, is transformed into the second thoracic segment, increasing its size and causing sensory bristles to develop. Moreover, the
haltere (T3) is transformed toward wing fate (T2), manifested by
increases in size and the development of bristles. The strength of
these transformations is increased when one copy of E(bx) also is removed. Mutation of one copy of nurf301 similarly enhances bx phenotypes. With one
copy of either the nurf3011, nurf3012 or a
deficiency that removes nurf301 -- Df(3L)3643 -- the
strength of the transformation is enhanced. nurf301 enhances both bx34e and bx8 mutations (Badenhorst, 2002).
Although NURF is required for expression of the homeotic genes in
imaginal discs, neither E(bx) nor nurf301 homozygous
mutant larvae display obvious homeotic transformations of the larval cuticle. The absence of mutant larval cuticle phenotypes is likely due
to the large maternal dowry of nurf301 transcript contributed to embryos. Larval cuticular patterning is established before these transcripts have dissipated. Attempts were made to generate embryos lacking the maternal nurf301 contribution through use of the dominant female sterile technique. Although germ-line clones were produced using the parental chromosome, it was not possible to recover germ-line clones using nurf3011. Like ISWI, NURF301 is required for ovary development (Badenhorst, 2002).
An important question is how NURF is recruited to target sites in vivo.
Four genes were shown in this study to be dependent on nurf301 for expression: Ubx, en, hsp26, and
hsp70. All contain multiple binding sites for the GAGA factor,
which is genetically required for their correct expression. On the Drosophila hsp70 and hsp26 promoters, (GA.CT)n cognate elements (to which the GAGA factor binds) are required for HSF-binding.
When these sequences are deleted, HSF-binding to transgenes in polytene
chromosomes is impaired, consistent with the defects seen in nurf301 mutant animals. It is therefore compelling that recent biochemical studies show that NURF and the GAGA factor bind to each other in crude extracts, and that purified NURF301 and GAGA factor interact directly in vitro. The principal interacting domains map to an N-terminal
region of NURF301 and a stretch flanking the Zn finger DNA-binding
motif of GAGA factor. These data suggest that NURF
is recruited by the GAGA factor through specific, direct interactions
with the NURF301 subunit, to catalyze local sliding of nucleosomes at
bx, en, hsp26, and hsp70 promoters,
increasing accessibility to sequence-specific transcription factors and
RNA polymerase II. Curiously, though, reduction of nurf301
levels fails to enhance phenotypes of mutations in Trithorax-like, the gene that encodes the GAGA factor (Badenhorst, 2002).
Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators; these proteins are required to maintain active states of Hox gene expression during development. A trithorax acetylation complex (TAC1) has been purified that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression (Petruk, 2001).
Polycomb group proteins (PcG) repress homeotic genes in cells where these
genes must remain inactive during Drosophila and vertebrate
development. This repression depends on cis-acting silencer sequences, called
Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known
sequence-specific DNA-binding PcG protein, binds to PREs, but pho
mutants show only mild phenotypes compared with other PcG mutants. pho-like, a gene encoding a protein with high similarity
to Pho, has been characterized. Pho-like binds to Pho-binding sites in vitro and pho-like; pho double mutants show more severe misexpression of homeotic genes than
do the single mutants. These results suggest that Pho and Pho-like act
redundantly to repress homeotic genes. The distribution of five
PcG proteins on polytene chromosomes was examined in pho-like, pho double
mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at
most sites in the absence of Pho and Pho-like. At a few chromosomal locations,
however, some of the PcG proteins are no longer present in the absence of Pho
and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein
complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not
participate in the anchoring of PcG complexes, but may be necessary for
transcriptional repression mediated through PREs. In contrast to Pho and
Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other
DNA-binding proteins implicated in PRE function, do not cause misexpression
of homeotic genes or reporter genes in imaginal discs (Brown, 2003).
pho homozygotes die as pharate adults with weak homeotic
transformations, while phol homozygotes survive and are
phenotypically normal adults. By contrast, phol; pho double mutants
die as third instar larvae and fail to pupate. Examination of phol,
pho larvae shows that the brain is smaller than normal, the discs are
misshapen and smaller than wild-type discs, and the salivary gland polytene
chromosomes are enlarged. The larger salivary gland polytene
chromosomes may be due to additional rounds of endoreplication in the double
mutants. To test whether phol functions in PcG repression, Ubx and Abd-B expression was examined in wing imaginal discs
from single and double mutants of phol and pho. As expected, no Ubx
or Abd-B expression was observed in wild-type or phol mutant wing
discs. pho mutants showed misexpression of Ubx in a few cells in the
wing pouch, but did not misexpress Abd-B. By contrast, phol; pho double mutants strongly misexpress Ubx and Abd-B in the wing disc. This suggests that Phol and Pho redundantly repress homeotic genes in imaginal
discs and can partially substitute for each other. Ubx
misexpression is confined to the wing pouch in phol; pho double
mutants; the lack of Ubx misexpression in more peripheral areas of
the disc possibly reflects downregulation by Abd-B, which is strongly
misexpressed in these regions of the disc (Brown, 2003).
Whether removal of phol during larval development
would also cause derepression of Ubx and Abd-B was tested by generating
clones of phol mutant cells in imaginal wing discs of pho
mutant larvae. In these experiments, phol mutant cells were
identified by the absence of a GFP marker. Strong misexpression of Ubx and
Abd-B was observed in double mutant cells in the wing pouch similar to the
misexpression observed in wing discs from the phol; pho double mutant
larvae. These observations suggest that either Phol or Pho is required
throughout development to keep homeotic genes repressed (Brown, 2003).
The Drosophila Polycomb Group (PcG) proteins are
required for stable long term transcriptional silencing of
the homeotic genes. Among PcG genes, esc is unique
in being critically required for establishment of PcG-mediated
silencing during early embryogenesis, but not for
its subsequent maintenance throughout development. Esc has been shown to be physically associated
with the PcG protein E(Z). Esc,
together with E(z), is present in a 600 kDa complex that is
distinct from complexes containing other PcG proteins. This Esc complex has been purified and it also
contains the histone deacetylase Rpd3 and the histone-binding
protein p55 (Chromatin assembly factor 1 subunit), which is also a component of the
chromatin remodeling complex NURF and the chromatin
assembly complex CAF-1. The association of Esc and
E(z) with p55 and Rpd3 is conserved in mammals. Rpd3 is required for silencing mediated by a
Polycomb response element (PRE) in vivo and E(z)
and Rpd3 are bound to the Ubx PRE in vivo, suggesting
that they both act directly at the PRE. It is proposed that histone
deacetylation by this complex is a prerequisite for
establishment of stable long-term silencing by other
continuously required PcG complexes (Tie, 2001).
The presence of Rpd3 in the Esc complex suggests that
histone deacetylation is an intrinsic activity of the Esc
complex and that Rpd3 is required for PRE-mediated
silencing. The related mammalian EED complex has been
shown to contain the Rpd3 homologs HDAC1 and HDAC2,
and immunoprecipitates containing this complex can
deacetylate a histone H4 tail-peptide in vitro. In yeast, Rpd3-dependent repression in vivo has
been shown to be associated with deacetylation of histones H4
and H3.
Which nucleosomes would be deacetylated by the Esc
complex? Histone deacetylation by yeast Rpd3 appears to be
highly localized, extending only one or two nucleosomes from
a site to which it is recruited. Since components of the Esc complex
are physically associated with the Ubx PRE in vivo, Esc-mediated
deacetylation may be restricted to nucleosomes
comprising and immediately adjacent to PREs. Nucleosomes
outside the PRE might also be targeted if the PRE has long-range
interactions with the promoter or if the Esc complex
itself also binds to the promoter or other regions outside the Ubx
PRE, a possibility that the data presented here do not rule out.
Although an effect is observed of several Rpd3 mutations on
silencing of a PRE-mini-white reporter, which is an extremely
sensitive assay, PcG phenotypes have not been reported for
Rpd3 mutants. A hypomorphic Rpd3 allele associated with the
insertion of a P-element transposon in the noncoding 5'
untranslated region has been analyzed in the most detail.
Homozygous mutant embryos derived from germline clones of
this allele do not exhibit PcG phenotypes, but have a pair-rule
phenotype similar to that of ftz mutants. Abundant ubiquitously
distributed Rpd3 RNA and protein of maternal origin are
detectable in early (0-2 hour) wild-type embryos, but are
reduced no more than fivefold in these Rpd3 mutant embryos
derived from germline clones. By stage 9-10, the level of
maternally derived Rpd3 RNA and protein is greatly
diminished. Localized zygotic expression of Rpd3 becomes
detectable in the brain and ventral nervous system of wild-type
embryos, but is not detectable in these mutant embryos, suggesting that this
Rpd3 allele may have a stronger effect on zygotic expression
than maternal expression. If Rpd3 protein derived from
maternally synthesized RNA is sufficient to promote
development of a normal cuticular phenotype, then it remains
possible these mutant embryos may contain sufficient
maternally derived protein to do so and that germline clones of
a true null Rpd3 allele would display PcG phenotypes.
Alternatively, it is possible that the function of Rpd3 in the
Esc complex is not absolutely essential for Esc-dependent
silencing or is redundant, i.e. when eliminated, it can be
compensated by another histone deacetylase, either one
normally associated with the Esc complex or a related one that
can associate with the complex in the absence of Rpd3. A
number of other histone deacetylases have been identified in
Drosophila and at least two are reported to be ubiquitously
distributed in the early embryo (Tie, 2001).
The 600 kDa Esc complex is distinct from complexes
containing PC and other PcG proteins. This suggests that the
Esc complex and other PcG complexes are likely to have
separate functions. Furthermore, in embryos lacking any
functional Esc protein, some weak residual Pc-dependent
silencing activity is still detected, also
supporting separate, if interdependent, functions. Similar
conclusions have been drawn for the homologous mammalian
PcG complexes, which have been reported to be expressed in temporally distinct stages of B cell
differentiation, further suggesting they have distinct functions. In Drosophila, derepression of
homeotic genes is detected slightly earlier in Esc mutants than
in other PcG mutants, raising the possibility
that Esc complex function might be required earlier than other
PcG complexes. However, unlike the apparent temporal
separation of the homologous complexes during mammalian B
cell development, both Esc- and PC-containing complexes are
present together throughout most of embryogenesis, before
Esc disappears, and E(z), like other PcG proteins, is required
continuously throughout development. The phenotypic
similarities between Esc, E(z) and other PcG mutants, the
genetic interactions among them and their common association
with PREs, suggests that their functions, however distinct at
the biochemical level, are interdependent (Tie, 2001).
What role might Esc-mediated histone deacetylation play
in PcG silencing? Given the critical early requirement for Esc,
Esc-mediated deacetylation of PRE-associated nucleosomes
might be an essential prerequisite for the initial binding of one
or more components of PRC1 or other PcG complexes to
PREs. A schematic model is presented for such a function
of the Esc complex in which Esc complex-mediated
deacetylation of PRE associated histones is a critical step in
establishing stable long-term PcG silencing. Alternatively, the
Esc complex may be required for events subsequent to the
initial binding of other PcG proteins to a PRE, perhaps for their
assembly into active silencing complexes or for interaction of
PRE-bound PcG complexes with the promoter. Indeed,
repression of a reporter gene by a tethered GAL4-Pc fusion
protein remains dependent on endogenous Esc and E(z) as
well as other PcG proteins. This indicates that,
at least for PC, constitutive binding to DNA does not bypass
the requirement for Esc and E(z). This also suggests that
while the biochemical evidence reveals no stable direct association of the
Esc complex with other PcG complexes, it remains possible
that there is a transient or less stable association in vivo that is
essential for establishing PcG silencing (Tie, 2001).
Polycomb Group complexes assemble at polycomb
response elements (PREs) in vivo and silence genes in the
surrounding chromatin. To study the recruitment of
silencing complexes, various Polycomb
Group (PcG) proteins have been targeted by fusing them to the LexA DNA
binding domain. When LexA-Pc, -Psc, -Ph or -Su(z)2
are targeted to a reporter gene, they recruit functional
PcG-silencing complexes that recapitulate the silencing
behavior of a PRE: silencing is sensitive to the state of
activity of the target chromatin. When the target is
transcriptionally active, silencing is not established but
when the target is not active at syncytial blastoderm, it
becomes silenced. The repressed state persists through
embryonic development but cannot be maintained in larval
imaginal discs even when the LexA-PcG fusion is
constitutively expressed, suggesting a discontinuity in the
mechanism of repression. These proteins also interact with
other PC-containing complexes in embryonic nuclear
extracts. In contrast LexA-Pho is neither able to silence
nor to interact with Pc-containing complexes. Analysis of
pho mutant embryos and of PRE constructs whose Pho-binding
sites are mutated suggests that, while Pho is
important for silencing in imaginal discs, it is not necessary
for embryonic PcG silencing (Poux, 2001).
These results show that several PcG proteins, targeted by fusion
to a DNA-binding domain, can recruit a repressive PcG
complex. Several new
conclusions can be drawn from these experiments. (1) The
recruitment of the silencing complex cannot occur before
blastoderm. The alpha1-tubulin-LexA-Pc construct reveals that
even when the protein is deposited in the egg during oogenesis,
as well as being zygotically expressed, it does not block the
initiation of transcription from the Ubx promoter. These results
suggest that the LexA-PcG protein cannot establish repression
at this early stage, just as the endogenous PcG proteins known
to be present in the normal pre-blastoderm embryo do not
prevent the initiation of Ubx transcription. Thus, PcG silencing
directed by a PRE or by LexA-Pc appears only to set in after
blastoderm. One possible explanation is that the assembly of a
functional PcG complex at the PRE or at the LexA-binding
sites is a multistep process that requires time and is not
accomplished until after blastoderm. Another interesting
possibility is that the state of the chromatin in nuclei whose
very rapid nuclear divisions are just beginning to slow down,
cannot yet support the establishment of PcG silencing, for
example, because the nucleosomes still bear the deposition-associated histone acetylation pattern. A similar argument
might explain why centric heterochromatin is not detectable
until blastoderm (Poux, 2001).
(2) The repression established by the LexA-PcG protein
is not unconditional but it is sensitive to the state of activity of
the target. Like a genuine PRE complex, the LexA-PcG protein
establishes silencing only in cells in which the reporter gene is
inactive, thus discriminating between active and inactive
chromatin targets. The later-acting Ubx H1 enhancer can still
function but only in the progeny of cells that were active at
early times. The fact that, like the endogenous PRE, the action of the
LexA-PcG protein distinguishes between active and silent
chromatin, indicates that the discrimination occurs after the
binding of the first PcG protein. The sensitive step could be the
assembly of a sufficient nucleus of PcG proteins or still later,
the involvement of other factors that effect the silencing. It has been shown that Pc-containing complex purified from
embryonic nuclear extracts can prevent chromatin remodelling
in vitro if it is bound to chromatin before the addition of
purified SWI/SNF complex but not if it is added
simultaneously or afterwards. If the
activation of the Ubx-lacZ reporter gene by the enhancers
involves the recruitment of the Drosophila SWI/SNF complex,
this observation could help to understand how the LexA
binding sites function as a genuine synthetic PRE possessing
at least one aspect of the cellular memory displayed by
endogenous PREs (Poux, 2001).
(3) Nevertheless, the LexA-binding sites do not constitute a fully
functional PRE. Silencing by LexA-PcG proteins is much less
effective during larval development. It is possible that the
activity of the Ubx H1 enhancer in imaginal discs is more difficult
to repress, e.g. because of very high activator concentration.
More likely, once the H1 enhancer is active, it is much more
difficult to repress by LexA-Pc induced at later times. It cannot be
explained, however, why neither daily heat shocks, nor
constitutive expression of LexA-Pc can maintain a continuity
between embryonic silencing and larval silencing. Repeated
heat shocks do eventually reduce the level of expression of the
reporter in imaginal discs but the memory of the early domains
of repression is lost. The apparent discontinuity in PcG
silencing between the embryo and the larva, suggests that there
might be a real mechanistic difference between the two states.
There are also differences in the
maintenance properties of embryos and larvae. One possible explanation is that additional proteins,
recruited at a true PRE but not by the LexA fusion proteins,
might be necessary for continuous repression at postembryonic
developmental stages (Poux, 2001).
PcG proteins Psc, Su(z)2 and Ph are as effective as Pc in
recruiting a silencing complex, implying that any one of the
'core' PcG proteins can reconstitute a silencing complex. In
contrast, LexA-Gaga and -Pho do not silence the reporter
gene. That LexA-Gaga does not silence is hardly surprising.
Many promoters, including the hsp70 promoter, the alpha1-tubulin
promoter or the Ubx promoter itself, contain Gaga-binding
sites but are not thereby targets for PcG silencing in vivo. The possibility that the LexA-Gaga fusion
protein is defective in some respect cannot be excluded, although it is able to bind
to the normal endogenous sites on polytene chromosomes and
participate in PcG complexes. Most likely, however, Gaga/Trithorax-like
factor by itself cannot recruit PcG complexes. It is supposed that,
like many other nuclear factors, Gaga can have either a
stimulating or a repressing activity, depending on the binding
of other proteins. In the chromatin context of the PRE,
however, Gaga interacts with PcG proteins to form a stable
complex (Poux, 2001).
The Pho protein binds to DNA sequences contained in the
bxd PRE and has been proposed as a major recruiter of PcG
complexes. Mutating the Pho binding sites in the bxd PRE causes loss of
silencing in imaginal discs. In the experiments carried out in this study, however, LexA-Pho is unable to silence the
reporter gene. Since LexA-Pho efficiently rescues pho
mutants in vivo, it is concluded that LexA-Pho is fully functional and that
Pho cannot, by itself, recruit PcG complexes able to silence
the reporter gene either in the embryo or in imaginal discs (Poux, 2001).
The ability of LexA fusion proteins to assemble PcG
complexes that can be targeted to LexA-binding sites allow for the exploration in vitro of the composition of the complexes. It is
important to note that what is seen in the immunoprecipitation
experiments is the bulk of PcG complexes that contain the
LexA-Pc protein. It is not possible to distinguish between complexes
recruited by the LexA-Pc protein at the LexA-binding site and
complexes formed at other PREs, to which the LexA-Pc
protein has been recruited. These experiments therefore do not
necessarily reflect the nature of the complex formed at the
reporter gene, which is limited to what components can be
directly or indirectly recruited by the LexA fusion protein.
Nevertheless, the in vitro binding experiments reveal the ability
of the LexA fusion protein to participate in complexes
containing other PcG proteins (Poux, 2001).
The binding experiments show that the four 'core' proteins
Pc, Psc, Su(z)2 and Ph are recruited to PcG complexes and
are themselves able to recruit repressive complexes. It cannot be
shown that all four proteins are simultaneously associated in one
complex but it is very likely that at least Pc, Psc and Ph can
participate in the same complex. Similar
experiments have also shown that Gaga factor is associated
with at least some PcG complexes, although
it is not itself able to recruit them to the reporter gene. In contrast,
Pho neither recruits any of the core PcG proteins nor
participates in the majority of PcG complexes present in
embryonic extracts. Conceivably, when the LexA-binding
domain is bound to DNA, it might hinder the access of other
PcG proteins to the Pho moiety. It is thought that this is unlikely
because even wild-type Pho does not seem to be associated with
PcG proteins. A PRE fragment containing three Pho binding
sites but no GAGA sites does not bind PC-containing complexes
in vitro, although it interacts readily with
endogenous Pho or LexA-Pho in the same embryonic extracts,
indicating that also endogenous Pho is not stably associated
with PcG complexes. Similarly, Pho is not detected in a
purified PRC1 complex (Poux, 2001).
There remains the possibility that Pho is not the protein that
binds in vivo to the Pho consensus binding sites in the PRE.
These sites
in the bxd PRE are important for silencing in imaginal discs. Formally, this does not exclude the
possibility that these are binding sites for some other protein
required for silencing. More likely, Pho does intervene at the
PRE but not as a direct recruiter of PcG complexes. Its role
might be to alter the chromatin conformation at the PRE and
facilitate the interactions between different components
necessary for effective silencing. Mammalian YY1 has been
shown to act as a repressor as well as an activator, depending
on the context. It
can interact directly with histone deacetylases and,
presumably, contribute directly to repression. In addition, it may be a component of some mammalian
PcG complexes. In Drosophila, Pho
might make a similar contribution to silencing, at least when
it is bound in the context of the PRE. The domain of YY1 that
interacts with HDAC is not conserved in Pho but a different
sequence motif might preserve the ability to interact with
Drosophila histone deacetylase complexes (Poux, 2001).
These experiments, which show no obvious Ubx derepression in
pho1/pho1 embryos and no derepression when all pho consensus sites in the PRE are mutated, support the idea that
neither Pho nor its binding sites play an essential role in
embryonic PcG silencing. A very
limited derepression of abdA and AbdB has been reported in pho mutant embryos. This effect, involving ectopic expression
in a few cells of PS5, might also be interpreted as an indirect
consequence of segmentation defects frequently observed in
pho mutants rather than a real
derepression. In any case, the available evidence excludes a
major role for Pho as a recruiter of PcG complexes. However, Pho-binding sites are necessary for silencing in
larval imaginal discs and pho function is
required for normal imaginal segmental identity. These results
suggest that this function is required to stabilize PcG silencing
in late larval and pupal development, and that its essential role
in embryonic development is unrelated to homeotic regulation. The establishment and maintenance of silencing complexes at
a PRE is clearly a complicated multistage process and the
nature of the initial recruiters is still unknown (Poux, 2001).
To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) was used to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. PcG proteins interact in vitro with GTFs. It is suggested that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription (Breiling, 2001).
For X-ChIP analysis of promoter regions, the following PcG target genes were chosen: Abdominal-B (Abd-B, B-promoter), iab-4, abdominal-A (abd-A, AI-promoter) and
Ultrabithorax (Ubx), all located in the Bithorax complex (BX-C), engrailed (en) and empty spiracles (ems). Also chosen were RpII140 (the subunit of RNA
polymerase II with relative molecular mass 140,000 [Mr 140K]) and brown (bw): these last two do not reside in PC binding sites on polytene chromosomes and thus are most probably not PcG regulated. Expression of these genes in Drosophila SL-2 culture cells was assessed by polymerase chain reaction with reverse transcription (RT-PCR) and it was found that Abd-B and RpII140 are transcribed whereas iab-4, abd-A, Ubx, en, ems and bw are inactive (Breiling, 2001).
Acetylation of histones H3 and H4 is considered to be a mark for ongoing transcription. Thus, the promoters of the genes were screened for the presence of
amino-terminally acetylated H4 and H3 by X-ChIP. Two antisera were used, one that recognizes H4 acetylated at lysine 12 and one or more other lysines, and one
that recognizes H3 acetylated at lysines 9 and/or 18. H4 was found generally acetylated across the promoter regions analysed, in some cases with reduced levels
in upstream and downstream regions. H3 is strongly acetylated in the active Abd-B and RpII140 promoters, whereas the
inactive loci (iab-4, abd-A, Ubx, en, ems and bw) showed a decrease (5-10 times less than the H3 signal in the active Abd-B and RpII140 promoters) or absence of acetylation both at the core promoters as well as downstream of the initiator. Thus, H3 is acetylated in the active but underacetylated in the inactive
promoters, whereas H4 acetylation shows no such changes. Acetylation of histones H3 and H4 seems to be regulated independently across the BX-C, consistent with
results in other systems (Breiling, 2001).
The same promoter regions were analyzed by X-ChIP using antibodies against the PcG proteins Polycomb (PC) and Polyhomeotic (PH), dHDAC1, TBP, TFIIB and TFIIF
(RAP 30 subunit, associated with RNA polymerase II). All six proteins were found in the core promoter regions (200 base pairs [bp] around the initiator) of the Abd-B, iab-4, abd-A, Ubx, en and ems transcription units. PC was found in most regions both upstream and downstream of the transcription start site (Breiling, 2001).
The major conclusion from this work is that promoters constitute a key target of PcG function. Evidence is provided that, unexpectedly, GTFs are retained at
PcG-repressed promoters and that PcG proteins may function through direct physical interactions with GTFs. This mechanism of transcriptional regulation may provide both transcriptional competence and the flexibility necessary for the rapid re-arrangement of patterns of gene expression in response to developmental signals. Thus, the presence of GTFs and some trxG proteins at PcG-repressed promoters would allow a relatively fast re-activation of these genes, as differentiation processes require. In this context, PcG proteins would need to be continuously present at target gene promoters to constitutively inhibit transcription, a prediction supported by the finding that PcG-repressed genes are re-expressed in cells depleted of PcG proteins by dsRNA interference (Breiling, 2001).
Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Drosophila Rpd3, referred to here by its alternative name HDAC1, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes (Chang, 2001).
To demonstrate that the effect of Hdac1 mutations is exerted at the level of expression of homeotic genes, the expressions were examined of Sex combs reduced (Scr) and Ultrabithorax (Ubx) proteins in wild-type and Pc mutant imaginal discs. Scr proteins normally are expressed at high levels in the first leg discs, but are not expressed in the second and third leg discs. In Pc4 mutant heterozygotes, however, Scr proteins also can be detected at low levels in second and third leg discs. Consistent with the increase in ectopic sex comb teeth, dramatic increases in the levels of Scr proteins are observed in the second and third leg discs from Pc4 mutant heterozygotes that were also heterozygous for any of the Hdac1 alleles except Hdac1326. In addition, Ubx proteins are marginally detectable only in the peripodial membranes of imaginal wing discs of wild-type or Pc4 mutant heterozygous larvae. In larvae heterozygous for both Pc4 and an Hdac1 mutation, high levels of Ubx proteins are observed in the medial sections of the wing discs proper. In contrast to the lack of ectopic Scr expression in Pc4 heterozygotes carrying the Hdac1326 allele, a much stronger effect on ectopic Ubx expression is observed; Ubx protein levels in both first and second leg discs are increased substantially. It is highly likely that the expanded Ubx expression reduces Scr expression, resulting in suppressed Pc phenotype (i.e., reduced numbers of ectopic sex comb teeth) in Pc4/Hdac1326 trans-heterozygotes. These results strongly suggest that Hdac1 acts cooperatively with Pc to repress homeotic genes during larval and pupal development (Chang, 2001).
Experiments also were performed to explore the role of Hdac1 in regulating the embryonic expressions of two homeotic genes, Abd-B and Ubx. Abd-B proteins normally are expressed in a graded fashion in the posterior part of ventral nerve cord, starting from parasegment 10 (PS10). Although this pattern is not altered in homozygous Hdac1303 mutants, significant levels of Abd-B proteins are observed in more anterior parasegments of homozygous Psce24 mutants. Much higher levels of ectopic Abd-B proteins are found in Psce24 Hdac1303 double mutants, indicating a synergistic effect of Hdac1 and Psc on Abd-B repression. Consistent (but less striking) effects also are observed on Ubx protein levels. The anterior boundary of the Ubx expression domain is PS5, with the exception of a small cluster of cells in the middle of PS4 that also express Ubx proteins. Although homozygous Psce24 mutants only show sporadic low levels of Ubx expression in more anterior parasegments, Psce24 Hdac1303 double mutants show significantly higher levels of ectopic Ubx expression in more cells. In PS5, more cells with higher levels of Ubx proteins are observed in the double mutants than in either of the single mutants. In contrast, Ubx expression is reduced substantially in the abdominal parasegments of the double mutants compared with that in the single mutants, presumably reflecting Ubx repression by more extensive ectopic expression of Abd-B and possibly ABD-A. These data indicate that Hdac1 is essential for homeotic gene silencing in embryos (Chang, 2001).
Given the genetic and biochemical interactions between Hdac1 and Pc, it might be anticipated that a fraction of HDAC1 would colocalize with Pc-G complexes on polytene chromosomes. Approximately 100 common binding sites have been identified for several Pc-G proteins. At least 70% of these sites (identified by staining with PSC mAbs) also stain with the HDAC1 antibody, including the Antennapedia complex at 84AB and the bithorax complex at 89E. These results suggest that HDAC1 proteins act together with a substantial fraction of the Pc-G complex. However, the relative intensities of the signals for PSC and HDAC1 at these sites do not always correlate, suggesting a regulatory, rather than a constitutive function. Furthermore, HDAC1 is much more widely distributed along the chromosomes than is PSC, consistent with its role in global gene regulation and/or chromatin structure (Chang, 2001).
The colocalizations of HDAC1 and PSC were examined further on polytene chromosomes from a transgenic line that carries a Ubx upstream cis-regulatory region (i.e., bxd-14) inserted at 62A. This insert contains a functional PRE and creates a new Pc-G-binding site. Staining with both PSC and HDAC1 antibodies reveals that a new PSC site coincides with a new HDAC1-binding site. This new binding site is beside an HDAC1 site present in the wild-type chromosome, creating a broader signal of HDAC1 at this site. These results strongly suggest that HDAC1 and Pc-G proteins are recruited to this ectopic PRE (Chang, 2001).
In both Drosophila and vertebrates, spatially restricted expression of HOX genes is controlled by the Polycomb
group (PcG) repressors. Mutants of a novel Drosophila PcG gene, Suppressor of zeste 12 [Su(z)12], exhibit very strong homeotic transformations. Su(z)12 function is required throughout development to
maintain the repressed state of HOX genes. Unlike most other PcG mutations, Su(z)12 mutations are strong
suppressors of position-effect variegation (PEV), suggesting that Su(z)12 also functions in heterochromatin-mediated
repression. Furthermore, Su(z)12 function is required for germ cell development. The Su(z)12 protein is highly conserved in vertebrates and is related to the Arabidopsis proteins EMF2, FIS2 and VRN2. Notably, EMF2 is a repressor of floral homeotic genes. These results suggest that at least some
of the regulatory machinery that controls homeotic gene expression is conserved between animals and plants (Birve, 2001).
Su(z)12 hemizygous mutant embryos derived from Su(z)12 mutant germ cells already show very extensive misexpression of Ubx at the
extended germ band stage. These animals showed severe homeotic phenotypes with all abdominal, thoracic and several head segments
transformed into copies of the eight abdominal segment. This phenotype is consistent with Abd-B being misexpressed in all segments.
The strong PcG phenotype of these Su(z)12 mutant embryos is comparable to that of embryos lacking esc or Pc function. Zygotically provided Su(z)12 function is sufficient to prevent the inappropriate activation of HOX genes; Su(z)12 -/+ heterozygotes obtained as the progeny
of Su(z)12 mutant germ cells and a wild-type sperm develop into wild-type-looking adults (Birve, 2001).
The requirement for Su(z)12 at later developmental stages was tested by generating Su(z)12 mutant clones in imaginal discs. Assays were performed for HOX gene silencing in such clones by monitoring the expression of the HOX genes Ubx and Abd-B in the imaginal wing disc (where they are normally stably repressed) using antisera against their protein products. In these experiments, the Su(z)12 mutant cells were identified by the absence of a GFP-expressing marker gene. In addition, the Minute technique was used to generate Su(z)12-/Su(z)12-clones that carry two copies of a wild-type Minute allele [i.e., Su(z)12- M+/ Su(z)12- M+], which gives them a growth advantage relative to their Su(z)12- M+/ Su(z)12+ M- neighbors (Birve, 2001).
Cell clones of the different Su(z)12 alleles were examined 96 hours after clone induction. Su(z)12 mutant clones show strong misexpression of both Ubx and Abd-B in most mutant cells. In summary, the PcG phenotypes observed with several Su(z)12 alleles suggest that Su(z)12 is needed throughout development to keep HOX genes repressed. Moreover, these results support the allele classification obtained by the analysis of germ-line clones; namely, that Su(z)122 and Su(z)125 are hypomorphic alleles whereas Su(z)121 and Su(z)124 appear to be stronger alleles (Birve, 2001).
The kinetics of HOX gene derepression was examined in Su(z)121 and Su(z)124 mutant clones by analyzing Ubx expression 24, 48 and 72 hours after clone induction. The Minute technique was used in these experiments. 24 hours after clone induction, Ubx is still tightly repressed. 48 hours after clone induction, Su(z)121 mutant clones show misexpression of Ubx protein in the wing pouch but Ubx is still stably silenced in other parts of the wing disc. In Su(z)124 mutant clones, Ubx is still stably silenced 48 hours after clone induction except in a few clones in the center of the pouch where weak Ubx signal was detected. Finally, 72 hours after clone induction, repression of Ubx is lost in most Su(z)121 and Su(z)124 mutant clones in the pouch, in the latter case Ubx is still silenced in some parts of the disc. This slow and gradual loss of silencing is comparable to the kinetics of HOX gene derepression in Pc, Pcl, Scm or Sce mutant clones (Birve, 2001).
Database searches show that the Su(z)12 protein is highly conserved in vertebrates and, strikingly, that Su(z)12-related proteins also exist in plants. In contrast, the worm and yeast genomes do not seem to encode Su(z)12-related proteins. The function of the highly conserved human homolog of Su(z)12, HsSU(Z)12, is not known but EMF2, FIS2 and VRN2, the three Su(z)12-related proteins in Arabidopsis, have been identified as regulators in plant development. One characteristic feature of all these proteins is a single classical C2H2 zinc finger similar to the fingers found in sequence-specific DNA-binding proteins. Attempts to show any DNA-binding activity of a polypeptide containing the Su(z)12 zinc finger have failed so far. A second stretch of amino acids that is conserved between Su(z)12, HsSU(Z)12, EMF2, VRN2 and FIS2 is located C-terminal to the zinc finger. This part of the protein has been termed the VEFS box [VRN2-EMF2-FIS2-Su(z)12 box]. The predicted protein products encoded by Su(z)123 and Su(z)124 lack both the zinc finger and the VEFS box, whereas the protein encoded by Su(z)121 is predicted to contain the zinc finger but lacks the VEFS box (Birve, 2001).
During late embryogenesis, the expression domains of homeotic genes are maintained by two groups of ubiquitously
expressed regulators: the Polycomb repressors and the Trithorax activators. It is not known how the activities of the
two maintenance systems are initially targeted to the correct genes. Zeste and GAGA are sequence-specific DNA-binding proteins that are Trithorax group activators of the homeotic gene Ultrabithorax. Zeste and GAGA DNA-binding sites at the proximal promoter are also required to maintain, but
not to initiate, repression of Ubx. Furthermore, the repression mediated by Zeste DNA-binding site is abolished in zeste null embryos. These data
imply that Zeste and probably GAGA mediate Polycomb repression. A model is presented in which the dual transcriptional activities of Zeste and
GAGA are an essential component of the mechanism that chooses which maintenance system is to be targeted to a given promoter (Hur, 2002).
Zeste, GAGA and a third transcription factor, NTF-1 (Grainy head), activate promoter constructs of the Ubx gene in embryos via an intermingled cluster of sites between nucleotides -200 to -31. However, the constructs that were used in these experiments contain only a small subset of the Ubx cis regulatory region, and while they reproduce many features of Ubx expression, they do not respond to Polycomb repression when inserted at many chromosomal locations. Consequently, they have not permitted a rigorous analysis of the role of the proximal promoter factors in maintaining repression. To address this question, larger constructs have been used that contain the 22 kb of DNA upstream of the Ubx mRNA start site. These constructs do not suffer from significant position effect variation; they more closely approximate the expression pattern of the endogenous Ubx gene than the shorter constructs; they maintain efficient repression in late embryos as shown by the lack of ß-galactosidase reporter gene expression in more anterior and posterior regions, and they are genetically under the control of PcG genes (Hur, 2002).
Deletion of nucleotides -200 to -31 essentially abolishes transcription from the large Ubx promoter constructs, indicating a crucial role for factors binding to the proximal promoter. To determine the role of each factor separately, three constructs were prepared, each containing binding sites for either Zeste, GAGA or NTF-1 inserted between the deletion end points of the above construct. Importantly, biochemical, in vivo u.v. crosslinking, and genetic experiments strongly suggest that the DNA-binding sites used in these constructs are recognized only by their cognate factor, and not by any other sequence-specific DNA-binding activities. Binding sites for each factor separately activate transcription of the large constructs during late embryogenesis. Strikingly, constructs containing only GAGA- or Zeste-binding sites at the proximal promoter are not expressed in the anterior or posterior of the embryo, whereas constructs bearing only NTF-1 sites are strongly transcribed in these terminal regions (Hur, 2002).
Ectopic expression of Ubx in anterior and posterior regions is generally caused by a failure of the initiating repressors or the Polycomb maintenance system. One interpretation of this result is that Zeste and GAGA are required for at least one form of repression, while NTF-1 is not. It is also possible, however, that Zeste and GAGA are not repressors. Instead, it may be that they are unable to activate expression in anterior or posterior regions, even though they are expressed at similar levels throughout the embryo. To distinguish between these two possibilities, constructs were examined that contained either Zeste and NTF-1 sites or GAGA and NTF-1 sites. These constructs are expressed in the central region of the embryo; but, importantly, they are not significantly expressed in anterior or posterior regions. Since NTF-1 can activate Ubx transcription in these terminal regions, the absence of terminal expression is consistent with GAGA and Zeste directly repressing transcription in addition to their activation function (Hur, 2002).
To establish decisively if Zeste and GAGA are repressors, it was desirable to use a genetic test. Unfortunately, GAGA is a lethal gene and a broadly acting regulator required for expression of transcription factors that regulate Ubx in early embryos. Thus, it has not been possible to determine genetically whether GAGA is a direct repressor of Ubx. By contrast, zeste is a largely redundant gene. zeste null embryos and flies are essentially wild type, and the endogenous Ubx gene is expressed normally in these animals; but because the 22UZ transgenes lack the cis regulatory elements through which factors that redundantly share the function of zeste act, these transgenes should be regulated by zeste (Hur, 2002).
Consistent with this idea, transgenes containing only Zeste sites at the proximal promoter fail to express in zeste mutant embryos, whereas constructs containing only GAGA or NTF-1 binding sites are expressed in this same genetic background. Thus, this genetic experiment confirms that Zeste bound at the proximal promoter is required to activate transcription of the 22UZ constructs in the normal domain of Ubx expression. To test the role of Zeste in repression, constructs containing binding sites for both Zeste and NTF-1 at the proximal promoter were compared in wild type and zeste mutant embryos. In the normal domain of Ubx expression, these constructs are expressed at similar levels in mutant and wild-type embryos. Importantly, these constructs are derepressed in anterior and posterior regions of embryos lacking zeste. Thus, Zeste actively represses transcription in terminal regions of the embryo via binding sites at the proximal promoter (Hur, 2002).
To distinguish if Zeste is required for the initiation or the maintenance of repression, expression of the 22UZ ZESTE/NTF-1 construct was examined at an earlier stage. In embryos that lack zeste, the 22UZ ZESTE/NTF-1 transgene is almost fully repressed in anterior and posterior regions at this earlier stage. Only weak derepression is observed in a few isolated cells. Thus, the transiently expressed factors that initiate repression in the early embryo must be active, and the extensive derepression observed later must be due to a failure in the maintenance system (Hur, 2002).
The PcG genes are an essential part of system that maintains repression of the endogenous Ubx gene. To confirm that these genes also act on these transgenes, the 22UZ Zeste and 22UZ GAGA constructs were crossed into PcG mutant embryos. Both transgenes are derepressed in late stage embryos lacking the Polycomb gene. Similar results were obtained in embryos lacking another PcG gene, extra sex combs. Thus, Zeste -- and probably also GAGA -- act together with the Polycomb system to maintain repression of Ubx (Hur, 2002).
It is suspected that GAGA and Zeste have redundant, overlapping functions in maintaining repression because the 22UZ Native construct, which contains Zeste, GAGA and NTF-1 sites, is not derepressed in zeste mutant embryos, which contrasts with the behavior of the 22UZ ZESTE/NTF-1 construct. Such redundancy in repression would parallel the known redundancy between these two transcription factors in activating Ubx in the central portions of the animal, and helps explain the previous lack of evidence that Zeste and GAGA are repressors (Hur, 2002).
The data presented in this paper are consistent with the earlier genetic data that suggested that some trxG and PcG proteins may have dual activities. Further support for this idea comes from recent biochemical experiments that have shown that GAGA is complexed with two PcG proteins in Drosophila nuclear extracts and Zeste is part of a multisubunit complex that contains Polycomb. In addition, PcG proteins are frequently associated in vivo with promoter regions that include Zeste or GAGA DNA recognition sites, including the Ubx proximal promoter examined in this paper. Most PcG proteins do not recognize specific DNA sequences; thus, the interaction with Zeste and GAGA may serve to recruit PcG proteins to promoters (Hur, 2002).
But is it essential that some proteins, such as Zeste and GAGA, participate in both repression and activation, or is it mere coincidence? This joint participation may be essential. At the transition between the initiating repressors and the Polycomb system, one possibility is it that Polycomb proteins are recruited to or activated on only those genes that are bound by initiating repressors; the initiating repressors may physically bind to PcG proteins to recruit them. However, Polycomb repression can be established on Ubx promoter constructs that lack initiating repressors elements, provided that initiating enhancer elements are also absent. In other words, at the transition between the establishment and maintenance of the Ubx expression pattern, the Polycomb systems reads the absence of activation, rather than the presence of repression or repressors (Hur, 2002).
Endogenous Zeste protein binds to Ubx promoter constructs in vivo whether they are transcribed or not. It is suggested that in the early embryo in the cells in which Ubx is activated, Zeste is complexed, directly or indirectly, with initiating activators on the Ubx promoter. These complexes mask surfaces on Zeste that would otherwise be bound by components of the Polycomb system. By contrast, in those cells where Ubx is not activated, Zeste is still bound to the promoter but is not a part of an activating complex. Surfaces on Zeste protein would then be exposed and could serve as the signal that the Polycomb system reads to initiate the maintenance phase of repression. The dual activities of Zeste and GAGA could be a key to understanding this fascinating regulatory mechanism (Hur, 2002).
Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, evidence is provided for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, in vivo evidence is provided that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues either PEV modification, telomeric silencing of SAS2-deficient yeast cells, or lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, the observation that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels (Grienenberger, 2002).
Direct evidence for a role of Chm in Hox gene silencing was obtained from the examination of Ubx expression in imaginal discs. Whereas Ubx is not detected in the columnar epithelium of a wild-type wing disc, derepression is observed in few cells from discs heterozygous for Pc, and a more extended activation occurs in discs heterozygous for both chm and Pc. These results confirm that Chm and PcG proteins act together to repress Hox genes. No misexpression of Ubx could be detected, however, in discs from chm homozygous larvae. Thus, chm can be classified as an enhancer of PcG mutations instead of a novel PcG gene (Grienenberger, 2002).
The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. The epigenetic activator Ash1 is a multi-catalytic histone
methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns (Beisel, 2002).
Polycomb group (PcG) proteins play important roles in
maintaining the silent state of HOX genes. Recent studies have
implicated histone methylation in long-term gene silencing. However, a
connection between PcG-mediated gene silencing and histone methylation
has not been established. This study reports the purification and
characterization of an EED-EZH2 complex, the human counterpart of the
Drosophila ESC-E(Z) complex. The
complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, it is shown that
H3-K27 methylation colocalizes with, and is dependent on, E(z)
binding at an Ultrabithorax (Ubx) Polycomb
response element (PRE), and that this methylation correlates with
Ubx repression. Methylation on H3-K27 facilitates binding of
Polycomb (Pc), a component of the PRC1 complex, to histone H3
amino-terminal tail. Thus, these studies establish a link between
histone methylation and PcG-mediated gene silencing (Cao, 2002).
To understand the functional relation between E(z)-mediated H3-K27
methylation and HOX gene silencing, a study was carried out of E(z) binding, H3-K27 methylation, and recruitment of PC, a core component of the PRC1
complex, to the major PRE of the Ubx gene in
S2 tissue culture cells by chromatin immunoprecipitation (ChIP). Consistent with the involvement of E(z) in H3-K27 methylation, ChIP
analysis of a 4.4-kb region that includes this PRE showed
precise colocalization of E(z) binding and H3-K27 methylation. In contrast, similar colocalization was not observed for
mK9, indicating that H3-K9 methylation, or at least K9-dimethylation,
is independent of E(z) binding. To further verify the importance of E(z)
binding for H3-K27 methylation, attempts were made to disrupt Esc-E(z)
complex activity using RNA interference (RNAi). It was reasoned that
depletion of the Esc protein, a direct binding partner of E(z) and a
component of the Esc-E(z) complex, would result in disruption of PRE
binding by E(z). Depletion of Esc with RNAi results in greatly reduced PRE binding by E(z), loss of H3-K27 methylation, and concomitant loss
of PC binding. Depletion of PC in S2 cells has
been shown to result in derepression of Ubx.
Therefore, these data collectively suggest that the Esc-E(z) complex is
critical not only for H3-K27 methylation, but also for PC binding to
the PRE region, and that H3-K27 methylation is associated with
Ubx repression (Cao, 2002).
To examine the relation between E(z) binding, H3-K27 methylation, and
Ubx gene repression in vivo, wing imaginal discs were dissected from homozygous E(z)61 larvae that had
been either reared continuously at 18°C or shifted from 18° to
29°C ~48 hours before dissection, and analyzed E(z) binding and
H3-K27 methylation in the same Ubx PRE region by ChIP. Consistent with previous studies demonstrating disruption of polytene chromosome binding by both E(z)61 and PC proteins at 29°C, ChIP analysis showed loss of E(z)61 and PC binding to this PRE at restrictive temperature.
In addition, H3-K27 methylation colocalizes with E(z) binding at
permissive temperature, but is lost along with E(z) binding at 29°C.
In contrast, similar changes in H3-K9 methylation were not observed
under the same conditions. Under normal conditions,
Ubx is not expressed in wing discs due to PcG-mediated
silencing. Similar inactivation of an
E(z) temperature-sensitive allele during larval
development has been shown to result in derepression of Ubx
in wing discs. Thus, Ubx PRE-associated
nucleosomes appear to be targeted by E(z)-mediated H3-K27 methylation,
which correlates with PC binding and repression of Ubx.
Collectively, these data suggest that H3-K27 methylation plays an
important role in the maintenance of Ubx gene silencing (Cao, 2002).
Polycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd) PRE contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, DNA binding activities have been identified that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)(3) repeats for MHS-70 binding in vitro. Mutation of d(GA)(3) repeats within MHS-70 in the context of the 5.1-kb bxd PRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)(3) repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)(3) repeats, the Trl/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations in Trl and psq enhance homeotic phenotypes of ph, indicating that Trl/GAGA factor and Psq are enhancers of Polycomb that have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of the bxd PRE by d(GA)(n) repeat binding activities mediates PcG-dependent silencing (Hodgson, 2001).
Repression and activation of the expression of homeotic genes are maintained by proteins encoded by the Polycomb group (PcG) and trithorax group (trxG) genes. Complexes formed by these proteins are targeted by PcG or trxG response elements (PREs/TREs), which share binding sites for several of the same factors. The repressive class II PcG complex PRC1 has more than 30 protein subunits, including 5 that have been genetically defined as PcG proteins: Polycomb (Pc), Posterior sex combs (PSC), Polyhomeotic (Ph), dRING1, and, at substoichiometric levels, sex combs on midlegs (SCM).
GAGA factor and Zeste bind specifically to PREs/TREs and have been shown to act as both activators and repressors. Purified proteins and complexes have been reconstituted from recombinant subunits to characterize the effects of GAGA and Zeste proteins on PcG function using a defined in vitro system. Zeste directly associates with the PRC1 core complex (PCC) and enhances the inhibitory activity of this complex on all templates, with a preference for templates with Zeste binding sites. GAGA does not stably associate with PCC, but nucleosomal templates bound by GAGA are more efficiently bound and more efficiently inhibited by PCC. Thus Zeste and GAGA factor use distinct means to increase repression mediated by PRC1 (Mulholland, 2003).
To demonstrate directly that GAGA enhances template recognition by PCC, a recruitment assay was developed. Ubx5S DNA, consisting of a portion of the Ubx promoter with known high-affinity Zeste binding sites, was biotinylated, assembled into chromatin, and immobilized on streptavidin-coated magnetic beads. Nucleosomal arrays were incubated with either buffer only or GAGA for 15 min at 30°C prior to addition of PCC. Following a 20-min binding period, array-bound beads and all material bound to them were separated magnetically from unbound protein. Western blot analysis demonstrates that PCC and GAGA components bound to the nucleosomal array-bead complex and that PCC association is increased on arrays bound by GAGA. GAGA and PCC bind only minimally to unconjugated beads. Increasing amounts of competitor nucleosomal array result in a loss of PCC association with the array-bead complex, but do not affect PCC association with the GAGA array-bead complex. These results demonstrate that PCC has a higher affinity for nucleosomal templates bound by the GAGA factor. Prebinding the GAGA{Delta}POZ protein does not lead to increased recruitment of PCC (Mulholland, 2003).
These experiments show that a template prebound by GAGA factor is more efficiently bound and repressed by PCC than an unbound template. GAGA factor might recruit or stabilize PCC binding by directly interacting with its subunits, or GAGA might alter the template in a manner that favors PCC binding. GAGA factor oligomers have been shown to be able to bind multiple templates simultaneously, bringing them together. Binding by GAGA factor might create a network of templates that is more efficiently bound and recognized by PCC than an individual template might be (Mulholland, 2003).
In the defined in vitro system used in this study, both Zeste and GAGA factor can enhance the activity of a PRC1 core complex to repress remodeling of a nucleosomal template. Zeste binds directly to these PcG proteins to generally increase their repressive function, and prebinding GAGA factor to the template recruits the PRC1 core to that template. Previous genetic and mechanistic studies have suggested that regulation of PRC1 repression is a complicated process involving targeting by sequence-specific DNA binding proteins, covalent modification of histone tails, and perhaps targeting by siRNAs. The differences in function of GAGA and Zeste suggest that their role in PcG repression is more complex than previously suspected. One hypothesis for how sequence-specific factors establish PcG repression is that they create a binding surface with greater affinity for PRC1. Although these experiments with GAGA factor are consistent with this hypothesis, the experiments with Zeste suggest that additional mechanisms contribute to targeting by sequence-specific factors. Zeste binds tightly to the core components of PRC1 and enhances their activity even when templates do not contain targeting sequences. This might be important in facilitating the ability of PRC1 repression to spread away from PRE elements, and thus may facilitate the repression of large domains by PRC1 (Mulholland, 2003).
Although originally identified as an activator, Zeste can also function in vivo as a repressor. For example, in zeste mutant flies, a transgene containing the Ubx promoter modified to contain only Zeste binding sites is derepressed in the anterior and posterior segments of the embryo. Two distinct substitution mutants of zeste express proteins that repress rather than activate the white gene but retain activator function required for transvection, suggesting that Zeste has both inherent activation and repression activities that can be separated. It is possible that, when incorporated into PRC1, Zeste is configured so as to only display surfaces responsible for repression (Mulholland, 2003).
It is widely believed that PcG activity is targeted and maintained throughout the course of development by multiple systems. For instance, ESC/E(z) can methylate H3 K27, and PC can bind to this modification, suggesting that a methylation mark might also play a key role in targeting PRC1 and/or in regulating the spread of PRC1 activity. The combined effects of factors such as GAGA that target PRC1 activity, factors such as Zeste that augment PRC1 activity, and other systems such as those for covalent modification of histones might be necessary for faithful maintenance of PRC1 association with a template. It is likely that further mechanisms, such as RNAi, also contribute (Mulholland, 2003).
These multiple mechanisms might be additive or synergistic. Additionally, redundancy between them would provide a fail-safe scheme for maintenance of repression. For instance, if methylation at H3 K27 and increased function by Zeste each were sufficient to establish repression by PRC1, then repression could be established even if one or the other were to fail. Consistent with this hypothesis of redundant function, experiments were performed in which both Zeste and GAGA were present; no significant additive or synergistic effects on PCC function was seen. The establishment of defined in vitro systems, such as used here, will aid in unraveling the connections between the different mechanisms that contribute to regulation of PcG function (Mulholland, 2003).
Chromatin structure plays a critical role in the regulation of transcription. Drosophila GAGA factor directs chromatin remodeling to its binding sites. Drosophila FACT (facilitates chromatin transcription), a heterodimer of dSPT16 and dSSRP1, is associated with GAGA factor through its dSSRP1 subunit, binds to a nucleosome, and facilitates GAGA factor-directed chromatin remodeling. Moreover, genetic interactions between Trithorax-like encoding GAGA factor and spt16 implicate the GAGA factor-FACT complex in expression of Hox genes
Ultrabithorax, Sex combs reduced, and Abdominal-B. Chromatin immunoprecipitation experiments indicate the presence of the GAGA factor-FACT complex in the regulatory regions of Ultrabithorax and Abdominal-B. These data illustrate a crucial role of FACT in the modulation of chromatin structure for the regulation of gene expression (Shimojima, 2003).
GAGA factor-dFACT complex was identified by co-immunoprecipitation with epitope tagged GAGA factor. GST pull-down assays show that GAGA factor makes a direct contact with dFACT through its dSSRP1 subunit. Gel electrophoresis mobility shift assays reveal that dFACT binds to the nucleosome. Furthermore, dFACT stimulates GAGA factor-directed chromatin remodeling in the embryonic extract of
Drosophila. Based on these data, the following model is proposed for
GAGA factor-directed site-specific chromatin remodeling. The GAGA factor-dFACT complex binds to a GAGAG sequence on DNA. dFACT binds to
nucleosome and stimulates chromatin remodeling. This allows remodeling in a
GAGA factor binding site-dependent manner. Because human FACT binds to
histones H2A and H2B (Orphanides, 1999), and the yeast SPN complex enhances DNase I sensitivity of nucleosome in a region where H2A and H2B contact the DNA
(Formosa, 2001), it is most likely that FACT binds to DNA at the entry and exit site of the nucleosome through its HMG subunit SSRP1, and then acts to destabilize and remove the H2A/H2B dimers to facilitate chromatin remodeling. However, the
H2A/H2B dimers remain associated with the FACT-nucleosome complex
through SPT16 such that they can quickly rebind to the H3/H4 tetramer when
required. In support of this model, an acidic amino acid stretch found in
histone-interacting proteins such as nucleoplasmin and NAP1 is conserved in
the C-terminal tail of SPT16. Furthermore, H2B (and probably H2A) has been shown to turn over more rapidly than H3 and H4 during transcription (Shimojima, 2003).
The most interesting finding in this study is the involvement of the GAGA
factor-dFACT complex in the regulation of gene expression. The anterior
transformation of T3 and A6 in Deltaspt16 Trl double heterozygotes
and the binding of the GAGA factor-dFACT complex to the bxd region of Ubx and the iab-6 element of Abd-B in vivo indicate that the complex contributes to the epigenetic maintenance of Hox gene expression. Based on these data, the following scheme is envisioned for the maintenance of the active state. The GAGA factor-dFACT complex induces chromatin remodeling in the regulatory regions of various GAGA factor-dependent genes and potentiates transcription. Whereas the expression of ftz and hsp70 is transient, the active state is maintained in Hox genes such as Ubx, Scr, and Abd-B with the aid of other trx group gene products (Shimojima, 2003).
What is the mechanism underlying the maintenance? Among trx group proteins, BRM constitutes an SWI/SNF-type chromatin remodeling complex. This type of chromatin remodeler possesses a unique ability to act on condensed mitotic chromatin. A sequence-specific regulator, Zeste, has been shown to recruit the BRM complex to its target sites. Functionally distinct chromatin remodeling induced by the GAGA-dFACT and Zeste-BRM complexes may be important to keep the active state through many rounds of cell cycle. In addition to the GAGA factor-dFACT and the BRM complexes, three trx group protein complexes have been identified to date. One is TAC1 consisting of Trx, dCBP, and Sbf1, which acetylates core histones in nucleosomes. Mutations in trx or nejire encoding dCBP have been shown to reduce the expression of Ubx. The others are ASH1 and ASH2 complexes. ASH1 also has been known to interact directly with dCBP. These data suggest that acetylation of core histones or other proteins plays a crucial role in the maintenance of the active state. In support of this hypothesis, hyper-acetylation of H4 has been shown to be a heritable epigenetic mark of the active state. The finding that a counteracting Pc group complex ESC/E(Z) contains histone deacetylase RPD3 is also consistent with this hypothesis. Chromatin remodeling induced by the GAGA factor-d-FACT and the Zeste-BRM complexes might be essential for maintenance of the hyperacetylated state of H4 (Shimojima, 2003).
In Drosophila, the Polycomb group (PcG) of genes is required for the maintenance of homeotic gene repression during development. The Drosophila ortholog of the products of the mammalian Ring1/Ring1A and Rnf2/Ring1B genes has been characterized. Drosophila Ring corresponds to Sex combs extra, a previously described PcG gene. Ring/Sce is expressed and required throughout development and the extreme Pc embryonic phenotype due to the lack of maternal and zygotic Sce can be rescued by ectopic expression of
Ring/Sce. This phenotypic rescue is also obtained by ectopic
expression of the murine Ring1/Ring1A, suggesting a
functional conservation of the proteins during evolution. In addition,
Ring/Sce binds to about 100 sites on polytene chromosomes, 70% of which
overlap those of other PcG products such as Polycomb, Posterior sex combs and
Polyhomeotic, and 30% of which are unique. Ring/Sce interacts
directly with PcG proteins, because it occurs in mammals (Gorfinkiel, 2004).
Drosophila is part of a protein complex that monoubiquitinates nucleosomal histone H2A. Reducing the expression of mammalian Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis has demonstrated colocalization of Drosophila Ring with ubiquitinated H2A at the polycomb response elements and
promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of Drosophila Ring in SL2 tissue culture cells by RNA interference results in loss of H2A ubiquitination concomitant with derepression of Ubx. These studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing (H. B. Wang, 2004).
The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. Recent findings have identified PcG proteins both as targets for modification by the small ubiquitin-like modifier (SUMO) protein and as catalytic components of the SUMO conjugation pathway. This study found that the SUMO-conjugating enzyme Ubc9 binds to Scm and that this interaction, which requires the Scm C-terminal sterile α motif (SAM) domain, is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx), and efficient PRE recruitment requires an intact Scm SAM domain. Global reduction of sumoylation augments binding of Scm to the PRE. This is likely to be a direct effect of Scm sumoylation because mutations in the SUMO acceptor sites in Scm enhance its recruitment to the PRE, whereas translational fusion of SUMO to the Scm N terminus interferes with this recruitment. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced, decreased expression of Ubx and partial haltere-to-wing transformation phenotypes were observed. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE (Smith, 2011).
The Extra sex combs (ESC) protein is a Polycomb group (PcG) repressor that is a key noncatalytic subunit in the ESC-Enhancer of zeste [E(Z)] histone methyltransferase complex. Survival of esc homozygotes to adulthood based solely on maternal product and peak ESC expression during embryonic stages indicate that ESC is most critical during early development. In contrast, two other PcG repressors in the same complex, E(Z) and Suppressor of zeste-12 [SU(Z)12], are required throughout development for viability and Hox gene repression. A novel fly PcG repressor, called ESC-Like (ESCL), is described whose biochemical, molecular, and genetic properties can explain the long-standing paradox of ESC dispensability during postembryonic times. Developmental Western blots show that ESCL, which is 60% identical to ESC, is expressed with peak abundance during postembryonic stages. Recombinant complexes containing ESCL in place of ESC can methylate histone H3 with activity levels, and lysine specificity for K27, similar to that of the ESC-containing complex. Coimmunoprecipitations show that ESCL associates with E(Z) in postembryonic cells and chromatin immunoprecipitations show that ESCL tracks closely with E(Z) on Ubx regulatory DNA in wing discs. Furthermore, reduced escl+ dosage enhances esc loss-of-function phenotypes and double RNA interference knockdown of ESC/ESCL in wing disc-derived cells causes Ubx derepression. These results suggest that ESCL and ESC have similar functions in E(Z) methyltransferase complexes but are differentially deployed as development proceeds (Wang, 2006).
ESC and E(Z), and their homologs, are functional partners in the chromatin of plants, invertebrates, and mammals. Working together, they control a diverse array of developmental processes, including flower and seed differentiation in Arabidopsis thaliana, germ line development in Caenorhabditis elegans, X chromosome inactivation in mice, and Hox gene repression in flies and mammals. Recent studies show that this partnership reflects a requirement for ESC in potentiating the histone methyltransferase activity of E(Z) (Wang, 2006).
In light of this functional interdependence, a paradox is presented by developmental studies in Drosophila melanogaster, which show that ESC is primarily needed during early embryogenesis, whereas E(Z) is required throughout embryonic, larval, and pupal development. Analysis of ESCL, which can replace ESC in E(Z) HMTase complexes in vitro, provides a plausible solution to this puzzle. ESCL expression is largely complementary to that of ESC, peaking during later developmental stages, and functional studies show that ESCL is partially redundant with ESC in imaginal tissues. These results, together with prior genetic data that address esc time of action, indicate that ESC predominates in embryos, whereas both ESCL and ESC make functional contributions during postembryonic development (Wang, 2006).
Phenotypic analyses of esc loss-of-function mutants provided the original evidence that the primary time of ESC action is during embryogenesis. Although complete loss of esc+ product is embryonic lethal and yields wholesale misexpression of Hox genes, it was shown that maternally provided esc+ product provides sufficient function during embryogenesis to enable zygotically null esc– animals to survive to adulthood. These esc– adults are fertile, healthy, and phenotypically normal except for minor homeotic transformations such as extra sex combs on the meso- and meta-thoracic legs. In contrast, animals that are zygotically null for any other PcG subunit of the ESC-E(Z) complex or PRC1 fail to survive beyond early pupal stages, with most dying by the embryonic/L1 stage (Wang, 2006).
Additional experiments with a conditional esc allele further delimited the main time of ESC function to a period of mid-embryogenesis extending from about the onset of gastrulation (about 3 h at 25°C) until germ band shortening (approximately 9 to 12 h). An independent study that measured phenotypic rescue by a heat-inducible esc+ transgene confirmed that the time of ESC action begins at about 3 h of embryogenesis. These genetically determined times of esc+ function coincide with the accumulation of ESC protein, which peaks during mid-embryogenesis and declines by the end of embryogenesis (Wang, 2006).
However, full consideration of the genetic evidence also indicates that ESC does contribute to postembryonic PcG repression, particularly in imaginal tissues. Analysis of esc– larvae showed modest defects in Hox gene repression in imaginal discs as well as in the central nervous system. In particular, this study attributed the extra sex combs phenotype of esc– larvae to misexpression of the Scr Hox gene in the T2 and T3 leg discs. In addition, production of extra sex combs from patches of esc– tissue generated by somatic recombination during larval development indicates that the time of ESC action extends into the larval period, at least in leg discs (Wang, 2006).
A postembryonic role is consistent with the detection of ESC on the Ubx gene in wing discs and with the overlapping roles of ESC and ESCL in Ubx repression in disc-derived MCW12 cells. This result might explain why esc– wing discs did not produce homeotic phenotypes even after sufficient passage to ensure depletion of maternal esc+ product; presumably, both ESC and ESCL would need to be disrupted in this tissue to yield robust Hox misexpression. Finally, although it is much less abundant at late developmental times than in embryos, ESC is detected by Western blotting in larval and pupal extracts. Thus, the genetic and molecular data together indicate that ESC does function during postembryonic stages, albeit with a more modest overall contribution than its critical role in embryos (Wang, 2006).
These considerations imply that the developmental division of labor between ESC and ESCL is not simply that ESC functions only in embryos and ESCL takes over for subsequent stages. Rather, although ESC does predominate early, as evidenced by the global loss of H3 K27 methylation in esc– embryos, postembryonic development appears to involve both ESC and ESCL. It was originally hypothesized that late developmental functions of the esc locus might be executed by an esc+ isoform distinct from the embryonic version. The current data confirm that multiple ESC-related proteins do operate during fly development, with a late-acting version supplied by a second copy of the esc gene (Wang, 2006).
The functional context for ESCL during postembryonic development is presumably as a subunit in E(Z)-containing complexes with histone methyltransferase activity. The fact that ESCL can assemble in place of ESC and restore HMTase activity to a reconstituted E(Z) complex indicates that the biochemical roles of ESCL and ESC are similar. ESCL/ESC functional overlap could reflect a mixture of postembryonic E(Z) complexes, with some containing ESCL and others containing ESC. The simplest version of this scenario would entail four-subunit postembryonic HMTase complexes similar to the embryonic core complex of E(Z), SU(Z)12, NURF-55, and ESCL or ESC. However, postembryonic E(Z) complexes have yet to be purified, so their molecular compositions are not yet known. In fact, there is evidence that larval E(Z) complexes may differ from embryonic E(Z) complexes in features besides the ESCL/ESC subunit. For example, the SIR2 histone deacetylase has been reported to associate with larval but not embryonic E(Z) complexes. Much remains to be determined about postembryonic E(Z) complexes, including subunit compositions and characterization of presumed HMTase activity (Wang, 2006).
Although the catalytic subunit E(Z) contains the conserved SET domain, studies on fly, worm, and mammalian homologs reveal that the ESC subunit is also critical for HMTase function. The single loss of ESC from the fly complex or loss of its homolog, MES-6, from the C. elegans complex yields subcomplexes with little or no HMTase activity in vitro. In agreement with this, genetic removal of ESC eliminates most or all methyl-H3 K27 in fly embryos, loss of MES-6 eliminates most or all methyl-H3 K27 in worm germ lines and early embryos, and loss of EED removes most or all methyl-H3 K27 from embryonic mouse cells. The mechanism by which ESC and its relatives potentiate the activity of HMTase complexes is not known. An in vitro study argues against a role for fly ESC in mediating stable contacts with nucleosome substrate. In contrast, loss of ESC by RNA interference in fly S2 cells leads to dissociation of E(Z) from chromatin targets (Wang, 2006).
A biochemical analysis of the human EED-EZH2 complex (also called PRC2) has revealed an intriguing difference in the HMTase depending upon the subtype of EED subunit present in the complex. Multiple isoforms of EED are expressed in HeLa cells that differ in the extents of their N-terminal tails through use of alternative translation start sites. Incorporation of particular EED isoforms into EZH2 complexes can shift the enzyme specificity so that K26 of histone H1 is methylated in addition to H3 K27. Taken together with other studies, this suggests that EED is a regulatory subunit that can influence both substrate specificity and catalytic efficiency of the HMTase (Wang, 2006).
In light of this finding, it seems possible that ESCL-E(Z) complexes might also have HMTase activity with altered lysine specificity. However, both ESCL-E(Z) and ESC-E(Z) recombinant complexes showed similar specificities for H3 K27 in H3/H4 tetramers and no methylation of mammalian histone H1 by either of these recombinant complexes was detected in vitro. It is noted that the human H1 K26 methylation site is embedded in an ARKS sequence, which is also present surrounding H3 K27. This sequence is not conserved in Drosophila histone H1, suggesting that the ability of certain EZH2 complexes to methylate H1 may not be conserved in the fly system. However, there may well be other relevant methylation substrates besides histone H3 and it remains possible that alternative ESC isoforms could alter lysine specificities for these other substrates (Wang, 2006).
Based upon their temporal expression profiles, it seems clear that esc and escl have distinct functions in a developmental context. Their temporal division of labor is most clearly demonstrated by esc– escl+ embryos, which show extreme homeotic transformations accompanied by dramatically reduced levels of methylated H3 K27. This division could be entirely a consequence of differential transcriptional controls built into their divergent promoters. That is, ESC and ESCL could be functionally identical proteins that are just expressed at peak levels at different times. Alternatively, the two proteins may possess intrinsic differences that are also important during development but are not revealed by the assays applied so far. One possibility is that ESC and/or ESCL may play a role in methylation of nonhistone proteins. The only nonhistone proteins yet identified that fly E(Z) complexes can methylate are two subunits of the core complex itself, E(Z) and SU(Z)12. It is not clear if this self-methylation is functionally relevant and, in any case, it occurs at comparable levels with the ESC- and ESCL-containing recombinant complexes (Wang, 2006).
It is also possible that ESC and ESCL could differ in contributions to E(Z) complexes besides HMTase activity. These other functions could include interacting with and recruiting histone deacetylases, mediating physical interactions with PRC1 components, recruiting E(Z) complexes to target loci, and influencing the way E(Z) complexes interact with other (non-K27) histone tail modifications. Indeed, there is evidence for differential association of histone deacetylases with E(Z) complexes at embryonic versus larval stages, which parallels temporal changes in ESC and ESCL abundance. At the same time, ESC and ESCL functions must overlap enough to account for the sufficiency of either one to maintain Ubx repression in at least some postembryonic cells (Wang, 2006).
Definitive answers will require promoter swap experiments in which ESCL is placed under control of the ESC promoter and vice versa, to determine which combinations provide genetic rescue of esc and escl mutations in vivo. Along with this approach, a complete understanding of the developmental role of ESCL will require generation of escl mutant alleles and systematic analysis of the phenotypic consequences of escl loss of function (Wang, 2006).
The Polycomb and trithorax groups of genes control the maintenance of homeotic gene expression in a variety of organisms. A putative participant in the regulation of this process is the murine RYBP (Ring and YY1 Binding Protein). Sequence comparison between different species has identified the homologous gene in Drosophila, the dRYBP gene. Whether dRYBP participates in the mechanisms of silencing of homeotic genes expression was investigated. dRYBP expression, examined by RNA in situ hybridisation, was found ubiquitously throughout development. Moreover, a polyclonal anti-dRYBP antibody was generated that recognises the dRYBP protein. dRYBP protein is nuclear and expressed maternally and ubiquitously throughout development. To study the transcriptional activity of dRYBP, a fusion protein was generated containing the entire dRYBP protein and the GAL4 DNA binding domain. This fusion protein functions, in vivo, as a transcriptional repressor throughout development. Importantly, this repression is dependent on the function of the Polycomb group genes. Furthermore, using the GAL4/UAS system, dRYBP was over-expressed in the haltere and the wing imaginal discs. In the haltere discs, high levels of dRYBP repress the expression of the homeotic Ultrabithorax gene. This repression is Polycomb dependent. In the wing discs, dRYBP over-expression produces a variety of phenotypes suggesting the overall miss-regulation of the many putative genes affected by high levels of dRYBP. Taking together, these results indicate that dRYBP is able to interact with PcG proteins to repress transcription suggesting that the dRYBP gene might belong to the Polycomb group of genes in Drosophila (Bejarano, 2005).
The mouse homologous gene, RYBP, was identified in a two-hybrid screen for murine Ring1 interacting proteins. RYBP family members include the human YEAF1 homologous gene and the murine and human YAF2 gene coding for structurally related proteins. Although very similar in sequences, they seem to have different functions as transcriptional regulators of the hGABP gene, i.e. YAF2 positively regulates the transcriptional activity of hGABP but YEAF1 negatively regulates this activity (Sawa, 2002; Bejarano, 2005).
dRYBP is expressed maternally and throughout development in all the nuclei of the embryo and the imaginal discs cells. The murine RYBP gene is also expressed ubiquitiously in the mouse embryo (Garcia, 1999). The ubiquitous and nuclear pattern of dRYBP expression coincides with the pattern of expression of the Polycomb group proteins so far described (Bejarano, 2005).
When dRYBP is tethered to DNA sequences, it is able to repress the transcriptional state of minigene reporter constructs. Moreover, GALDB-dRYBP transcriptional repression function requires the products of at least the Pc, Sce and pho genes, suggesting that GALDB-dRYBP represses transcription by interacting with PcG protein complexes. The Pho protein (homologous to mouse YY1) is able to bind DNA in a sequence specific manner and it has been proposed to recruit the PcG complexes to DNA. However, the results show that the transcriptional repression function of GALDB-dRYBP cannot be achieved in the absence of Pho protein. Although silencing in these experimental conditions could formally result solely from the interaction of dRYBP with Pho, the need of Pho to execute the transcriptional repression may also suggest that in the process of maintenance of homeotic gene expression, the Pho protein serve other functions than the recruitment of PcG complexes to DNA (Bejarano, 2005).
Additional evidence for the transcriptional repressor function of dRYBP comes from the experiments of over-expression of dRYBP using the GAL4/UAS system. UbxGAL4/UASdRYBP halteres show partial transformation towards wing which is correlated with the repression of UBX expression in the haltere imaginal discs due to high levels of dRYBP. The partial transformation of the haltere towards wing is not fully understood. It is speculated that the over-expression of dRYBP may also affect genes involved in proliferation that act downstream the Ubx gene. The repressive effect is Polycomb dependent, suggesting that dRYBP transcriptional repression function needs the interaction with Polycomb proteins. Moreover, although no changes have been detected in the levels of engrailed expression, some of the phenotypes observed in enGAL4/UASdRYBP flies are indicative of engrailed repression, revealing again the repressor effect of dRYBP over expression (Bejarano, 2005).
A model has been proposed in which RYBP protein, through its interaction with DNA-binding proteins like YY1, function as a ‘bridge’ to ensure interactions of DNA and non-DNA binding proteins in multimeric protein complexes. It is not yet known if dRYBP serves a similar bridging function in Drosophila. The YY1 protein (homologous to Drosophila Pho) is able to bind DNA in a sequence specific manner and directly interacts with dRYBP. It is speculated that dRYBP, serves a similar bridging function, bridging between DNA binding proteins like Pho and the multimeric PcG complexes. Further work and mutations in the dRYBP gene will be necessary to define whether dRYBP serves this putative bridge function (Bejarano, 2005).
dRYBP over-expression in the wing produces homeotic and non-homeotic phenotypes indicative of miss regulation of a variety of genes. High levels of dRYBP in the wing (i.e. sdGAL4/UASdRYBP flies) produces, among others, transformation towards haltere with the corresponding expression of the Ubx protein in the wing cells, i.e. outside its normal domain of expression. This effect could seem opposite to the repressor effect observed when dRYBP is tethered to DNA (GALDBdRYBP) or when dRYBP is over expressed under the control of the UbxGAL4 line. However, interference with the assembling/recruting of the PcG and trxG complexes either because of sequestration of PcG/trxG proteins, perturbation of the PcG/trxG balance or disruption of the cross regulatory interactions between PcG proteins could perhaps explain the observed expression of UBX protein in the wing disc due to over-expression of dRYBP. Alternatively, over abundance of dRYBP or dRYBP containing complexes might lead to a unique target gene repretoire that lead to the effects observed. Finally, the cross regulatory interactions between the genes patterning the wing, that are perhaps being miss regulated by the high levels of dRYBP could also explain the range of phenotypes observed in the wing due to over expression of dRYBP (Bejarano, 2005).
In conclusion, these results show that dRYBP protein is nuclear, maternal and ubiquotiously expressed throughout development. The results also show that dRYBP functions, in a Polycomb dependent manner, as a transcriptional repressor, suggesting that dRYBP is able to interact with the PcG proteins to repress transcription and therefore might belong to the Polycomb group of genes of Drosophila. Finally, the study of the multiple phenotypes produced by high levels of dRYBP in the wing might be indicative of the involvement of dRYBP on the regulation of many genes as also described for the PcG genes in Drosophila (Bejarano, 2005).
Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. In contrast, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter (Comet, 2006).
The present work suggests two complementary mechanisms for promoter silencing by PcG proteins. (1) The data show directly that PcG proteins recruited at a PRE can spread over several kilobases along the flanking chromatin. Therefore, promoters located within short distances from PREs might be silenced by PcG spreading and interference with the transcription machinery. However, PcG spreading induced by the Ubx PRE did not extend beyond few kilobases in these experiments, and ChIP on chip also showed limited extension of PcG protein binding from known PREs. This limited spreading might depend on genomic sequences or proteins bound to them that might attenuate chromatin association of PcG proteins. Thus, spreading alone might not be sufficient for silencing promoters located several tens of kilobases away, as in the case of the Ubx gene, suggesting that additional mechanisms allow PcG proteins to gain access at distant promoters. It was found that pairing of two Su(Hw) insulators can induce promoter association of PcG complexes without PcG-mediated coating of the insulated domain. (2) This suggests an additional mechanism of PRE-dependent promoter silencing, whereby PREs located at large distances from their promoters might contact them via looping of intervening domains. This looping might be favored by natural regulatory elements present at these loci, which might play a role similar to the pair of Su(Hw) insulators used in this study (Comet, 2006).
The endogenous distribution of PcG proteins might reflect spreading from a PRE into the flanking genomic region as well as their ability to bypass insulators. At the two endogenous target loci en and ph, where PREs are located in the promoter region, the distribution of PC and PH suggests spreading from the PREs. The distribution of PC and PH was characterized at Ubx, a locus where the PRE is over 20 kb upstream from the Ubx promoter. In addition to Ubx, this region contains the bxd locus, driving the production of noncoding transcripts. PC and PH binding shows a peak at the bxd transcription start site downstream to the PRE, in addition to the previously described peaks corresponding to the PRE and the Ubx promoter. Furthermore, binding of PH and PC drops between the bxd peak and the Ubx promoter and rises again at the promoter. This distribution is consistent with spreading from the PRE for short-distance chromatin silencing, and direct targeting of PRE bound PcG complexes to the downstream promoter to drive silencing over larger distances (Comet, 2006).
The sharp transitions in PcG protein binding detected at insulators are surprising, especially considering that the PRC1 complex is larger than 1 MDa, a size equivalent to several nucleosomes. The block in PcG spreading might depend on a physical barrier imposed by protein complexes tightly bound to the insulator. The bypass of the insulated domain might be explained by topological features imposed by insulators on three-dimensional chromatin folding. The Su(Hw) and Mod(mdg4) proteins that regulate the Su(Hw) insulator are organized into discrete “insulator bodies” in the cell nucleus. PcG proteins are also organized into “PcG bodies” that might be the sites of PRE-mediated silencing. A single Su(Hw) insulator located near a PRE might thus exclude the downstream domain from the PcG body associated to the PRE. A second insulator paired with the first one in the insulator body might bring the downstream promoter at the PRE-associated PcG body, while excluding from it the intervening chromatin domain. This type of regulation of three-dimensional chromatin folding by insulator elements might modulate gene expression at a number of loci in Drosophila and other species (Comet, 2006).
Polycomb group proteins are transcriptional repressors that control many developmental genes. The Polycomb group protein Enhancer of Zeste has been shown in vitro to methylate specifically lysine 27 and lysine 9 of histone H3 but the role of this modification in Polycomb silencing is unknown. This study shows that H3 trimethylated at lysine 27 is found on the entire Ubx gene silenced by Polycomb. However, Enhancer of Zeste and other Polycomb group proteins stay primarily localized at their response elements, which appear to be the least methylated parts of the silenced gene. These results suggest that, contrary to the prevailing view, the Polycomb group proteins and methyltransferase complexes are recruited to the Polycomb response elements independently of histone methylation and then loop over to scan the entire region, methylating all accessible nucleosomes. It is proposed that the Polycomb chromodomain is required for the looping mechanism that spreads methylation over a broad domain, which in turn is required for the stability of the Polycomb group protein complex. Both the spread of methylation from the Polycomb response elements, and the silencing effect can be blocked by the gypsy insulator (Kahn, 2006).
The experiments described in this study show clearly that all three PcG proteins tested, Pc, Psc, and E(z), are preferentially located at the PREs. This specificity is clearest and most sharply delineated in the case of Psc and E(z). In the case of PC, the peaks centered at the PREs are much broader, including secondary peaks, and although the binding detected at other Ubx regions decreases to low values, it never reaches the level seen at control sites such as the white gene that possess no PRE. The second basic conclusion from these experiments is that, in contrast to the localization of PcG proteins, the H3 me3K27 profile forms a broad domain that includes the entire Ubx transcription unit and upstream regulatory region. The third important observation is that, contrary to previously published accounts, the PREs themselves appear to contain the lowest levels of me3K27 of the entire domain. This surprising result will be considered first. The lack of apparent methylation at the PREs does not depend on the antibody used or on the level of cross-linking. Comparable results were obtained with two different anti-me3K27 antibodies and with anti-me3K9. Furthermore, the fact that a similar result was obtained with antibody against total histone H3 or histone H2B suggests that nucleosomes are underrepresented at the bxd PRE core. The result is not because of lack of accessibility to the histone or to the epitope: GAGA factor bound to the PRE appears as easily accessible as GAGA factor bound to the Ubx promoter. Salt extraction of the PcG complexes before cross-linking does not qualitatively change the me3K27 binding profile (Kahn, 2006).
In sum, careful quantitative analysis of ChIP indicates that while PcG proteins are principally localized at the PRE, the histone H3 methylation they produce is distributed over the entire Ubx gene. It is evident from this and from the undermethylation of the PRE core that that K27 methylation does not, by itself, recruit PcG complexes. This does not preclude an important role for methylation in PcG binding and silencing but suggests that the relationship between the two requires a more dynamic model (Kahn, 2006).
PREs have been shown to recruit PcG complexes and to produce new binding foci detectable in polytene chromosomes. It is not surprising therefore to find the three PcG proteins tested are associated with the two Ubx PREs. A much smaller peak in microarray profiles for all three proteins can be discerned in the vicinity of the Ubx 3'-exon but its significance is unknown. More surprising was the striking difference between the distributions of Psc and E(z) and that of Pc. E(z) and Psc belong to two different complexes that do not co-precipitate (except in the very early embryo) but are both recruited to the PRE. Pc and Psc are core components of the PRC1-type of PcG complex yet, while Psc is detected almost exclusively at the PREs, Pc has a much broader distribution peaking at the PREs but tailing over considerable distances along the Ubx gene and regulatory regions. The simplest interpretation of this is that a second type of complex containing Pc but not Psc is recruited by a different mechanism to the rest of the Ubx sequences. Alternatively, the same complex, containing both proteins is involved in both cases but the nature of the chromatin contact is different, such that in one case both proteins are well cross-linked to the chromatin but in the second case only Pc is efficiently cross-linked (Kahn, 2006).
Just as striking is the fact that, although the E(z) complex is responsible for the H3 K27 methylation spread over the entire Ubx gene, the E(z) protein is found localized at the PREs. It is concluded that the E(z) complex methylates the Ubx domain by a hit-and-run type of mechanism. Because the methylation is stable, the E(z) complex needs only visit each nucleosome once on the average every cell cycle. It is noted that E(z)-dependent histone H3 K27 dimethylation is highly abundant and widely distributed in the genome but E(z) complexes are not associated with it. Where then does the E(z) that methylates PcG target genes come from? While more complicated scenarios may be imagined, the simplest one involves the E(z) complex bound at the PRE (Kahn, 2006).
It is supposed that the PcG complexes are recruited to PREs by DNA-binding proteins independently of histone methylation. To methylate the entire Ubx domain, the E(z)/ESC histone MTase complex might then detach from the PRE and slide along the chromatin from one nucleosome to the next to survey the entire domain. However, it more likely that both the Pc and the E(z) complexes assembled at the PRE remain associated with the PRE sequences, where they are detected, but that the whole PRE assembly loops over to scan the entire region, methylating all accessible nucleosomes. Such looping models were originally proposed to be mediated by sites of weak PcG complex formation. In a modern version of this type of model, the looping activity would be mediated by the distinct affinity of the Pc protein for histone H3, which is greatly increased by K27 methylation. These affinities would mediate transient interactions of the complexes bound at the PRE with the surrounding chromatin and allow continuous scanning and methylation of unmethylated or hemimethylated nucleosomes (Kahn, 2006).
In such a model, ChIP experiments would always detect a strong PcG presence at the PRE but PcG interactions with the rest of the repressed gene would be distributed over a region, which is very large in the case of the Ubx gene, smaller in the case of the YGPhsW transposon, hence the signal detected at any one site would be weaker in proportion to the extent of the methylated domain. In addition, the contacts between the PcG complex and the rest of the silenced gene would be much more transient than contacts with the PRE. Together, these considerations would explain why ChIP assay gives such low values for PcG proteins over the rest of the methylated domain (Kahn, 2006).
The looping mechanism proposed for the PRE-bound complex strongly resembles that suggested for the interaction between the Locus Control Region and β-globin genes or for enhancer-promoter interactions. Like these interactions, the silencing of a promoter by the PRE is blocked by insulator elements. In transposon constructs, the insertion of a gypsy Su(Hw) insulator between PRE and promoter blocks the spread of methylation. At present, the mechanism of insulator action is not clear and how the block to methylation is achieved is unknown. It is possible that the insulator element produces topological constraints that prevent the PRE-bound complexes from looping beyond the insulator. This would be consistent with the observation that a significant level of Pc presence becomes detectable over the yellow gene when the insulator block is lifted (Kahn, 2006).
Although the data argue against a principal role of histone methylation in the recruitment of Polycomb proteins to their response elements, it seems to be important for both transcriptional repression and stable association of PcG proteins with chromatin. Loss of catalytic E(z) function eventually results in derepression of HOX genes and dissociation of PcG proteins from polytene chromosomes. It is speculated that once the me3K27 domain is established, modified nucleosomes will pave the way for looping interactions of the PRE-bound PcG proteins with the parts of the silenced gene including promoter or enhancer regions. Silencing might then result from hit-and-run interactions with either or both, possibly even resulting in methylation of the associated factors. Alternatively or in addition, trimethylation of K27 and possibly K9 may directly interfere with the signaling cascade of consecutive histone modifications that guide the multistep process of transcription initiation and elongation. Since histone methylation is thought to persist through cell division its immediate presence at the very beginning of the subsequent interphase might win the time necessary for the full assembly of PcG complexes on the PREs before competing transcription has taken over (Kahn, 2006).
Homeotic genes contain cis-regulatory trithorax response elements (TREs) that are targeted by epigenetic activators and transcribed in a tissue-specific manner. The transcripts of three TREs located in the Drosophila homeotic gene Ultrabithorax mediate transcription activation by recruiting the epigenetic regulator Ash1 to the template TREs. TRE transcription coincides with Ubx transcription and recruitment of Ash1 to TREs in Drosophila. The SET domain of Ash1 binds all three TRE transcripts, with each TRE transcript hybridizing with and recruiting Ash1 only to the corresponding TRE in chromatin. Transgenic transcription of TRE transcripts restores recruitment of Ash1 to Ubx TREs and restores Ubx expression in Drosophila cells and tissues that lack endogenous TRE transcripts. Small interfering RNA-induced degradation of TRE transcripts attenuates Ash1 recruitment to TREs and Ubx expression, which suggests that noncoding TRE transcripts play an important role in epigenetic activation of gene expression (Sanchez-Elsner, 2006). Please note that the Sanchez-Elsner paper has been retracted (see retraction report)
Histone acetyltransferase (HAT) complexes have been linked to activation of transcription. Reptin is a subunit of different chromatin-remodeling complexes, including the TIP60 HAT complex (see Tip60). In Drosophila, Reptin also copurifies with the Polycomb group (PcG) complex PRC1, which maintains genes in a transcriptionally silent state. Genetic interactions have been demonstrated between reptin mutant flies and PcG mutants, resulting in misexpression of the homeotic gene Scr. Genetic interactions are not restricted to PRC1 components, but are also observed with another PcG gene. In reptin homozygous mutant cells, a Polycomb response-element-linked reporter gene is derepressed, whereas endogenous homeotic gene expression is not. Furthermore, reptin mutants suppress position-effect variegation (PEV), a phenomenon resulting from spreading of heterochromatin. These features are shared with three other components of TIP60 complexes, namely Enhancer of Polycomb, Domino, and dMRG15. It is concluded that Drosophila Reptin participates in epigenetic processes leading to a repressive chromatin state as part of the fly TIP60 HAT complex rather than through the PRC1 complex. This shows that the TIP60 complex can promote the generation of silent chromatin (Qi, 2006).
It is proposed that Reptin acts as a subunit of the TIP60 HAT complex to generate a repressive chromatin state. This is a novel activity of a HAT complex previously shown to promote transcription. This study shows that Reptin copurifes with the Polycomb complex PRC1. This prompted an investigation of whether the biochemical interaction with PRC1 was accompanied by a genetic interaction. It was shown that Reptin and PRC1 components genetically interact to regulate expression of the Hox gene Scr. However, Reptin also interacts with a PcG gene product not associated with the PRC1 complex, Pcl. Although no interactions were detected between reptin heterozygous mutants and several PREs tested, a PRE from the Ubx gene is derepressed in reptin homozygous mutant cells. This shows that Reptin contributes an essential function to the activity of this PRE. However, unlike most PcG genes, reptin homozygous mutants do not derepress endogenous Hox gene expression. It appears that repression of endogenous Hox genes is more complex and not as sensitive to the loss of Reptin as the Ubx PRE. In contrast to most PcG genes, reptin mutants suppress PEV. Interestingly, derepression of the Ubx PRE also occurs in embryos mutant for other suppressors of PEV, indicating that this PRE may be highly sensitive to the chromatin environment in its vicinity. Since reptin mutants suppress PEV and fail to derepress endogenous Hox gene expression, reptin is not considered a bona fide PcG gene, and it is found unlikely that Reptin protein contributes an essential function to the PRC1 complex. In fact, the biochemical activities ascribed to PRC1 can be reconstituted either with recombinant dRing1/Sce or with four core components whose activity can be further enhanced by the DNA-binding proteins Zeste and GAGA (Qi, 2006).
Given that Reptin is present in TIP60 complexes in mammals and recently was shown to be a component of a Drosophila TIP60 complex, the possibility is considered that the genetic interactions observed with PcG genes are due to the presence of Reptin in the fly TIP60 complex. The products of two previously characterized Drosophila genes, E(Pc) and domino, are also present in the TIP60 complex. Strikingly, E(Pc) and domino mutants share with reptin the ability to genetically interact with PcG genes and suppress PEV. E(Pc) is an unusual PcG gene that has very minor effects on Hox gene expression, and unlike most PcG genes, modifies PEV. In both yeast and humans, E(Pc) homologs form a core complex with Esa1 (TIP60) and Yng2 (ING3) that is sufficient for the nucleosomal acetylation of histones H4 and H2A by the NuA4 complex. That such an integral NuA4/TIP60 complex component displays phenotypes similar to reptin mutants suggests that Reptin functions through the fly TIP60 complex (Qi, 2006).
Domino protein is similar to p400 and to SRCAP in mammals and to Swr1 in yeast. Swr1 has recently been shown to exchange the variant histone H2A.Z (Htz1 in yeast) for H2A in nucleosomes. Intriguingly, an involvement of Htz1 (H2A.Z) in controlling the spreading of silenced chromatin has recently been demonstrated in yeast. Exchange of variant histones may be a conserved feature of chromatin regulation since a recent report demonstrates that Drosophila H2Av behaves genetically as a PcG gene and suppresses PEV. Domino exchanges phosphorylated and acetylated H2Av for unmodified H2Av after DNA damage. However, no change was found in binding of H2Av to polytene chromosomes prepared from domino mutant larvae (Qi, 2006).
Dendritic fields are important determinants of neuronal function. However, how neurons establish and then maintain their dendritic fields is not well understood. Polycomb group (PcG) genes are required for maintenance of complete and nonoverlapping dendritic coverage of the larval body wall by Drosophila class IV dendrite arborization (da) neurons. In esc, Su(z)12, or Pc mutants, dendritic fields are established normally, but class IV neurons display a gradual loss of dendritic coverage, while axons remain normal in appearance, demonstrating that PcG genes are specifically required for dendrite maintenance. Both multiprotein Polycomb repressor complexes (PRCs) involved in transcriptional silencing are implicated in regulation of dendrite arborization in class IV da neurons, likely through regulation of homeobox (Hox) transcription factors. Genetic interactions and association between PcG proteins and the tumor suppressor kinase Warts (Wts) is demonstrated, providing evidence for their cooperation in multiple developmental processes including dendrite maintenance (Parrish, 2007).
Dendrite arborization patterns are a hallmark of neuronal type; yet how dendritic arbors are maintained after they initially cover their receptive field is an important question that has received relatively little attention. The Drosophila PNS contains different classes of sensory neurons, each of which has a characteristic dendrite arborization pattern, providing a system for analysis of signals required to achieve specific dendrite arborization patterns. Class IV neurons are notable among sensory neurons because they are the only neurons whose dendrites provide a complete, nonredundant coverage of the body wall. This study found tha the function of Polycomb group genes is required specifically in class IV da neurons to regulate dendrite development. In the absence of PcG gene function, class IV dendrites initially cover the proper receptive field but subsequently fail to maintain their coverage of the field. Time-lapse analysis of dendrite development in esc or Pc mutants suggests that a combination of reduced terminal dendrite growth and increased dendrite retraction likely accounts for the gradual loss of dendritic coverage in these mutants. Maintenance of axonal terminals in class IV da neurons is apparently unaffected by loss of PcG gene function, suggesting that PcG genes function as part of a program that specifically regulates dendrite stability (Parrish, 2007).
Establishment of dendritic territories in class IV neurons is regulated by homotypic repulsion, and this process proceeds normally in the absence of PcG function. In PcG mutants, class IV neurons tile the body wall by 48 h AEL, similar to wild-type controls. However, beginning at 48 h AEL, likely as a result of reduced dendritic growth and increased terminal dendrite retraction, class IV neurons of PcG mutants gradually lose their dendritic coverage. In contrast, the axon projections and terminal axonal arbors of PcG mutants show no obvious defects. Although an early role for PcG genes in regulating axon development cannot be ruled out, MARCM studies showed that PcG genes are required for the maintenance of dendrites but not axons in late larval development. Thus, different genetic programs appear to be responsible for the establishment and maintenance of dendritic fields, and for the maintenance of axons and dendrites (Parrish, 2007).
It is well established that PcG genes participate in regulating several important developmental processes including expression of Hox genes for the specification of segmental identity. In comparison, much less is known about the function of PcG genes in neuronal development. Studies of the expression patterns of PcG genes and the consequences of overexpression of PcG genes suggest that PcG genes may affect the patterning of the vertebrate CNS along the anterior-posterior (AP) axis, analogous to their functions in specifying the body plan. A recent study demonstrates that the PcG gene Polyhomeotic regulates aspects of neuronal diversity in the Drosophila CNS. The current study now links the function of PcG genes to maintenance of dendritic coverage of class IV sensory neurons. Thus it will be interesting to determine whether PcG genes play a conserved role in the regulation of dendrite maintenance (Parrish, 2007).
Since Hox genes function in late aspects of neuronal specification and axon morphogenesis, it seems possible that regulation of Hox genes by PcG genes may be important for aspects of post-mitotic neuronal morphogenesis, including dendrite development. The PcG genes esc and E(z) are required for proper down-regulation of BX-C Hox gene expression in class IV neurons. The timing of this change in BX-C expression corresponds to the time frame during which PcG genes are required for dendritic maintenance. Furthermore, post-mitotic overexpression of BX-C genes in class IV da neurons, but not other classes of da neurons, is sufficient to cause defects in dendrite arborization, thus phenocopying the mutant effects of PcG genes. Finally, it was found that Hox genes are required cell-autonomously for dendrite development in class IV neurons, and loss of Hox gene function causes defects in terminal dendrite dynamics that are opposite to the defects caused by loss of PcG genes. Therefore, it seems likely that PcG genes regulate dendrite maintenance in part by temporally regulating BX-C Hox gene expression (Parrish, 2007).
Several recent studies have focused on the identification of direct targets of PcG-mediated silencing, demonstrating that PcG genes regulate expression of distinct classes of genes in different cellular contexts. During Drosophila development, PRC proteins likely associate with >100 distinct loci, and the chromosome-associate profile of PRC proteins appears dynamic. Therefore, identifying the targets of PcG-mediated silencing in a given developmental process has proven difficult. Thus far, alleles of >20 predicted targets of PcG-mediated silencing have been analyzed for roles in establishment or maintenance of dendritic tiling and a potential role has been found for only Hox genes. Future studies will be required to identify additional targets of PcG-mediated silencing in regulation of dendrite maintenance (Parrish, 2007).
PcG genes are broadly expressed, so it seems likely that interactions with other factors or post-translational mechanisms may be responsible for the cell type-specific activity of PcG genes. Indeed, PcG genes genetically interact with components of the Wts signaling pathway to regulate dendrite development specifically in class IV neurons. Based on the observations that wts mutants also show derepression of Ubx in class IV neurons and that Wts can physically associate with PcG components, it seems likely that Wts may directly or indirectly influence the activity of PcG components. In proliferating cells, Wts phosphorylates the transcriptional coactivator Yorkie to regulate cell cycle progression and apoptosis, demonstrating that Wts can directly influence the activity of transcription factors. In support of a possible role for Wts directly modulating PcG function, several recent reports have documented roles for phosphorylation in regulating PcG function both in Drosophila and in vertebrates. Thus, it is possible that some of the components involved in PcG-mediated silencing are regulated by Wts phosphorylation. Alternatively, association of Wts with PcG proteins may facilitate Wts-mediated phosphorylation of chromatin substrates (Parrish, 2007).
The tumor suppressor kinase Hpo regulates both establishment and maintenance of dendritic tiling in class IV neurons through its interactions with Trc and Wts, respectively, but how Hpo coordinately regulates these downstream signaling pathways is currently unknown. Similar to mutations in wts, mutations in PcG genes interact with mutations in hpo to regulate dendrite maintenance but show no obvious interaction with trc, consistent with the observation that PcG gene function is dispensable for establishment of dendritic tiling. Although it is possible that different upstream signals control Hpo-mediated regulation of establishment and maintenance of dendritic tiling, the nature of such signals remain to be determined. Another possibility is that the activity of the Wts/PcG pathway could be antagonized by additional unknown factors that promote establishment of dendritic tiling (Parrish, 2007).
In addition to their interaction in regulating dendrite maintenance, PcG genes and wts interact to regulate expression of the Hox gene Scr during leg development. This finding suggests that the Hpo/Wts pathway may play a general role in contributing to PcG-mediated regulation of Hox gene expression. The presence of ectopic sex combs provides a very simple and sensitive readout of wts/PcG gene interactions and should form the basis for conducting large-scale genetic screens to identify other genes that interact with wts or PcG genes and participate in this genetic pathway (Parrish, 2007).
Drosophila Polycomb group (PcG) and Trithorax group (TrxG) proteins are responsible for the maintenance of stable transcription patterns of many developmental regulators, such as the homeotic genes. ChIP-on-chip assay was used to compare the distribution of several PcG/TrxG proteins, as well as histone modifications in active and repressed genes across the two homeotic complexes ANT-C and BX-C. The data indicate the colocalization of the Polycomb repressive complex 1 [PRC1; containing the four PcG proteins Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs (Psc), and dRing/Sex combs extra (Sce)] with Trx and the DNA binding protein Pleiohomeotic (Pho) at discrete sequence elements as well as significant chromatin assembly differences in active and inactive regions. Trx binds to the promoters of active genes and noncoding transcripts. Most strikingly, in the active state, Pho covers extended chromatin domains over many kilobases. This feature of Pho, observed on many polytene chromosome puffs, reflects a previously undescribed function. At the hsp70 gene, it was demonstrated in mutants that Pho is required for transcriptional recovery after heat shock. Besides its presumptive function in recruiting PcG complexes to their site of action, these results now uncover that Pho plays an additional role in the repression of already induced genes (Beisel, 2007).
This work used two Drosophila tissue culture lines to map the distribution of chromatin proteins required for the transcriptional maintenance of the HOX genes. Although compromising on the precise developmental identity, the tissue culture cells provided a biochemically tractable homogeneous material, which currently would be difficult to obtain from whole animals. This choice was important to obtain the sharply delineated ChIP profiles, which show a highly significant correlation to mapped genetic elements in the two homeotic complexes. As such, the protein patterns obtained seem to reflect a valid situation as found in material from whole animals. In addition, the ChIP profiles uncovered a new function of Pho, which could be confirmed in whole animals (Beisel, 2007).
The results for SF4 cells are consistent with data that used a Schneider cell derivative for ChIP studies. PRC1 binds to discrete sequence elements, whereas H3K27me3 covers large genomic domains, including genic and intergenic regions. These observations indicate that H3K27me3 cannot be solely responsible for PRC1 targeting. How these H3K27 methylated domains influence HOX gene expression and whether the broad methylation pattern is the cause or consequence of gene silencing remains unclear. H3K27me3 may prevent the binding of activating protein factors as e.g., chromatin remodeling complexes and/or prevent the establishment of activating histone modifications. To this regard, a complementary pattern of H3K27me3 and H4ac, which is present in active gene regions, was detected (Beisel, 2007).
Several lines of evidence suggest that PcG proteins propagate their silencing effect by the direct interaction with the promoter region, which results in the inhibition of transcription initiation. In agreement with that, all promoter regions of the silent ANT-C HOX genes are occupied by PRC1. However, the Ubx promoter, which is silent in both cell lines, as well as the silent AbdB transcription units in Kc cells, are devoid of PRC1. Here, probably the numerous PREs, which are occupied by PRC1 in the Ubx and AbdB domains, build up a special chromatin structure that maintains the silent transcription state (Beisel, 2007).
In agreement with the observed H3K27me3 pattern in Drosophila cells, in mammalian Hox clusters inactive domains are covered by H3K27 and active domains are found entirely covered by H3K4 methylation. In contrast, the distribution of the enzymes setting the histone marks are completely different. In Drosophila E(Z), Trx, and Ash1 are bound at discrete sequence elements, whereas the mammalian homologues EZH2 and MLL1 localize to extended regions coincident with the methylation signals. MLL1 acts as a functional human equivalent of yeast Set1. Both proteins colocalize with RNA Pol II at the transcription start site of highly expressed genes and catalyze the trimethylation of H3K4 at this location. Only at active Hox genes MLL1 reveals a different binding behavior covering entire active chromatin domains. In contrast, the current data shows that Trx also localizes to promoter regions of silent HOX genes and does not show the spreading behavior of MLL1 but appears at additional discrete sites. A complete colocalization of Trx with PRC1 sites was observed at silent genes, i.e., in this expression state no obvious competition is taking place with regard to binding sites (Beisel, 2007).
The comparison of the AbdB gene with the Dfd gene shows that the maintenance of the active state can be performed in alternative ways. The absence of PcG complexes does not seem to be a prerequisite of the active state as observed at the promoter of Dfd in this study and at regulatory regions of Ubx in imaginal discs (Beisel, 2007).
In the active AbdB domain Ph stays bound in a minor but significant amount, and Psc is present in the active Dfd intron. In this regard, Ph and Psc could serve as recruiting platforms for other PRC1 subunits in case of the gene switching to the off state. However, both proteins have been reported to be associated with active genes. Consistent with this, Ph was also observed in the proximal part of both homeotic complexes binding actively transcribed non-HOX genes. The function of this binding behavior remains elusive (Beisel, 2007).
The transcription of noncoding RNAs (ncRNAs) seem to play an important, although diverse, role in the regulation of the BX-C. Noncoding transcription found through the bxd PRE is crucial for Ubx repression and transcription through Mcp overlaps with AbdB transcription in the embryo. NcRNA transcription in the AbdB domain coincides with an active AbdB gene indicates a nonuniversal, gene specific function for ncRNAs in the BX-C (Beisel, 2007).
In the silent state PRC1 is bound to all PREs in the AbdB domain and might be recruited by the action of sequence-specific factors like Pho and the E(Z) histone methyltransferase activity, which may also mark the entire domain as being inactive. In the active AbdB domain, ncRNA transcription may directly influence the binding of of PRC1 and E(Z) or may trigger the enzymatic activity of Trx. Consistent with this scenario, Trx has been shown to bind single-stranded DNA and RNA in vitro. The switch of Trx into an activating mode could lead to the methylation of histones and/or other proteins setting positive transcriptional marks and modulate their activity, respectively. In this case, the displacement of PcG proteins could be directly caused by the Trx action. The binding of Trx to the promoter regions of the active AbdB transcription units could either be caused by (transient) chromatin looping events bridging Trx-bound PREs with the promoters, or Trx could be recruited independently to the active HOX promoters by interaction with RNA Pol II, similar to MLL1, which is recruited to actively transcribed genes in mammalian cells. Trx- and TAC1-interacting histone acetyltransferases may then be responsible for setting epigenetic marks that maintain the active transcription state. Trx has been shown to be required for transcription elongation and it is localized in the gene body of active Ubx, caused by the interaction with elongation factors. In contrast, other studies that investigated the distribution of PcG and TrxG proteins at the active and repressed Ubx gene in imaginal discs found the same restricted Trx profile did the current study, namely Trx binding at discrete sites. These differences may be explained by the different Trx antibodies used. Trx is most probably proteolytically processed like human MLL which results in two fragments that form a heterodimeric complex. This raises the intriguing question whether the complete heterodimeric Trx complex might get recruited to the promoter and upon gene induction the N-terminal fragment tracked along the gene body together with elongation factors, whereas the C-terminal fragment stayed at the promoter (Beisel, 2007).
Pho maps were generated to investigate its role in the recruitment of PRC1. However, the distribution of Pho suggests that the protein also functions in the gene body of actively transcribed genes. The immunostaining of polytene chromosomes revealed that Pho seems not only to be limited to HOX gene control but plays a general role in gene regulation. The colocalization of Pho with strong signals of active Pol II on polytenes together with the effect of a pho-null mutation on the recovery of induced hsp70 indicates that Pho may be directly involved in the rerepression of highly active genes (Beisel, 2007).
It is difficult to imagine that the spreading of Pho is the result of the ability of this protein to bind sequence specifically to DNA. Instead, a model is proposed in which Pho either acts directly at the Pol II elongation complex or it interacts with a remodeling complex, carrying it along the chromatin fiber. In this line, Pho has been shown to interact with BRM and dINO80, two nucleosome remodeling complexes. Interestingly, heat-shock gene transcription is independent of BRM but involves the recruitment of the TAC1 complex, possibly through multiple interactions with the elongating Pol II complex. The simultaneous action of Trx and Pho at heat-shock genes is striking and might resemble their antagonistic functions at HOX genes. Further studies are necessary to unravel the exact molecular mechanism of Pho in this process (Beisel, 2007).
The GAGA factor (GAF), encoded by the Trithorax like gene (Trl) is a multifunctional protein involved in gene activation, Polycomb-dependent repression, chromatin remodeling and is a component of chromatin domain boundaries. Although first isolated as transcriptional activator of the Drosophila homeotic gene Ultrabithorax (Ubx), the molecular basis of this GAF activity is unknown. This study shows that dmTAF3 (also known as BIP2 and dTAFII155), a component of TFIID, interacts directly with GAF. Mutations were generated in dmTAF3; in Trl mutant background, they affect transcription of Ubx leading to enhancement of Ubx phenotype. These results reveal that the gene activation pathway involving GAF is through its direct interaction with dmTAF3 (Chopra, 2008)
GAF has been shown to be a transcriptional activator of many genes. Recent studies suggested that both GAF and TFIID are necessary for formation of the appropriate chromatin structure at the hsp26 promoter indicating a mechanism in which GAF binding precedes and contributes to the recruitment of TFIID. However, the question of how GAF leads to the recruitment of TFIID remained unanswered. The finding that GAF interacts directly with dmTAF3 reveals a possible mechanism for how GAF could recruit the TFIID complex to carry out transcriptional activation. It is known that GAF carries out functions other than the activation of transcription. Since GAF does not interact with large number of transcription factors directly, as is evident from genome scale interaction screen, it is likely that recruitment of transcription machinery to activate a promoter is mediated through specific factors, and the current results show that dmTAF3 plays a major role in this. Colocalization and ChIP results also suggest that interaction of GAF with dmTAF3 is very likely to be dependent on the genomic context. This may reflect the fact that such loci may be involved in functions that do not require activator proteins, for example, the loci where GAF functions as repressor protein and recruits Polycomb group members. GAF functions that are independent of dmTAF3 would also include chromatin domain boundary elements where GAF is known to play a role (Chopra, 2008)
The fact that a 3-fold reduction in dmTAF3 expression is sufficient to enhance the phenotype of Ubx mutation in a sensitized background -- heterozygous for the Trl gene encoding GAGA factor -- is functional evidence that TAF3 is a direct partner of GAF in the activation pathway. The effect is likely to be at the level of transcription as shown by the modified expression of a Ubx-lacZ transgene in dmTAF3 and Trl mutant background and confirmed by the reduced level of UBX in the double mutant context. A simple model proposes that GAF contributes to Ubx transcription by its binding to specific sites (via its Zinc fingers) near the promoter and then recruits the transcriptional machinery by interaction of its BTB/POZ domain with dmTAF3
GAF can remodel the nucleosomes with the help of NURF complexes and facilitate access to activator or repressor components to such remodeled cis elements. The bound GAF can interact with dmTAF3 which can help recruit TFIID complex and maintain active state of the target gene. In contrast, when a target gene needs to be repressed the bound GAF could recruit PcG complexes to maintain repressed state. GAF binding sites have been found in promoters as well as in PREs. The interaction of GAF either with transcription factors like dmTAF3 or with PcG repressor proteins such as LOLAL, raises the possibility that GAF functions like a switch that could recruit either activator or repressor complexes at a target promoter and then maintain the transcriptional state. In cell types in which a gene needs to be active, GAF bound at the promoter sites would interact with activators and maintain the active state. By contrast, in cell types where a promoter needs to be silenced, GAF would interact with PcG proteins, associated with even distant PREs, by a looping mechanism, and bring about a repressive chromatin context, which probably involves histone tail modifications. It is likely that additional DNA binding factors and their interacting partners contribute to these cross talks of delicately regulated loci with activation and repression machinery. Further studies will be needed to understand this complex network of regulatory events (Chopra, 2008)
Development of the fruit fly Drosophila depends in part on epigenetic regulation carried out by the concerted actions of the Polycomb and Trithorax group of proteins, many of which are associated with histone methyltransferase activity. Mouse PTIP is part of a histone H3K4 methyltransferase complex and contains six BRCT domains and a glutamine-rich region. This study describes an essential role for the Drosophila ortholog of the mammalian Ptip (Paxip1) gene in early development and imaginal disc patterning. Both maternal and zygotic ptip are required for segmentation and axis patterning during larval development. Loss of ptip results in a decrease in global levels of H3K4 methylation and an increase in the levels of H3K27 methylation. In cell culture, Drosophila ptip is required to activate homeotic gene expression in response to the derepression of Polycomb group genes. Activation of developmental genes is coincident with PTIP protein binding to promoter sequences and increased H3K4 trimethylation. These data suggest a highly conserved function for ptip in epigenetic control of development and differentiation (Fang, 2009).
The establishment and maintenance of gene expression patterns in
development is regulated in part at the level of chromatin modification
through the concerted actions of the Polycomb and trithorax family of genes
(PcG/trxG). In Drosophila, Polycomb and Trithorax response elements
(PRE/TREs) are cis-acting DNA sequences that bind to Trithorax or Polycomb
protein complexes and maintain active or silent states, presumably in a
heritable manner. In mammalian cells however, such PRE/TREs have not been
conclusively identified. Polycomb and Trithorax gene products function by
methylating specific histone lysine residues, yet how these complexes
recognize individual loci in a temporal and tissue specific manner during
development is unclear. Recently, a novel protein, PTIP (also
known as PAXIP1), was identified that is part of a histone H3K4 methyltransferase complex and binds to the Pax family of DNA-binding proteins
(Patel, 2007). PTIP is essential for assembly of the histone methyltransferase (HMT) complex at a Pax
DNA-binding site. These data suggest that Pax proteins, and other similar
DNA-binding proteins, can provide the locus and tissue specificity for HMT
complexes during mammalian development (Fang, 2009).
In mammals, the PTIP protein is found within an HMT complex that includes
the SET domain proteins ALR (GFER) and MLL3, and
the accessory proteins WDR5, RBBP5 and ASH2. This PTIP containing complex can methylate lysine 4 (K4) of histone H3, a modification implicated in epigenetic activation and maintenance of gene expression patterns. Furthermore, conventional Ptip-/- mouse embryos and conditionally inactivated Ptip-/- neural stem cell derivatives show a marked decrease in the levels of global H3K4 methylation, suggesting that PTIP is required for some subset of H3K4 methylation events (Patel, 2007). The PTIP
protein contains six BRCT (BRCA1 carboxy terminal) domains that can bind to
phosphorylated serine residues. This is consistent with the observation that PAX2 is serine-phosphorylated in response to inductive signals. In mammals,
PAX2 specifies a region of mesoderm fated to become urogenital epithelia at a
time when the mesoderm becomes compartmentalized into axial, intermediate and
lateral plate. These data suggest that PTIP provides a link between tissue
specific DNA-binding proteins that specify cell lineages and the H3K4
methylation machinery (Fang, 2009).
To extend these finding to a non-mammalian organism and address the
evolutionary conservation of Ptip, it was asked whether a Drosophila
ptip homolog could be identified and if so, whether it is also an
essential developmental regulator and part of the epigenetic machinery. The
mammalian Ptip gene encodes a novel nuclear protein with two
amino-terminal and four carboxy-terminal BRCT domains, flanking a
glutamine-rich sequence. Based on the number and position of the BRCT domains and the glutamine-rich domain, the Drosophila genome contains a single ptip homolog. To understand the function of Drosophila ptip in development, a ptip mutant allele was characterized that contained a piggyBac transposon insertion between BRCT domains three and four. Maternal and zygotic ptip mutant embryos exhibited severe patterning
defects and developmental arrest, whereas zygotic null mutants developed to
the third instar larval stage but also exhibited anterior/posterior (A/P)
patterning defects. In cell culture, depletion of Polycomb-mediated repression
activates developmental regulatory genes, such as the homeotic gene
Ultrabithorax (Ubx). This derepression is dependent on trxG
activity and also requires PTIP. Microarray analyses in cell culture of
Polycomb and polyhomeotic target genes indicate that many,
but not all, require PTIP for activation once repression is removed. The
activation of PcG target genes is coincident with PTIP binding to promoter
sequences and increased H3K4 trimethylation. These data argue for a conserved
role for PTIP in Trithorax-mediated epigenetic imprinting during development (Fang, 2009).
Embryonic development requires epigenetic imprinting of active and inactive
chromatin in a spatially and temporally regulated manner, such that correct
gene expression patterns are established and maintained. This study shows that Drosophila ptip is essential for early embryonic
development. In larval development, ptip
coordinately regulates the methylation of histone H3K4 and demethylation of
H3K27, consistent with the reports that mammalian PTIP complexes with HMT
proteins ALR and MLL3, and the histone demethylase UTX. In wing
discs, ptip is required for appropriate A/P patterning by affecting
morphogenesis determinant genes, such as en and ci. These
data demonstrate in vivo that dynamic histone modifications play crucial roles
in animal development and PTIP might be necessary for coherent histone coding.
In addition, ptip is required for the activation of a broad array of
PcG target genes in response to derepression in cultured fly cells. These data
are consistent with a role for ptip in trxG-mediated activation of
gene expression patterns (Fang, 2009).
Early development requires ptip for the appropriate expression of
the pair rule genes eve and ftz. The characteristic
seven-stripe eve expression pattern is regulated by separate enhancer
sequences, which are not all equally affected by the loss of ptip. The complete absence of en expression at the extended germband stage also indicates the dramatic effect of ptip mutations on transcription. The
characteristic 14 stripes of en expression depends on the correct
expression of pair rule genes, which are clearly affected in ptip
mutants. However, the maintenance of en expression at later stages
and in imaginal discs is regulated by PREs and PcG proteins.
If ptip functions as a trxG cofactor, then expression of en
along the entire A/P axis in the imaginal discs of ptip mutants might
be due to the absence of a repressor. This might explain the surprising
presence of ectopic en in the anterior halves of imaginal discs from
zygotic ptip mutants. This ectopic en expression is likely
to result in suppression of ci through a PcG-mediated mechanism. Yet,
it is not clear how en is normally repressed in the anterior half,
nor which genes are responsible for derepression of en in the
ptip mutant wing and leg discs (Fang, 2009).
The direct interaction of PTIP protein with developmental regulatory genes
is supported by ChIP studies in cell culture. Given the structural and
functional conservation of mouse and fly PTIP, mPTIP was expressed in fly cells; it can localize to the 5' regulatory regions of many PcG
target genes that are activated upon loss of PC and PH activity. Consistent
with the interpretation that a PTIP trxG complex is necessary for activation
of repressed genes, mPTIP only bound to DNA upon loss of Pc and
ph function. In the Kc cells, suppression of both Pc and
ph results in the activation of many important developmental
regulators, including homeotic genes. A recent report details the genome-wide
binding of PcG complexes at different developmental stages in Drosophila and
reveals hundreds of PREs located near transcription start sites.
Strikingly, most of the genes found to be activated in the Kc cells after PcG
knockdown also contain PRE elements near the transcription start site (Fang, 2009).
In vertebrates, PTIP interacts with the Trithorax homologs ALR/MLL3 to
promote assembly of an H3K4 methyltransferase complex. The tissue and locus
specificity for assembly may be mediated by DNA-binding proteins such as PAX2
(Patel, 2007) or SMAD2 (Shimizu, 2001), which
regulate cell fate and cell lineages in response to positional information in
the embryo. In flies, recruitment of PcG or trxG complexes to specific sites
also can require DNA-binding proteins such as Zeste,
DSP1, Pleiohomeotic and Pipsqueak. Whereas PcG complexes have been purified and described in detail, much less is known about the Drosophila trxG
complexes. Purification of a trxG complex capable of histone acetylation
(TAC1) revealed the proteins CBP and SBF1 in addition to TRX. By
contrast, the mammalian MLL/ALL proteins are components of large multi-protein
complexes capable of histone H3K4 methylation. Although the mutant analysis, the reduction of H3K4 methylation and the dsRNA knockdowns in Kc cells all suggest that Drosophila ptip has trxG-like activity and hence might be a suppressor of PcG proteins, a more definitve biochemical analysis awaits the generation of antibodies and the delineation of in vivo DNA-binding sites for PTIP and its associated proteins at specific target genes (Fang, 2009).
Mammalian PTIP is also thought to play a role in the DNA damage response
based on its ability to bind to phosphorylated p53BP1. PTIP also binds preferentially to the P-SQ motif, which is a good substrate for the ATR/ATM cell cycle checkpoint regulating kinases. Several reports demonstrate that PTIP is part of a RAD50/p53BP1 DNA damage response complex, which can be separated from the MLL2 histone H3K4 methyltransferase complex. Both
budding and fission yeast contain multiple BRCT domain proteins that are
involved in the DNA damage response, including Esc4, Crb2, Rad9 and Cut5. All
of these yeast proteins have mammalian counterparts. However, neither the
fission nor budding yeast genomes encodes a protein with six BRCT domains and
a glutamine-rich region between domains two and three, whereas such
characteristic PTIP proteins are found in Drosophila, the honey bee,
C. elegans and all vertebrate genomes. These comparative genome
analyses suggest that ptip evolved in metazoans, consistent with an
important role in development and differentiation (Fang, 2009).
In summary, Drosophila ptip is an essential gene for early
embryonic development and pattern formation. Maternal ptip null
embryos show early patterning defects including altered and reduced levels of
pair rule gene expression prior to gastrulation. In cultured cells PTIP
activity is required for the activation of Polycomb target genes upon
derepression, suggesting an important role for the PTIP protein in
trxG-mediated activation of developmental regulatory genes. The conservation
of gene structure and function, from flies to mammals, suggests an essential
epigenetic role for ptip in metazoans that has remained
unchanged (Fang, 2009).
Trimethylation of histone H3 at lysine 27 (H3-K27me3) by Polycomb repressive complex 2 (PRC2) is a key step for transcriptional repression by the Polycomb system. Demethylation of H3-K27me3 by Utx and/or its paralogs has consequently been proposed to be important for counteracting Polycomb repression. To study the phenotype of Drosophila mutants that lack H3-K27me3 demethylase activity, UtxΔ), a deletion allele of the single Drosophila Utx gene, was created. UtxΔ homozygotes that contain maternally deposited wild-type Utx protein develop into adults with normal epidermal morphology but die shortly after hatching. By contrast, UtxΔ homozygotes that are derived from Utx mutant germ cells and therefore lack both maternal and zygotic Utx protein, die as larvae and show partial loss of expression of HOX genes (Ubx and Abd-B) in tissues in which these genes are normally active. This phenotype classifies Utx as a trithorax group regulator. It is proposed that Utx is needed in the early embryo to prevent inappropriate installment of long-term Polycomb repression at HOX genes in cells in which these genes must be kept active. In contrast to PRC2, which is essential for, and continuously required during, germ cell, embryonic and larval development, Utx therefore appears to have a more limited and specific function during development. This argues against a continuous interplay between H3-K27me3 methylation and demethylation in the control of gene transcription in Drosophila. Furthermore, this analyses do not support the recent proposal that Utx would regulate cell proliferation in Drosophila as Utx mutant cells generated in wild-type animals proliferate like wild-type cells (Copur, 2013).
The Polycomb group of proteins (PcG) is important for transcriptional repression and silencing in all higher eukaryotes. In Drosophila, PcG proteins are recruited to the DNA by Polycomb-group response elements (PREs), regulatory sequences whose activity depends on the binding of many different sequence-specific DNA-binding proteins. Previous studies have shown that that a binding site for the Sp1/KLF family of zinc-finger proteins is required for PRE activity (Brown, 2005). This study reports that the Sp1/KLF family member Spps binds specifically to Ubx and engrailed PREs, and that Spps binds to polytene chromosomes in a pattern virtually identical to that of the PcG protein, Psc. A deletion of the Spps gene causes lethality late in development and a loss in pairing-sensitive silencing, an activity associated with PREs. Finally, the Spps mutation enhances the phenotype of pho mutants. It is suggested that Spps may work with, or in parallel to, Pho to recruit PcG protein complexes to PREs (Brown, 2010).
Spps binds polytene chromosomes in a pattern comparable with the PcG protein Psc and, as shown by chromatin immunoprecipitation, is bound to both the en and Ubx PREs in S2 cells and in larvae. Furthermore, a mutation in Spps abrogates PRE activity in a mini-white assay, and enhances the phenotypes seen in a pho mutant. It is suggested that Spps acts either with or in parallel to Pho to recruit PcG protein complexes to the DNA. This result is particularly interesting in lieu of the recent report that the PcG protein Scm is recruited to the DNA independently of Pho (Wang, 2010). It has been speculated that Scm is in a complex with another PRE-DNA-binding protein, and it was shown that, like Pho, Scm plays a role in recruitment of PRC1 and PRC2 to the PRE. It will be interesting to explore whether Spps or another Sp1/KLF family member recruits Scm to the DNA (Brown, 2010).
The mammalian homologues of Pho and Spps, YY1 and Sp1 are extremely versatile proteins. Their activities can be changed from repressor to activator or vice versa depending on the cellular and binding site context. The activity of both these proteins is sensitive to the influence of many different co-repressors and co-activators. Both factors have been shown to bend DNA. Finally, YY1 and Sp1 have also been shown to interact directly (see Li, 2008). It is intriguing that such proteins bind to the PREs of Drosophila genes. Given that PREs may mediate the action of both the Polycomb and Trithorax group proteins, DNA-binding/recruitment proteins with such versatility and adaptability could be one way to facilitate the change from repression to activation. In fact, there is a report that Pho and Phol, in addition to their association with PREs, are bound to regions of chromatin with active histone modifications (Schuettengruber, 2009
). Finally, a single Pho-binding site in a PRE in the even-skipped gene has been shown to be important for both activation and repression, dependent on the context (Fujioka, 2008). It will be interesting to explore whether Spps also has a dual role in gene regulation (Brown, 2010).
RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function
Ubx regulation: Table of contents
moira mutations suppress the derepression phenotypes caused by mutations in another Pc group gene, Polycomblike. moira mutant clones in the haltere differentiate large bristles, characteristic of the anterior wing margin, and often lead to absence or duplication of halteres. Homozygous mor mutations in the posterior wing result in a distorted wing shape; the venation is disrupted and large socketed bristles appear along the posterior wing margin. Leg clones result in the femur and tibia being short and twisted and enlargement of the tarsal segment. Clones of the head cause the shape of the head to be abnormal in the dorsal region and sometimes cause the ocellus to be abnormal or absent. Embryos homozygous for moira mutations have defects in head structures, including truncated lateralgraten and defects in the mouth hooks and dorsal bridge. The first and second midgut constrictions are shifted posterior to their wild-type positions (Brizuela, 1997).
A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq was performed in two different embryonic cell lines; AGO2 was found to localize to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. It is proposed that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome (Moshkovich, 2011).
This study provides the first evidence for an Argonaute protein functioning directly on euchromatin to effect changes in gene expression. The genome-wide binding profile of AGO2 displays striking overlap with insulator proteins. Genetic analysis revealed that AGO2, independent of its catalytic activity, promotes Fab-8 insulator activity. Like known insulator proteins, AGO2 also associates with promoters and can oppose PcG function. Genome-wide AGO2 recruitment to chromatin is dependent on CTCF and CP190 binding and may be partially achieved via looping interactions among cis-regulatory regions and promoters. It is proposed that AGO2 may act to facilitate or stabilize looping that is needed to partition the genome into independent transcriptional domains (Moshkovich, 2011).
These results suggest that the main function of AGO2 on chromatin resides in euchromatin and not in heterochromatin. Immunofluorescence localization of AGO2 on polytene chromosomes and cell lines indicates exclusion from heterochromatic and HP1-enriched regions. Furthermore, the majority of chromatin-associated AGO2 resides in nonrepetitive euchromatic but not repeat-rich regions, as determined by genome-wide ChIP-seq. It is suggested that the role of AGO2 in RNAi-dependent silencing of TEs occurs primarily at the post-transcriptional level and that AGO2 harbors a second RNAi-independent activity to promote chromatin insulator function (Moshkovich, 2011).
Several observations suggest that AGO2 chromatin association is mainly, if not exclusively, independent of the RNAi pathway. First, AGO2 chromatin association does not correspond to regions of the genome that produce high levels of endo-siRNAs, which are dependent on Dcr-2 and AGO2. Second, AGO2, but not Dcr-2, is required for Fab-8 insulator function. Finally, a catalytically inactive AGO2 protein, which is defective for RNAi, retains the ability to associate with chromatin and is functional with respect to both TrxG function and Fab-8 insulator activity (Moshkovich, 2011).
An intriguing question raised by these findings is whether or not the functions of AGO2 in RNAi and chromatin insulator activity are completely distinct. CP190 mutants were found to remain competent for silencing, suggesting that AGO2 chromatin association is not required for RNAi. Nevertheless, it remains possible that chromatin-associated AGO2 is loaded with siRNA. Future work will address how AGO2 subcellular localization and seemingly disparate functions in RNAi and chromatin insulator activities are regulated (Moshkovich, 2011).
A unique positive role for AGO2 but not other RNA silencing factors was identified in Fab-8 insulator function. Importantly, a catalytically inactive mutant form of AGO2 expressed at wild-type levels retains insulator activity, further suggesting that the RNAi pathway is dispensable for Fab-8 insulator function. A significant fraction of AGO2 resides in the nucleus, and physical interaction is observed between AGO2 and CP190. This interaction is insensitive to RNaseA, suggesting that RNA does not mediate the interaction between AGO2 and CP190. It remains possible that AGO2 can interact with siRNA or other RNA while associated with the insulator complex, although there is no evidence to support this hypothesis (Moshkovich, 2011).
This study shows that chromosomal looping in the Abd-B locus is dependent on CTCF, CP190, and AGO2. Confirming and extending previous studies, it was found that the Abd-B RB promoter interacts frequently with Fab-7, Fab-8, and the iab-8 enhancer and, moreover, that the Fab-8 region also contacts Fab-7 as well as multiple Abd-B promoters. Currently, the significance of insulator protein promoter association is unclear, but insulators may be thus situated to control looping interactions between promoters and cis-regulatory elements. Depletion of CP190 or CTCF reduces these high-frequency looping interactions, and loss of this specialized chromatin configuration could result in disassociation of AGO2. Given this possibility, AGO2 may act to detect the insulator-dependent conformation of this locus (Moshkovich, 2011).
AGO2 is recruited to chromatin insulator sites as well as noninsulator sites in a CTCF/CP190-dependent manner. It is speculated that AGO2 chromatin association with insulator sites could result from physical interactions with CP190 complexes, while AGO2 recruitment to other sites may be achieved at least in part by chromatin looping mediated by CP190 and CTCF. In fact, it was recently shown that PcG proteins can be transferred from a PRE to a promoter as a result of intervening insulator-insulator interactions. Once recruited to chromatin, AGO2 could perform a primarily structural function to promote or stabilize the frequency of CTCF/CP190-dependent looping interactions (Moshkovich, 2011).
AGO2 appears to promote Fab-8 insulator activity independently of an effect on gypsy insulator body localization. Previous work showed that both the gypsy class and CTCF/CP190 insulators colocalize to insulator bodies, suggesting that these subnuclear structures may be important for both gypsy and Fab-8 activities. However, since Fab-8 activity is not affected by RNA silencing components that disrupt gypsy insulator body localization, this subnuclear structure appears to be dispensable for Fab-8 function. Recent work indicates that the BX-C harbors multiple redundant cis-regulatory elements that can maintain looping interactions of this locus, suggesting that the configuration of the BX-C may not require a nuclear scaffold such as the gypsy insulator body (Moshkovich, 2011).
AGO2 mutations suppress the Polycomb phenotype, indicating that AGO2 behaves similarly to trxG genes and opposes PcG function. A previous study proposed that RNA silencing factors promote long-range PRE-dependent chromosomal pairing as well as PcG body formation but did not examine AGO2. This study found that the AGO251B-null mutation has no effect on Fab-X PRE pairing-dependent silencing on sd as assayed in that study, and genetic results suggest that AGO2 is unlikely to promote PRE-dependent interactions or PcG body formation, which are both positively correlated with PcG function. Interestingly, it has recently been shown in the case of AGO2-associated Fab-7 and Mcp boundary elements that long-range interactions are dependent on insulator sequences and not PREs. Future studies will elucidate the complex interplay between PcG and insulator organization as well as the role of AGO2 in the regulation of these structures (Moshkovich, 2011).
It remains to be seen whether Drosophila AGO2 euchromatin association and function may be conserved in other organisms. In Caenorhabditis elegans, the nuclear NRDE RNAi pathway can block transcriptional elongation of Pol II on a target transcript when treated with exogenous complementary dsRNA. Interestingly, this negative transcriptional effect is contemporaneous with an increase in H3K9me3. Whether the Argonaute protein NRDE-3/WAGO-12, which lacks Slicer activity, associates with euchromatin to effect this repression is not yet known. Furthermore, the C. elegans Argonaute Csr-1, loaded with 22G endo-siRNAs antisense to mRNAs of holocentric chromosomes, may serve as chromosomal attachment points to promote efficient chromosome segregation. Recently, it has been shown that Schizosaccharomyces pombe Ago1 participates in surveillance mechanisms to prevent readthrough transcription of mRNA. However, the majority of Ago1 associates with heterochromatic regions, and it is not clear thus far whether Ago1 directly associates with euchromatin or acts post-transcriptionally. An emerging theme from studies of RNAi in various model systems is that genome integrity and control of gene expression may be achieved by multiple yet overlapping mechanisms (Moshkovich, 2011).
The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. This study reports that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. It was demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells, known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes were dissected. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. It is concluded that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut (Schaub, 2013).
The analysis of org-1 expression and function during visceral mesoderm development defined this gene as a new and essential lineage specific regulator of circular visceral muscle founder cell identities and midgut patterning in Drosophila. The data add new insights into the developmental regulatory mechanisms responsible for the diversification of the circular visceral muscle founder cell lineage and midgut morphogenesis (Schaub, 2013).
The initial expression of org-1 occurs in the segmented trunk visceral mesoderm (TVM), where it is coexpressed with tin, bap, bin and Alk. It has been documented that the induction of tin and bap in the dorsal mesoderm involves the combined binding of Smad proteins (Medea and Mad) and Tin to Dpp-responsive enhancers of the tin and bap genes, whereas the segmental repression of bap is mediated by binding of the sloppy paired (slp) gene product. Genetic analysis of org-1 has shown that org-1 is activated downstream of tin but independently of bap and bin, and that dpp provides the key signals for its induction. This suggests a regulatory mechanism analogous to that of bap, in which the combined binding of Smads and Tin activates a Dpp-responsive org-1 enhancer, whereas Wg activated Slp is required for its mutual segmental repression (Schaub, 2013).
The similarities in the early expression patterns of bap, bin, Alk and org-1 in the trunk visceral mesoderm primordia raise the question of the contribution of org-1 to the early development of the TVM as such. Whereas bap and bin are crucially required for the specification of the trunk visceral mesoderm and visceral musculature, loss of org-1 function, like the loss of Alk, has no obvious impact on the specification of the early TVM. Therefore, it is notable that during the subdivision of the visceral mesoderm primordia into founder and fusion-competent myoblasts (cFCs and FCMs), org-1 expression is extinguished in the FCMs and only sustained in the cFC lineage of the circular visceral musculature. This lineage-specific restriction and maintenance of org-1 expression crucially depends on Jeb mediated Alk/Ras/MAPK signaling and points toward a possible cFC lineage specific function of org-1. The genetic analysis demonstrates that org-1 is not required for cFC specification, but plays a decisive role in the induction of the visceral mesoderm specific expression of patterning genes in the founder cells of the circular musculature. Thus, org-1 is critical for the processes of cell fate diversification that provide individual fields of cells along the anteroposterior axis of the visceral mesoderm with their specific identities (Schaub, 2013).
Proper anteroposterior patterning of the trunk visceral mesoderm and the formation of localized organizer fields are prerequisites for eliciting the morphogenetic events that shape the midgut. The formation of these organizer fields depends on the appropriate spatial expression domains of the homeotic selectors Scr, Antp, Ubx and abd-A, the secreted factors dpp and wg, as well as the zinc finger proteins opa and tsh, which are required for the formation of the midgut constrictions as well as the gastric caeca. The regulatory mechanisms responsible for the establishment of the spatial, temporal and tissue-specific expression patterns of these genes in the TVM are only partially understood. Genetic and molecular analyses with the FoxF gene bin, which is expressed in all trunk visceral mesoderm precursors and their descendents, have demonstrated that bin is a direct upstream regulator of dpp in PS7 and is also required for the expression of wg in PS8 of the TVM. Thus, Bin serves as an essential TVM-specific competence factor in conjunction with the dpp/wg signaling feedback loop. The current findings have defined Org-1 as an additional tissue-specific regulator with an even broader range of downstream patterning genes in the TVM, but with a narrower spatial range of action. org-1 acts specifically within the visceral muscle founder cell lineage as a positive regulator upstream of opa, tsh, Ubx, dpp as well as wg (Schaub, 2013).
This combination of genetic data and functional enhancer analyses provides convincing evidence that both dpp and wg are direct transcriptional targets of Org-1 in the cFCs. Prior dissections of the dpp visceral mesoderm (VM) enhancer had shown that it is also regulated by the direct binding of Ubx, Exd, dTCF (a Wg effector) and Bin, and that minimal synthetic variants that contain only the binding motifs for Ubx, Exd, Bin, and dTCF within conserved sequence contexts (which happen to include the Org-1 motif) are active as VM enhancers. Likewise, the wgXC enhancer fragment integrates Org-1 with the direct regulatory inputs of Abd-A as well as CREB and Smad (Mad/Medea) proteins mediating Dpp signaling (Schaub, 2013).
Org-1 is the first transcription factor known to be required for Ubx expression in PS7 of the visceral musculature. Extensive work on an Ubx visceral mesoderm CRM (UbxRP) indicated that dpp and wg regulate Ubx through indirect autoregulation. Of note, in bin embryos, which also lack visceral mesodermal dpp and wg expression, Ubx is still expressed. Genetic data show that the UbxRP element, while requiring org-1, is not directly regulated by Org-1, since mutation of its four predicted T-Box binding sites did not have any effects. Taking into account that no UbxRP reporter activity was detected in the cFCs at pre-fusion stages, it is suggested that UbxRP represents a late enhancer element and responds to dpp and wg only after they are activated by Org-1 in the founder cells. To clarify whether the regulation of Ubx by Org-1 is direct or indirect, the identification and dissection of a founder cell specific CRM will be required (Schaub, 2013).
tsh and opa were described as homeotic target genes of Antp in PS4-6 (tsh) and PS4-5 (opa) as well as of abd-A in PS8 (tsh) and PS9-12 (opa) of the visceral musculature. The current data show that tsh and opa expression is already activated in the respective cFCs of the visceral parasegments where it requires org-1. The later activation of tsh in PS8 during muscle fusion follows the org-1 dependent founder cell specific initiation of wg in PS8, which acts upstream of tsh. Thus it was conceivable that the regulation of tsh by org-1 is indirect. However, ectopic activation of wg in an org-1 loss of function background is not able to rescue tsh expression and Antp and abd-A expression is not altered upon loss of org-1. These observations suggest that Org-1 acts directly on tsh and opa, e.g., via functional cooperation with Antp and Abd-A, respectively, during the early activation of tsh and opa in the founder cells (Schaub, 2013).
It was reported that the absence of Jeb/Alk signaling causes loss of dpp expression in the founder cells in PS7 of the visceral mesoderm. In light of the current findings that org-1 loss-of-function produces a similar phenotype, and of the previous demonstration that org-1 expression is downstream of Jeb/Alk, this observation could simply be explained by the action of a linear regulatory cascade from Jeb/Alk via org-1 towards dpp. Alternatively, Jeb/Alk may provide additional inputs towards dpp (and other patterning genes) in parallel to org-1, which could explain the slightly stronger phenotype of Alk as compared to org-1 mutations with respect to dpp. A possible candidate for an additional effector of Jeb/Alk signals in this pathway is extradenticle (exd), which is known to be required for normal dpp expression in PS7 of the visceral mesoderm, presumably through direct binding of Exd in a complex with Hox proteins and Homothorax (Hth) to a PS7-specific enhancer element (a derivative of which was used in this study). Like org-1, exd is also needed for the expression of tsh and wg in the visceral mesoderm (Additionally, it represses dpp in PS4-6 through sequences not contained in the minimal PS7 enhancer). It is thought that Exd complexed with Hox proteins and Hth increases the binding preference of these Hox complexes for specific binding sites within visceral mesodermal enhancers of their target genes (Schaub, 2013).
Since exd is expressed in both founder and fusion-competent cells in the visceral mesoderm, it is unlikely that it fulfills its roles in the regulation of dpp, wg, and tsh in the founder cells as a downstream gene of org-1. However, it is known that Exd requires nucleocytoplasmic translocation for it to be functiona and, interestingly, it has been shown that Jeb/Alk signals trigger nuclear localization of Exd specifically in the cFCs of the visceral mesoderm. Because nuclear Exd appears to be hyperphosphorylated as compared to cytoplasmic Exd, nuclear translocation of Exd may be triggered by Alk-mediated phosphorylation. Alternatively, Jeb/Alk signals may induce the expression of hth in the cFCs and Hth could then translocate Exd to the nuclei, as has been shown in other contexts. This would be compatible with the observation that Hth is upregulated in the founder cells in an org-1-independent manner (Schaub, 2013).
The combined data show that Jeb/Alk signals exert at least two parallel inputs towards patterning genes in the cFCs, which are the induction of org-1 and the nuclear translocation of Exd. Taken altogether, a model is suggested in which combinatorial binding of Org-1, nuclear Exd/Hth and the homeotic selector proteins to the corresponding visceral mesoderm specific CRMs is required for the initiation of lineage specific expression of opa, tsh, dpp, Ubx and wg in the founder cells of the respective parasegments. As shown in the examples of dpp (PS7) and wg (PS8), accessory Bin is required for the activation as a general visceral mesodermal competence factor, whereas Dpp and Wg effectors mediate autoregulatory stabilization of their expression (Schaub, 2013).
Extensive work has shown that during somatic muscle development individual founder myoblasts acquire distinct identities, which are adopted by the newly incorporated nuclei upon myoblast fusion, thus leading to the morphological and physiological diversification of the differentiating muscles. It is proposed that the same principle is active during visceral muscle development. In this view, Org-1 acts as a muscle identity factor in both the somatic and visceral mesoderm. In the visceral mesoderm, Org-1 helps diversifying founder cell identities and, after myoblast fusion, their differential identities are transmitted to the respective differentiating circular gut muscles. The activation of downstream targets of this identity factor in the developing muscles leads to the observed morphogenetic differentiation events of the midgut and the establishment of the signaling center in PS7/8 that is also required for Dpp and Wg mediated induction of labial in the endodermal germ layer. As is the case for identity factors in the somatic muscle founders, Org-1 in the visceral mesoderm acts in concert with other, spatially restricted activities such as Hox factors and signaling effectors to achieve region-specific outputs. The main difference is that, in the trunk visceral mesoderm, Org-1 is present in all founder cells whereas in the somatic mesoderm this identity factor (like others) is expressed in a particular subset of founder myoblasts. Thus, in contrast to the somatic mesoderm, the spatial expression of Org-1 does not contribute to its function in visceral muscle diversification and instead, it solely relies on spatially-restricted co-regulators during this process (Schaub, 2013).
The pool of trunk visceral mesodermal fusion-competent cells contributes to the formation of both circular and longitudinal midgut muscles, depending on whether they fuse with resident founder cells of the trunk visceral mesoderm or with founders that migrated in from the caudal visceral mesoderm. The restricted expression of the identity factor Org-1 in the founder myoblasts in the trunk visceral mesoderm and its exclusion from the FCMs represents an elegant mechanism to ensure that the respective patterning events only occur in the developing circular musculature but not in the longitudinal muscle fibers, which extend as multinucleate syncytia throughout the length of the midgut (Schaub, 2013).
Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, a Drosophila H3 histone mutant was generated with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2 (containing Esc, Su(z)12, E(z) and Nurf55), suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, these data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis (Yung, 2015).
This report has established a H3S28A histone mutant in Drosophila. In theory, this mutation could have two different effects on the polycomb system. (1) It could be that PcG proteins are not evicted from H3K27me3-binding sites in the absence of H3S28ph, and thus, PcG target genes might become ectopically repressed or (2) the mutation at H3S28 or the absence of H3S28ph could compromise PcG functions, resulting in derepression of PcG target genes. No evidence was found for the first possibility, although it is formally possible that H3S28 is phosphorylated under certain developmental conditions or in response to particular stimuli to counteract polycomb silencing. Instead, the data point to an inhibition of PRC2 activity by the H3S28A mutation. This inhibition is independent of active H3K27 demethylation by dUtx. Besides, RNAi against Aurora B kinase and hence depletion of H3S28ph did not hamper polycomb silencing. On the other hand, H3S28A nucleosomes proved to be a suboptimal substrate for in vitro PRC2 HMT activity. Although a 3D structure of the human Ezh2 SET domain is available, the exact contribution of the hydroxyl group of H3S28 for H3K27 methylation is difficult to deduce from the available data. vSET, the only other protein capable of H3K27 methylation in the absence of PRC2 subunits, does not require H3S28 for catalysis, whereas it does use H3A29 to define substrate specificity. Clearly, more work will be required to determine the exact structural and biochemical role of H3S28 in PRC2 catalysis. Consistent with the in vitro HMT assays, in vivo the H3S28A mutant exhibits defects in H3K27 methylation and shows similar, though milder, Hox derepression profiles and transformation phenotypes to those observed in H3K27R mutant flies (Yung, 2015).
Interestingly, the 'KS' module is frequently found in Ezh2 substrates other than K27S28 of histone H3. These include K26S27 of human histone H1 variant H1b (H1.4), K38S39 of the nuclear orphan receptor RORα, and K180S181 of STAT3. Whether these serine residues act similarly to H3S28 to support methylation of the adjacent lysine residue remains unknown. Of note, some other Ezh2 substrates can be methylated despite the lack of a 'KS' module. These include K26 of mouse histone H1 variant H1e, K49 of STAT3, and K116 of Jarid2, where the lysine residue is followed by an alanine, glutamate, and phenylalanine, respectively. Moreover, the link between peptide sequence and enzymology of Ezh2 was shown to differ in non-histone substrates. Hence, the role of serine following the Ezh2 methylation target amino acid might not be extrapolated to all other Ezh2 substrates and should be tested individually (Yung, 2015).
Previous reports revealed discrepancies in Drosophila PcG protein localization on mitotic chromosomes depending on staining protocols and tissue types. Nonetheless, live imaging of Pc-GFP, Ph-GFP, and E(z)-GFP in early Drosophila embryos has suggested that the majority of these PcG components are dissociated from mitotic chromosomes. Because stress-induced H3S28ph evicts PcG complexes during interphase, one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. Whereas loss of Ph from mitotic chromosomes was observed in WT background, significant Ph association was not observed in H3S28A mutant condition. The reduced levels of H3K27me3 in the H3S28A mutant could contribute to this observation. Alternatively, other mechanisms might operate to dissociate the majority of PcG proteins during mitosis (Yung, 2015).
The establishment of the histone replacement system in Drosophila has proven to be an important tool to complement functional characterization of chromatin modifiers. Whereas depletion of H3K27 methylation, either by mutation of the histone mark writer E(z) or by mutation of the histone itself in the H3K27R mutant, leads to similar loss of polycomb-dependent silencing, other histone mutations revealed different phenotypes than the loss of their corresponding histone mark writers. For example, H3K4R mutations in both H3.2 and H3.3, hence a complete loss of H3K4 methylation, did not hamper active transcription. Also, the loss of H4K20 methylation upon H4K20R mutation unexpectedly supports development and does not phenocopy cell cycle and gene silencing defects reported upon the loss of the H4K20 methylase PR-Set7. In this study, by comparing the phenotype of Aurora B knockdown and H3S28A mutation in vivo, together with in vitro HMT assay, the requirement of the unmodified H3S28 residue is specifically attributed to supporting PRC2 deposition of H3K27 methylation (Yung, 2015).
Whereas the published data suggest that H3S28 phosphorylation might be important for eviction of PcG components for derepression of PcG target genes upon stimulatory cues, the data reveal a so far unacknowledged function of the unphosphorylated state of H3S28. This study shows that serine 28 is required to enable proper methylation of H3K27 by PRC2 and thus to establish polycomb-dependent gene silencing. Serine 28 of histone H3 is universally conserved in species that display canonical PRC2-dependent silencing mechanisms. Given the fact that no major mitotic defects are found upon its mutation, it is proposed that the major role of this residue is to ensure optimal PRC2 function while facilitating the removal of polycomb proteins in response to signals that induce phosphorylation (Yung, 2015).
Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. This study reports that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation (Tsai, 2017).
Disruptions of normal Hox gene expression can lead to severe morphological defects revealing a link between the regulation of Hox expression and pattern formation. This study explored these links focusing on the impact of microRNA regulation on the expression of the Drosophila Hox gene Ultrabithorax (Ubx) during haltere development. Through the combination of bioinformatic and transcriptomic analyses the miR-310/313 cluster (miR-310C) as a candidate regulator of Ubx. Several experiments confirm this. First, miR-310C and Ubx protein show complementary expression patterns in haltere imaginal discs; second, artificial activation of miR-310C expression in haltere discs leads to Ubx-like phenotypes. Third, expression of a fluorescent reporter bearing Ubx 3'UTR sequences is reduced when co-expressed with miR-310C. Fourth, deletion of miR-310C leads to Ubx upregulation and changes the array of mechanosensory sensilla at the base of the haltere. Fifth, artificial increase of Ubx levels within the miR-310C expression domain phenocopies the mechanosensory defects observed in miR-310C mutants. It is proposed that miR-310C-mediated repression delimits Ubx fine-grain expression contributing to the sculpting of complex morphologies in the Drosophila haltere. This work reveals a novel role of microRNA regulation in the control of Hox gene expression with impact on morphology (Kaschula, 2018).
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Ubx regulation: Table of contents
Ultrabithorax:
Biological Overview
| Evolutionary Homologs
| Targets of activity
| Protein Interactions
| Posttranscriptional regulation
| Developmental Biology
| Effects of Mutation
| References
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