trithorax
The Drosophila gene trithorax is required for the normal expression of a number of the homeotic
genes in the bithorax complex (BX-C) and the Antennapedia complex (ANT-C). Flies homozygous for
trx mutations exhibit segmental identity transformations similar to those caused by loss-of-function
mutations in the homeotic genes Sex combs reduced, Ultrabithorax, abdominal-A,
and Abdominal-B. A molecular characterization of the trx locus is presented and shown to be
necessary for normal levels of Antennapedia, Ubx, and abd-A protein accumulation.
Interestingly, the loss of trx function differentially affects the expression of these proteins; Ubx protein
levels are greatly reduced, Abd-A protein levels are reduced to a lesser extent, and Antp protein levels
are only slightly reduced. P-element mediated transformation using 34 kb of genomic DNA containing
the 25 kb trx transcription unit identifies all sequences necessary for normal trx function and limits to relatively small regions the
5' and 3' flanking sequences that could be used in a regulatory capacity. The
primary transcription unit is differentially spliced to produce two large transcripts of 12 and 15 kb that
have different developmental profiles (Breen, 1991).
The regulatory control of trx has not been analyzed. It seems likely however that early zygotic expression is under control of the dorsal-ventral system, perhaps repressed by Dorsal.
trithorax regulates the Antennapedia and Bithorax complexes.
trx is required for normal expression of multiple homeotic genes of the bithorax and Antennapedia complexes (BX-C and ANTP-C). The expression of the BX-C genes Ultrabithorax, abdominal-A, Abdominal-B and the ANTP-C genes Antennapedia, Sex combs reduced and Deformed are all influenced by trx . Each of the BX-C and ANTP-C genes exhibits different tissue-specific, parasegment-specific and promoter-specific reductions in their expression. Each of these genes appear to have different requirements for trx in different spatial contexts in order to achieve normal expression levels. Those components of homeotic gene expression patterns for which trx is dispensable require other
factors, possibly those encoded by other trithorax group genes (Breen, 1993).
Sex combs reduced
DNA fragments in the Scr regulatory region are important for regulation of Scr by Polycomb and trithorax group gene products. When DNA fragments containing these regulatory sequences are subcloned into P-element vectors containing a white minigene, transformants containing these constructs exhibit mosaic patterns of pigmentation in the adult eye, indicating that white minigene expression is repressed in a clonally heritable manner. The amount of white minigene repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci (Gindhart, 1995).
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 maintenance 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 for 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, 1994).
The Fab-7 boundary functions to ensure the autonomous activity of
the iab-6 and iab-7 cis-regulatory domains in the
Drosophila Bithorax Complex from early embryogenesis through to the
adult stage. Although Fab-7 is required only for the proper
development of a single posterior parasegment, it is active in all tissues and
stages of development that have been examined. In this respect, Fab-7
resembles conventional constitutive boundaries in flies and other eukaryotes
that act through ubiquitous cis-elements and trans-acting factors.
Surprisingly, however, the constitutive activity of
Fab-7 is generated by combining sub-elements with developmentally restricted boundary function. In vivo evidence is provided that the
Fab-7 boundary contains separable regions that function at different
stages of development. These findings suggest that the units (domains) of
genetic regulation that boundaries delimit can expand or contract by switching
insulator function off or on in a temporally regulated fashion (Schweinsberg, 2004).
The minimal Fab-7 boundary defined in the ftz:hsp70-lacZ
and wEN:mini-white enhancer blocking assays is 1.2 kb in
length. It
extends from the minor nuclease hypersensitive site on the
proximal side to the iab-7 PRE (which corresponds HS3) on the
distal side and includes two major chromatin-specific nuclease hypersensitive
regions, HS1 and HS2. The largest hypersensitive region, HS1, contains six
consensus GAGA factor binding sites arranged in three pairs, 1-2, 3-4 and 5-6.
The ubiquitously expressed GAGA factor is encoded by the
Trithorax-like (Trl) gene, and it
is thought to function in the formation and/or maintenance of the nucleosome
free regions of chromatin associated with a variety of cis-acting
elements in flies, including enhancers, promoters, Polycomb Response Elements
(PRES) and boundaries. Chromatin immunoprecipitation experiments demonstrate that
GAGA is associated with the Fab-7 boundary in vivo.
Moreover, the GAGA-binding sites in HS1 are important for boundary function.
The enhancer blocking activity of the
minimal 1.2 kb boundary is compromised in both the embryo and adult when GAGA
sites 1-5 are mutated. Although this finding indicates that GAGA (or another
protein that recognizes the GAGA consensus) is required for Fab-7
boundary activity throughout development, the GAGA sites are not functionally
equivalent. When only the centromere proximal pair, 1-2, are
mutated, blocking of the ftz UPS stripe enhancer in the ectoderm of
early embryos by the minimal Fab-7 boundary is weakened, but there is
no apparent effect on the blocking of either the ftz NE enhancer in
the CNS of older embryos or the w enhancer in adults. By contrast,
mutation of the central pair, 3-4, weakens blocking of the w enhancer
in the eye, but has little effect on the blocking of the ftz
enhancers in embryos (Schweinsberg, 2004 and references therein).
One interpretation of these results is that the constitutive boundary
activity of the Fab-7 element is generated by sub-elements whose
activities are developmentally restricted. In the studies reported here, this hypothesis was tested. Unlike other well characterized
boundaries, the constitutive activity of Fab-7 is generated by
combining a series of subelements that function at different stages of
development. This unexpected finding indicates that chromatin domains are not
always static units, but instead may be redefined by inactivating or
activating a boundary element such that the chromatin domain can expand to
include new genes or regulatory sequences, or alternatively contract
eliminating genes or regulatory sequences (Schweinsberg, 2004).
Ultrabithorax
To identify cis-acting sequences for functional reconstruction of regulation by both trithorax and Polycomb, the expression patterns have been examined in several Ubx-lacZ transgenes that carry upstream fragments (corresponding to a region of approximately 50 kb). A 14.5-kb fragment from the postbithorax/bithoraxoid region of Ultrabithorax exhibits proper regulation by both trithorax and Polycomb in the embryonic central nervous system. Trithorax or Polycomb can function independently through this upstream fragment to activate or repress the Ultrabithorax promoter, respectively. Deletion analysis of this fragment demonstrates that a 440-bp fragment contains response elements for both Trithorax and Polycomb. The integrity of the proximal promoter region is essential for trithorax-dependent activation, implicating a long-range interaction for promoter activation (Chang, 1995).
bithorax complex Transcription of bithorax complex genes (abd-A, Abd-B and Ubx) in the mesoderm and ectoderm is altered in strong trithorax mutants during germ band elongation, while the anteriorly expressed Antennapedia complex is affected only at later stages in embryonic development. In another mutant allele, expression of bithorax genes is normal, while expression of Antennapedia complex genes is reduced. Thus anterior and posterior trx effects are independently regulated (Sedkov, 1995). The earlier effect of trithorax represents a distinct expression domain of trithorax in the posterior region of the embryo, one required to maintain expression of the BX-C genes. At cellular blastoderm, Trithorax mRNA expression in D. virilis embryos is also confined to the posterior portion of the presumptive mesoderm. This
supports the idea that the specific BX-C-related expression domain is an essential and independent feature of the trithorax gene (Tillib, 1995).
In Drosophila, two classes of genes, the
trithorax group and the Polycomb group, are
required in concert to maintain gene expression by regulating chromatin
structure. Trithorax protein (Trx) binding elements have been identified
within the bithorax complex. Within the
bxd/pbx regulatory region these elements are functionally
relevant for normal expression patterns in embryos and they confer Trx
binding in vivo. Trx binding elements have been localized to three closely situated sites
within a 3-kb chromatin maintenance unit with a modular structure.
Results of an in vivo analysis have shown that these DNA fragments (each
~400 bp) contain both Trx- and Polycomb-group response elements (TREs
and PREs) and that in the context of the endogenous
Ultrabithorax gene, all of these elements are essential for
proper maintenance of expression in embryos. Dissection of one of these
maintenance modules has shown that Trx- and Polycomb-group responsiveness
is conferred by neighboring but separable DNA sequences, suggesting
that independent protein complexes are formed at their respective
response elements. The activity of this
TRE requires a sequence (~90 bp) that maps to within several tens of
base pairs from the closest neighboring PRE and the PRE activity
in one of the elements may require a binding site for Pho, the protein
product of the Polycomb-group gene
pleiohomeotic. These results show that long-range maintenance
of Ultrabithorax expression requires a complex element
composed of cooperating modules, each capable of interacting with both
positive and negative chromatin regulators (Tillib, 1999).
A PCR-linked immunoprecipitation procedure has been developed in order to discover Trx specific DNA binding sites. This procedure involves PCR amplification of DNA fragments retained in a pellet after immunoprecipitation of TRX-DNA complexes from embryonic nuclear
extracts using Trx-specific antibody.
Trx protein
localizes to 10 discrete fragments of the BX-C. The identification of
TRX binding regions within the regulatory DNA of all three genes of the
BX-C, (Ubx, abdominal-A [abd-A], and
Abdominal-B [Abd-B]), is striking because trx is required to maintain the expression levels of all
three genes in embryos. Next,
high-resolution mapping of the Trx binding sites was carried out. Since the sequence of the
entire BX-C is now available, the identified Trx binding sites were mapped to DNA fragments from
200 bp to 2,000 bp in size. Trx binding sites are found in several regulatory regions of the BX-C: abx,
bxd/pbx, iab-2, iab-3,
iab-4, iab-7, and iab-8. A
number of studies have defined PREs and
Pc protein binding sites in the BX-C. Comparison of
the data in this study with those of previous studies has shown that six Trx binding sites in
bxd/pbx, iab-3, iab-4, and
iab-7 regulatory regions are either within or very close to minimal PREs or PC binding sites identified previously. Interestingly, several signals are detected in the bxd/pbx
region, suggesting that there are multiple TRX binding sequences within this regulatory region (Tillib, 1999).
Two criteria
identify TRE and PRE activities within the reporter constructs:
maintenance of lacZ reporter expression patterns in embryos,
and trx- and PcG-dependent maintenance of white gene expression in the eyes of adult flies, including
their effects on pairing-sensitive repression of white.
Ultimately the results obtained with both reporter genes led to similar
conclusions, and the TRE and PRE activities of one module, fragment C of the Ubx promoter, were shown to
be conferred by neighboring but separable DNA sequences. An essential TRE and two distinct PREs were detected in this
central C module. Further analysis has shown that the TRE activity and TRX binding require a 90-bp region, which, based on its length, is
likely to bind more than just a single protein. This suggests that a
number of primary DNA proteins may be associated with the TRE in the C1
fragment. A gel-shift analysis of the protein binding properties of
this 90-bp TRE fragment suggests that this fragment contains two core
binding sequences that are required for apparent cooperative binding
by several nuclear proteins. These core sequences,
which are located on the boundary of the C1-B and C1-C fragments and in
the C1-D fragment appear to contribute to the formation of a large
multiprotein complex. [Note: The fragments referred to have the following locations in the bxd region of Ubx: C1 (nt 218,835 to 218,959); C2 (nt 218,960 to 219,088); C3 (nt 219,089 to 219,249). DeltaBC1-A, DeltaBC1-B, and DeltaBC1-C are deletions of nt 1 to 27, 28 to 61, and 86 to 122, respectively]. There is a direct correlation between
the sequences that are required to form this protein complex and those that are required for the Trx binding and TRE activity. Most
strikingly, the AACAA repeat in an addition fragment, the C1-D fragment, seems to be crucial
for forming this complex: it has been shown to be crucial for the TRE
activity, since complex formation is virtually abolished when the AC
residues are changed to TG. Since direct Trx
protein binding to DNA has not been established by using a number of
DNA binding assays and since the DNA binding protein complex in the C1
fragment does not contain Trx, it is suggested that Trx
binds to this TRE through interactions with a complex of primary DNA
binding proteins. At present, the identity of the
proteins that associate with these TREs is unknown. While it would not be
surprising to find that some are products of the trxG, it is
unlikely that the Gaga factor or the Zeste protein are the primary binding
proteins in this case, since the C1 element does not contain consensus binding sites for either of these proteins (Tillib, 1999).
This analysis suggested that the C2 and C3 fragments each contain a PRE.
Each of these elements, C2 and C3, is required to confer
pairing-sensitive repression on a white reporter gene. These
elements may be functionally different because their activities require
different sets of PcG proteins and because there is no
significant sequence similarity between them. Both PREs are apparently
also required, in concert, to provide full maintenance function in
embryos. This follows from the fact that while very strong anterior
overexpression occurs in embryos when the entire C fragment is deleted,
only moderate overexpression in PS5 results from the deletion of a
single PRE. In addition, one of these PREs, C3, may
contain a functionally important binding site for the PcG protein Pleiohomeotic (Pho), since deletion of this binding site abrogates both
pairing-sensitive repression and responsiveness of the white
reporter gene to three PcG mutations: pho,
Pcl, and Scm. Therefore, it is suggested
that the protein products of these three PcG genes may be
components of a putative PcG protein complex that is formed at the C3
PRE. In addition to the Pho binding sites, the C2 and C3 DNA fragments contain three consensus binding sites for the GAGA protein (Trithorax-like).
Further analysis is required to determine whether the deletion of GAGA
sites has an effect on PRE function. It is likely, however, that Pho
and GAGA are not the only primary DNA binding proteins in these PREs,
since the C2 PRE does not contain consensus Pho binding sites. These
proteins may be DNA-interacting components of particular subsets of PcG
complexes (Tillib, 1999).
The TREs and PREs in the bxd region of Ubx
are clustered in three closely situated maintenance modules, each
approximately 400 bp. Each module contains elements for both of these
opposing activities. The analysis has been focused on the Trx protein,
although it is possible that there are other positive maintenance
elements in this region that require the products of other
trxG genes. Similarly, since PRE function was analyzed only
in Trx binding regions, some PREs may have gone undetected. Despite
these limitations, several TRE- and PRE-containing modules were discovered in the bxd region of Ubx that are all
essential for proper Ubx expression, since deletion of any
one of the three modules leads to a significant loss of maintenance
activity in embryos. In the context of a natural Ubx
promoter and a large part of its regulatory region, these modules are
all essential in embryos with respect to PRE and TRE function. However,
when tested for effects on white gene expression, deletion
of individual elements does not completely abolish either eye color
variegation or the responsiveness to trx and PcG
mutations. These differences suggest that repression of
white expression in adults may not accurately reflect the
function of these elements in the context of the entire Ubx
gene. Thus, cooperative interactions among multiple PREs and TREs are
required for proper function of the Ubx gene, and these interactions may involve activities not reflected in assays with reporters unrelated to Ubx expression (Tillib, 1999).
Interestingly, trx function is found to be required for
Ubx expression not only in its normal domain of expression
in the posterior region of the embryo but also in the anterior region, when Ubx is overexpressed due to a deletion of PREs. There
are clear differences between the anterior and posterior regions of the
embryo with respect to both the effect of a trx mutation and the requirements of TREs for the expression patterns of these transgenes. (1) In trx mutants, the loss of expression in the
anterior is much more severe than it is in the posterior. (2)
Anterior expression is very strong when one of the three TREs is
deleted, and only a simultaneous deletion of two TREs leads to a
decrease of this expression that is comparable with the effect of a
trxB11 null mutation. In the posterior, however,
deletion of only one element mimics an almost complete loss of
trx-related activity, and deletion of two elements has no
further effect. This might be explained by a different mode of
functioning in the anterior versus posterior regions of the embryo.
Such a functional difference is also suggested by the
observation that different trx protein products, which
result from alternatively spliced mRNAs, may be required for the
maintenance of expression of the ANT-C genes (in the anterior region)
and not for maintenance of BX-C gene expression (in the posterior
regions). This is based on an analysis of the effects of different
trx alleles on the two homeotic complexes and on the finding
that the expression of one of the early trx RNAs is
spatially restricted to the posterior region where the BX-C genes are
expressed. Based on these observations, it has been concluded
that there are quite complex requirements for the activities of the
three maintenance modules in different cells. Functionally similar
maintenance units may regulate other genes in the BX-C as well, since
the other Trx binding regions detected are associated with either
PRE function, Pc protein binding sites, or both (Tillib, 1999).
The finding of discrete TRE and PRE sequences
argues against a direct competition between the proteins of these
opposing groups for binding sites on DNA, although the question of
whether they normally occupy their response elements simultaneously
within a given module remains open. In addition, some data suggest that the association of trxG and PcG proteins with a particular gene depends
on its transcriptional status: (1) the strength of Trx binding to
salivary gland polytene chromosomes at the site of a transcriptionally
active gene, such as fork head, is much higher than it is at
the location of the BX-C, which is silent in the salivary glands; (2) immunoprecipitation of Pc protein from
Drosophila cells has been found to be more abundant at silenced
genes than at activated genes of the BX-C; (3) when
transcription of a reporter gene is induced by GAL4, several PcG
proteins are displaced from the chromosomal site of insertion of a
Fab-7 transgene. Although this is not
directly related to trxG functioning, it indicates that PcG proteins
are not bound abundantly to actively transcribed genes, suggesting that
there might be quantitative or qualitative differences in bound trxG
and PcG protein complexes depending on the transcriptional activity of
a particular gene. This work suggests that the occupancy of TREs and
PREs may be independent rather than mutually exclusive. Since the
formation of functional activating or silencing complexes may depend on
and in turn lead to the maintenance of the on-off state of
Ubx expression in a particular tissue, it is suggested that the
occupancy of the TRE by a functional trxG complex alters, directly or
indirectly, the composition of nearby PRE complexes without necessarily
abolishing binding by PcG proteins (Tillib, 1999).
How do active trxG and PcG protein complexes function? There have as
yet been no specific biochemical activities associated with PcG
proteins. Most of the PcG proteins are associated with chromosomes, and
it is assumed that they act by forming repressive multiprotein
complexes that prevent active transcription. One of the functions of
trxG proteins may be simply to counteract the formation of these
repressive PcG complexes and thus to increase the accessibility of
enhancers to the neighboring regions of DNA. However, there is growing
evidence that the trxG represents a heterogeneous family of proteins
with diverse functions. Some of them, such as Trx, Ash1, Ash2, Gaga,
and Zeste, are associated with particular sites on polytene chromosomes, while others, such as Brm and Snr1,
are found in chromatin remodeling complexes that may not be associated
with specific chromosomal regions. There is some evidence that one of
the functions of trxG proteins may be to recruit chromatin remodeling
complexes to DNA. Gaga factor is required for the function of one
chromatin remodeling complex, the Drosophila NURF complex, and Trx has been shown to physically interact with Snr1, a component of the Drosophila SWI/SNF complex.
However, there is no evidence thus far that these interactions are
mediated through particular TREs. In addition, there is evidence that
Trx and its human homolog, ALL-1/HRX, may be involved directly in the
activation of promoters, since both of these proteins possess
transactivation activity in cells. Therefore, it
is likely that trxG proteins not only can counteract formation of
PcG-mediated repressive chromatin structure but may also play a more
direct role in maintaining transcription (Tillib, 1999 and references).
In conclusion, three discrete TRE/PRE modules have been identified in the Ubx regulatory
region. These modules are contained within a complex, 3-kb maintenance unit in which each detected element
is essential with respect to both PRE and TRE function. Furthermore, it has been found that Trx binds sequences in other regulatory regions of the BX-C
that are consistently associated with either PRE function, Pc protein
binding, or both; this suggests the possibility that similar maintenance
units are employed for regulation of other genes in the complex.
Functional dissection of one of these modules has shown that the TRE and
PRE activities can be ascribed to separable DNA elements, even though
they are located within tens of nucleotides of one another. This
proximity suggests that there may be some direct interaction between
protein complexes formed at these elements. In addition, the TREs and
PREs that have been identified do not contain extensive sequence
similarities, suggesting that they are bound by protein complexes of
different composition (Tillib, 1999).
Polycomb group (PcG) and trithorax group (trxG) proteins act in an epigenetic fashion to maintain active and repressive states of expression of the Hox and other target genes by altering their chromatin structure. Genetically, mutations in trxG and PcG genes can antagonize each other's function, whereas mutations of genes within each group have synergistic effects. This study showd in Drosophila that multiple trxG and PcG proteins act through the same or juxtaposed sequences in the maintenance element (ME) of the homeotic gene Ultrabithorax. Surprisingly, trxG or PcG proteins, but not both, associate in vivo in any one cell in a salivary gland with the ME of an activated or repressed Ultrabithorax transgene, respectively. Among several trxG and PcG proteins, only Ash1 and Asx require Trithorax in order to bind to their target genes. Together, these data argue that at the single-cell level, association of repressors and activators correlates with gene silencing and activation, respectively. There is, however, no overall synergism or antagonism between and within the trxG and PcG proteins and, instead, only subsets of trxG proteins act synergistically (Petruk, 2008).
Despite much interest, there is little understanding of how the epigenetic
TRE/PRE-containing MEs function. One key unresolved issue pertains to the organization of these complex transcription regulatory elements with regard to the response elements/binding sites of particular trxG and PcG proteins. Response elements for several PcG proteins were mapped in the bxd ME previously, and some PcG proteins were detected at this DNA element in ChIP assays. However, information about the association of trxG proteins in the bxd ME is very limited. Several Trx-dependent TREs have been mapped in the bxd ME. In addition, Trx and Ash1 proteins have been detected at the bxd ME in ChIP assays. Given the apparent functional heterogeneity of the trxG proteins, it is revealing that besides Trx, many other trxG genes are essential for functioning of the bxd ME. Two of the interacting genes, skd and kto, encode components of the Drosophila Mediator complex, so it is possible that their role in the functioning of the bxd ME relates to the transcription of some of the non-coding RNAs that are known to be transcribed through this element. Ash2 is a component of several purified MLL (a human homolog of Trx) protein complexes. The identification of an ash2 response element in the bxd ME suggests that a second putative Trx-containing MLL-like complex might reside at the bxd ME. The genes urd and sls have only been minimally characterized, mainly as suppressors of Pc phenotypes. Therefore, it is premature to speculate about their function at this element, although they clearly interact there in some capacity (Petruk, 2008).
Identification of multiple TREs and PREs within the same ME raises an important question with regard to potential interdependency or competition in the association of proteins from the same and different protein families. To address this, focus was placed on the fine mapping of response elements for several major trxG genes that are essential for functioning of the bxd ME: ash1, the brm component of the BRM chromatin remodeling complex, and the ETP gene Asx. These proteins or components of their protein complexes (i.e. Snr1, a component of BRM) can physically associate with Trx. Thus, finding their response elements either in DNA fragments that are juxtaposed to (brm and ash1) or the same as (Asx) the previously mapped trx response element is consistent with direct interactions of these proteins with Trx. It should be noted, however, that all these proteins are components of protein complexes other than the Trx complex TAC1. Nevertheless, this suggests that there might be interdependency in recruitment and/or association of these protein complexes at the bxd ME. However, the results indicate that this suggestion is only partially true. Binding of the components of the BRM complex and of another trxG protein, Kis, were not affected by elimination of Trx. However, the association of Ash1 and Asx at all their sites on the salivary gland polytene chromosomes is completely dependent on the presence of Trx. Previous results of the reciprocal experiments indicated that binding of Trx is strongly decreased in ash1 mutant animals. This suggests that Trx, Ash1 and Asx represent a special, and at least partially interdependent, set of trxG proteins. This also suggests, in contrast to the previously mentioned genetic studies, that not all trxG proteins are mutually dependent in their functioning (Petruk, 2008).
Close proximity or even overlap between some TREs and PREs in the bxd ME suggests the existence of potential competitive relationships with regard to the binding of these functionally opposing groups of proteins. Furthermore, some ChIP assays indicate that some trxG and PcG proteins can bind to the bxd ME of both the activated and silenced gene, suggesting a potential interaction of these proteins on DNA. This was tested by asking whether binding of the components of two major PcG complexes, PRC1 and PRC2, is affected by elimination of Trx. No significant change was detected in the number or intensity of immunostained bands for all tested PcG proteins on the polytene chromosomes of trx mutant larvae. This suggests that not only is the association of PcG proteins independent of Trx, but also that Trx is not essential for preventing binding of the PcG proteins to their response elements. This is an important conclusion because some genetic studies have proposed that the main function of Trx and Ash1 is to prevent silencing by the PcG proteins (Petruk, 2008).
An important issue in understanding the molecular mechanism of trxG/PcG
functioning is to correlate their association at MEs with the state of expression of their target genes. Although most of the existing data were obtained in cultured cells, two studies addressed this issue in Drosophila larval tissues. ChIP analysis in larval imaginal discs suggests that some trxG and PcG proteins are associated with the bxd ME irrespective of the status of gene expression. However, the results of another study suggest alternative association of Trx and Pc at the site of the endogenous BX-C on polytene chromosomes from both fat body and salivary glands, where BX-C is correspondingly activated or repressed. Ideally, to resolve this issue it is essential to investigate the association of PcG and trxG proteins with the ME in the same tissue at the single-cell level and at a gene of defined expression status. Such a test system was established. In this system the bxd-ME-containing transgene is either activated or repressed in cells within the same salivary gland. Direct visualization of the association of different proteins to the site of insertion of this transgene clearly indicates that major trxG and PcG proteins bind to the bxd ME in an alternative fashion. Importantly, using markers for activated and repressed transcription, it was possible to correlate binding of trxG and PcG proteins in a single cell with either the activated or repressed bxd transgene, respectively. The differences between these results and those of Papp (2006) might be explained by technical differences and by the fact that trxG and PcG proteins may behave differently in different tissues and/or in polyploid versus diploid cells. It is important to note that although the current analysis is limited to studies of a transgene, the detected alternative association of Trx and Pc on the bxd ME transgene correlates well with the results obtained at the endogenous BX-C on polytene chromosomes. It is concluded, therefore, that on a cell-by-cell basis, binding of trxG and PcG proteins is strictly dependent on the status of gene expression, in that they bind alternatively to the epigenetic regulatory elements of either activated or repressed target genes, respectively (Petruk, 2008).
In summary, this is the first work on the fine mapping of multiple TREs at any target gene. This is also the first assessment of mutual dependencies within the trxG group of activators and between the trxG and PcG of antagonistic proteins. It provides a glance of the enormously complex regulatory element that binds proteins with opposite transcriptional regulatory activities. The main conclusions of this study are that two major trxG proteins, Trx and Ash1, and the ETP protein Asx, constitute a specific subgroup of interacting proteins that depend on each other in their functioning at the bxd ME and throughout the genome. Although multiple trxG proteins are essential for epigenetic functioning of the bxd ME, their association with this element and other binding sites in the genome might not necessarily require Trx and associated proteins, as exemplified by the components of the BRM complex and Kis. The components of the major PcG complexes, PRC1 and PRC2, also associate with target genes independently of Trx, Ash1 and Asx. Another important conclusion of this work is that trxG and PcG proteins are associated with the bxd ME only at activated and repressed genes, respectively. It will be important to determine whether the choice between the establishment of trxG-mediated activation or PcG-mediated repression occurs only at very specific early stages of development, or whether it can also occur at later developmental stages (Petruk, 2008).
Additional genes regulated by trithorax
Maintenance but not initiation of engrailed (en) gene expression in the Drosophila embryo requires trithorax, required to maintain stable long-term expression of the homeotic genes throughout the development. en expression is dependent on trx in only a subset of embryonic cells normally expressing en, including specific cells
in the nervous system and the dorsal fat body cells surrounding the gonad. Loss of en expression in
the dorsal fat body is correlated with the sterility of en females that also carry trx mutations. In
addition, trx is required for normal en expression in the posterior compartment of the developing
wing, reflected in enhancement of en phenotypes in en adults that also carry trx mutations. trx
appears to be dispensable for maintenance of en expression in other embryonic cells (Breen, 1995).
The proximal promoter of engrailed does not direct expression in a tissue or stage-specific manner, but contains promoter activity which can be activated by nearby genomic enhancers. Three so-called pairing sensitive sites (PS) have demonstrated sensitivity to the actions of Polycomb and trithorax group genes (Kassis, 1994).
Trithorax also regulates the homeotic gene forkhead. A strong Trithorax binding site is found at the cytological location of the forkhead gene in salivary gland chromosomes. When a genetic element containing the forkhead upstream regulatory region is inserted into ectopic chromosomal regions, the Trithorax protein can be found localized to these ectopic sites associated with forkhead regulatory DNA (Kuzin, 1994).
The wing imaginal disc is subdivided into a dorsal and a ventral compartments. A new dominant
homeotic mutation, Dorsal wing1 (Dlw1), transforms ventral into dorsal compartment in heterozygotes. This phenotype is similar to one of the dominant phenotypes of Polycomb mutants. Dlw+ is required for the specification of dorsal compartment. Some genes of the Polycomb group act as negative regulators of Dlw+, while some genes of the trithorax group act as positive
regulators (Tiong, 1995).
The in vivo distribution of the E(Z) protein shows it to be ubiquitously present in embryonic and larval nuclei. In salivary gland polytenized nuclei, the identifiable E(Z) chromosome binding sites are a subset of those described for other Polycomb-group proteins, suggesting that E(Z) may also participate in Polycomb-group complexes. E(Z) binds to chromosomes in a DNA sequence-dependent manner, as illustrated by the creation of a new E(Z)-binding site at the location of a P element reporter construct that contains a Polycomb response element (PRE). This P element contains a 14.5 kb segment from the bxd/pbx Ubx regulatory region. The sequences of one null and three temperature-sensitive E(z) alleles are presented. These mutations diminish all chromosome binding of E(Z) protein. It is suggested that a Cys-rich region altered in these mutations functions as a DNA binding
domain. E(Z) binding is noticibly weaker in trx mutants. A reduced level of E(Z) chromosome binding may be due to alteration in the expression of one or more other proteins that are involved in E(Z) binding and does not necessarily imply a direct interaction between E(Z) and TRX (Carrington, 1997).
Both in vitro and in vivo transcription assays have been used to delineate the promoter for the 6-kb E74 mRNA. In vitro transcription of a series of 3' deletions defined the 3' in vitro promoter boundary at position +43. Additional 5'-flanking sequences, between -181
and -83, are necessary for efficient transcription in transfected Kc tissue
culture cells. Two transcription factors that interact with the E74 promoter, Zeste and
GAGA, were studied in DNA-binding assays. Zeste binds to two sites within the E74 promoter. These sites overlap with three of the six GAGA-binding sites. The Zeste- and GAGA-binding sites lie within domains identified by deletion mapping as cis-acting transcriptional control elements (Thummel, 1989).
Rapid induction of the Drosophila melanogaster heat shock gene hsp70 is achieved through the binding of heat shock factor (HSF) to heat shock elements (HSEs) located upstream of the transcription start site. The subsequent recruitment of several other factors, including Spt5, Spt6 and FACT, is believed to facilitate Pol II elongation through nucleosomes downstream of the start site. This study reports a novel mechanism of heat shock gene regulation that involves modifications of nucleosomes by the TAC1 histone modification complex. After heat stress, TAC1 is recruited to several heat shock gene loci, where its components are required for high levels of gene expression. Recruitment of TAC1 to the 5'-coding region of hsp70 seems to involve the elongating Pol II complex. TAC1 has both histone H3 Lys 4-specific (H3-K4) methyltransferase (HMTase) activity and histone acetyltransferase activity through Trithorax (Trx) and CREB-binding protein (CBP), respectively. Consistently, TAC1 is required for methylation and acetylation of nucleosomal histones in the 5'-coding region of hsp70 after induction, suggesting an unexpected role for TAC1 during transcriptional elongation (Smith, 2004).
Much of the genome is transcribed into long non-coding RNAs (ncRNAs). Previous data have suggested that bithoraxoid (bxd) ncRNAs of the Drosophila bithorax complex prevent silencing of Ultrabithorax (Ubx), and recruit activating proteins of the trithorax group to their maintenance elements. This study found that, surprisingly, Ubx and several bxd ncRNAs are expressed in non-overlapping patterns in both embryos and imaginal discs, suggesting that transcription of these ncRNAs is associated with repression, not activation, of Ubx. The data rule out siRNA or miRNA-based mechanisms for repression by bxd ncRNAs. Rather, ncRNA transcription itself, acting in cis, represses Ubx. The Trithorax complex TAC1 (containing Trx, Sbf1 and dCBP) binds the Ubx coding region in nuclei expressing Ubx, and the bxd region in nuclei not expressing Ubx. It is proposed that TAC1 promotes the mosaic pattern of Ubx expression by facilitating transcriptional elongation of bxd ncRNAs, which represses Ubx transcription (Petruk, 2006).
The Hox genes of the bithorax complex (BX-C) have spatially restricted expression patterns that vary within and between segments and tissues. Transcription factors encoded by segmentation genes establish the patterns of the Hox genes Ubx, abd-A, and Abd-B of the BX-C in embryos. After the segmentation proteins decay, Hox expression patterns are maintained epigenetically by proteins of the trithorax group (trxG) and the Polycomb group (PcG). PcG genes maintain the silent state, whereas trxG genes maintain the active state of Hox genes. PcG and trxG proteins act through partially overlapping sets of response elements known as maintenance elements (Petruk, 2006).
One of the most startling discoveries of the genomic era has been that much of the genome is transcribed into non-coding RNAs (ncRNAs). Recent attention has focused on small interfering RNAs (siRNAs) and micro-RNAs (miRNAs) that modulate gene activity by an antisense mechanism termed RNA interference (RNAi), which interferes with mRNA stability or translation. However, the most abundant and least characterized class of ncRNAs are long and have mostly unknown functions (Petruk, 2006 and references therein).
The intergenic regions of the Hox genes in D. melanogaster produce many long ncRNAs that may regulate Hox gene coding sequences. Increasing attention has been directed to the role of transcription of MEs in the regulation of BX-C genes. Several ncRNAs are transcribed through a well-studied ME in the bxd regulatory region that lies between the Ubx and abd-A transcription units. The bxd ME regulates Ubx. Transcription through bxd precedes activation of Ubx coding RNAs (hereafter referred to as 'Ubx RNA', or simply as 'Ubx'), suggesting that ncRNAs might regulate Ubx. Transcription patterns of ncRNAs appear similar to those of the neighboring Hox genes and are collinear with regulatory domains along the chromosome. A synthesis of genetic and transgenic studies led to the idea that transcription of ncRNAs through MEs interferes with PcG-mediated silencing, perhaps by preventing recruitment of PcG proteins. A recent study suggests that transcription of MEs may recruit trxG proteins to maintenance elements. If indeed transcription of MEs simultaneously prevents PcG binding and establishes trxG binding, this could be a key element of Hox regulation. Clearly, this model requires that intergenic bxd RNAs are expressed in the same cells as Ubx. However double-labeling of intergenic and coding RNAs at high resolution has not been performed, so this attractive model has not been rigorously tested (Petruk, 2006 and references therein).
The trxG protein complex TAC1 plays important roles in maintaining expression of homeotic genes throughout embryogenesis. Recent attention has focused on the role of trxG proteins in histone modifications and in altering nucleosome positioning. TAC1 contains three proteins, Trx, Sbf1 and dCBP, and thus acetylates histones and methylates histone H3 at Lys-4 (H3-K4), due to the enzymatic activities of dCBP and the SET domain of Trx, respectively. How binding of Trx to MEs regulates expression of Hox genes is unclear. Because embryos have a mixture of cells expressing and not expressing each Hox gene, it has not been possible to determine precisely how trxG protein binding correlates with transcription of Ubx and bxd ncRNAs (Petruk, 2006).
This study shows that Ubx is repressed by bxd transcription. The bxd ncRNAs do not act by siRNA or miRNA-based mechanisms, but repress Ubx in cis through a transcription-dependent mechanism. Alternative association of TAC1 with either Ubx or bxd correlates with their transcription. TAC1 appears to be part of an interdependent network of general elongation factors that associate with active genes. It is suggested that a key role of TAC1 in establishing the mosaic pattern of Ubx expression is to promote elongation of bxd ncRNAs, which in turn represses expression of Ubx (Petruk, 2006).
An attractive notion has been that transcription of bxd ncRNAs, which precedes that of Ubx in embryos, facilitates correct spatial expression of Ubx. Previous studies showed that transcription through the ME could interfere with silencing, so it was proposed that bxd ncRNA transcription normally prevents recruitment of PcG proteins to the ME. However, the current experiments unambiguously demonstrate that Ubx and bxd ncRNAs are transcribed in different cells in embryos. The results also suggest that bxd ncRNAs do not facilitate Ubx expression in larval imaginal discs, as was recently proposed. Instead, transcription of ncRNAs correlates with repression of Ubx. It is possible that the abnormal transcription induced in previous studies interfered with transcription of ncRNAs in the BX-C, rather than with ME function, a possibility that can be tested experimentally. It will be interesting to use the system of sorting Ubx+ and Ubx− nuclei to examine binding of PcG proteins in nuclei where bxd ncRNAs either are, or are not, transcribed (Petruk, 2006).
The experiments rule out trans-repression by bxd ncRNAs, and instead support repression of Ubx in cis by transcription of these RNAs per se. A likely mechanism of this repression is transcriptional interference, since it was shown that ncRNA transcription extends into the region just upstream of the Ubx initiation site, which may well disrupt protein-DNA interactions required for Ubx initiation. However, this does not rule out promoter competition, and both of these mechanisms may contribute to the observed effects. Previous genetic studies and the results presented in this study show that bxd ncRNAs do not work by RNAi. An RNAi-based repression mechanism has been described for the miRNA produced by the iab-4 transcript, which directly interacts with the 3'-untranslated region of Ubx and prevents translation. This work showed that ectopic expression of iab-4 leads to homeotic phenotypes in the haltere, but do not show that loss of RNAi prevents this effect, nor has the effect of loss of function mutations of the iab-4 transcript been tested, so it remains to be seen if the iab-4 transcript is a bona fide miRNA (Petruk, 2006).
Since significant levels of bxd ncRNAs were not detected in imaginal discs, nor do they persist to late embryonic stages, they are unlikely be responsible for repression of Ubx throughout development. In fact, it has been reported that Trx is bound to the bxd ME in both wing and haltere discs, which have low and high levels of Ubx expression, respectively. The difference between binding of Trx to the bxd ME in embryos and in discs may be a consequence of the absence of transcription of bxd ncRNAs in discs and its presence in embryos, or to other uncharacterized differences between Ubx regulation in embryos and discs. Also, since Trx binds constitutively in some areas of the ME, Papp may have detected such binding in imaginal discs (Petruk, 2006).
Intergenic transcription also cannot explain repression of Ubx in the anterior of the embryo, where it is thought that hunchback and PcG genes set up and maintain the anterior boundary of Ubx expression. However, the pattern of bxd ncRNA transcription, which prefigures, in a complementary fashion, the mosaic pattern of Ubx expression within the parasegments of the embryonic trunk, appears to be essential for proper Ubx initiation. The Ubx pattern may then be maintained or modified at later embryonic stages through repression by other Hox proteins (i.e., abdA and AbdB) and by PcG genes. Thus, maintenance of Ubx expression likely requires multiple mechanisms that are employed at different developmental stages (Petruk, 2006).
The data support a role for Trx in transcriptional elongation as a mechanism for maintenance of a developmentally regulated gene. It has been argued that Trx does not have a direct role in activation of homeotic genes in Drosophila, but instead prevents repression of transcription by PcG proteins. However, the current data suggest that trx is required for recruitment of elongation factors and for efficient completion of transcripts. Therefore, maintenance of transcriptional activity by Trx may be a consequence of its role in elongation, and a block in elongation might lead to the establishment of PcG-mediated repression. Alternatively, Trx may be required only for normal levels of Hox gene expression, and not for maintenance of low levels of expression, a possibility consistent with at least some aspects of the trx mutant phenotype (Petruk, 2006).
This work strongly supports a general role for Trx and TAC1 in transcription, and agrees with previous findings that TAC1 relocates from other genes to the transcribed region of hsp70 following induction of the cellular stress response. The histone methyltransferase activity of Set1, the SET domain protein homologous to Trx, has a role in transcription, and MLL was suggested to play a similar role in mammals. It is suggested that this role is in transcriptional elongation, because Trx and elongation factors are co-ordinately recruited, because Trx binds downstream of the promoter more strongly to the 5’ than the 3’ end, and because transcripts extending to the 3’ end are more strongly affected by trx mutations, for both Ubx and bxd ncRNAs (Petruk, 2006).
TAC1 is also present at the promoter, and this is unaffected by mutations in elongation factors. Therefore, association of TAC1 with the promoter likely precedes the recruitment of elongation factors. Thus, TAC1 may play several distinct roles, one in initiation, another during the recruitment of the elongation complex and perhaps a third during subsequent elongation, where its ability to modify histones may be required for effective completion of long transcripts (Petruk, 2006).
This work provides the first direct evidence of the involvement of long ncRNAs in regulation of homeotic genes of Drosophila. Repression of Ubx is apparently mediated by expression of several intergenic ncRNAs in different germ layers of Ubx-expressing parasegments. TAC1 may be required for efficient read-through by Pol II into the region upstream of the Ubx initiation site, and as a result, for efficient repression of Ubx. Therefore, a direct link is proposed between elongation facilitated by the TAC1 epigenetic complex and repression of Ubx by intergenic transcription. A goal for the future will be to determine if other homeotic genes of Drosophila, and of other organisms, are also regulated by long ncRNAs whose expression is regulated by TAC1 proteins (Petruk, 2006).
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).
Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. This study shows that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. It is proposed that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing (Tie, 2009).
The major findings of this work are: (1) that Drosophila CBP acetylates H3K27; (2) that this acetylation requires TRX; and (3) that it prevents H3K27 trimethylation by E(Z) at Polycomb target genes and antagonizes Polycomb silencing. The remarkably complementary developmental profiles of H3K27ac and H3K27me3 (but not H3K27me2) during embryogenesis suggest that the deposition of H3K27me3, which increases steadily after ~4 hours with the onset of Polycomb silencing, occurs at the expense of a substantial fraction of the H3K27ac already present. This suggests that the establishment of Polycomb silencing might require active deacetylation of this pre-existing H3K27ac. The reciprocal effects of knockdown and overexpression of CBP and E(Z) on H3K27 trimethylation and acetylation in bulk chromatin further suggest that the two modifications constitute alternative chromatin states associated with active and inactive genes. Consistent with this, ChIP-chip experiments revealed that H3K27me3 and H3K27ac are mutually exclusive genome wide. Moreover, in S2 cells, the inactive abd-A gene does not have the H3K27ac modification in its promoter region, but acquires it upon RNAi knockdown of E(Z). It will be important to determine whether such a modification switch occurs genome wide after loss of E(Z) (Tie, 2009).
The ability of E(Z) overexpression to suppress the small rough eye
phenotype of CBP overexpressers further supports the conclusion that H3K27 trimethylation by E(Z) antagonizes H3K27 acetylation by CBP and suggests that deacetylation of H3K27 by RPD3, and possibly other deacetylases, might be a prerequisite for subsequent methylation by E(Z) and therefore important for reversal of an active state. Conversely, the ability of CBP and TRX overexpression to increase the global H3K27ac level at the expense of H3K27me3 suggests that either active demethylation of H3K27me3 by the H3K27-specific demethylase UTX (Agge, 2007; Lee, 2007; Smith, 2008), or histone replacement (Ahmad, 2002), might be a prerequisite to acetylation by CBP. Indeed, depletion of Drosophila UTX in vivo using a GAL4-inducible UTX RNAi transgene line results in an increase in H3K27me3, as previously reported (Smith, 2008), and in a marked decrease in H3K27ac. These data, together with the evidence of developmentally programmed reversal of Polycomb silencing, now suggest that the widely accepted stability of Polycomb silencing during development might be more dynamically regulated than previously appreciated (Tie, 2009).
This is the first report that CBP/p300 acetylates H3K27. Recombinant Drosophila CBP acetylates H3K27 and K18 in vivo and in vitro.
The greatly reduced H3K27ac levels in CBP-depleted S2 cells also strongly suggest
that CBP is the major H3K27 acetylase in Drosophila. The conservation
of H3K27 acetylation by human p300, together with the reported association of
CBP with the TRX homolog MLL in humans (Ernst, 2001), suggest that it is likely to play a similar role in antagonizing Polycomb silencing in mammals (Tie, 2009).
The genome-wide distribution of H3K27ac, as estimated from human
ChIP-chip experiments, appears very similar to that of H3K4me3. This suggests
that H3K27ac is much more widely distributed than just at Polycomb target
genes, which are estimated to number several thousand in mammalian cells and
hundreds in Drosophila. Although these numbers could grow with the
identification of additional Polycomb-silenced genes in additional cell types,
the recently reported strong correlation of H3K27ac with active genes suggests
that it plays an additional role(s) in promoting the transcription of active
genes, including those that are never targets of Polycomb silencing. (Note
that the H3K27ac at non-Polycomb target genes will not be directly affected by
global changes in H3K27me3.) Interestingly, like H3K27me3, H3K27ac appears on
the transcribed regions of Polycomb target genes, which might reflect a role for
H3K27ac in facilitating transcriptional elongation, and, conversely, a role
for H3K27me3 in inhibiting elongation. In addition to its anti-silencing role
in preventing H3K27 trimethylation, H3K27ac may also serve as a signal for
recruitment of other proteins with additional enzyme activities that alter
local chromatin structure further to facilitate or promote transcription.
Prime candidates are those containing a bromodomain, a conserved
acetyl-lysine-binding module present in several dozen chromatin-associated
proteins, including a number of TrxG proteins that also antagonize Polycomb
silencing (Tie, 2009).
The results presented in this study provide new insight into how TRX and CBP
function together to antagonize Polycomb silencing. Robust H3K27 acetylation
by CBP is dependent on TRX, suggesting that H3K27ac plays a crucial role in the
anti-silencing activity of TRX. Consistent with this, preliminary genetic
evidence suggests that the Polycomb phenotypes caused by TRX overexpression
are dependent on CBP, as they are suppressed by RNAi knockdown of CBP. The nature of this dependence is currently unknown, but could involve targeting of CBP by TRX or regulation of the H3K27 acetylation activity of CBP by TRX (Tie, 2009).
The physical association of TRX and CBP and the widespread coincidence of
H3K27ac and H3K4me3 sites in the human ChIP-chip data further suggest that the
two modifications might be coordinately executed by TRX and CBP. However, the
results also raise the possibility that H3K4 trimethylation by TRX itself
might be less important for antagonizing Polycomb silencing than H3K27
acetylation. This possibility is also suggested by the discovery of
Polycomb-silenced genes in ES and human T cells that contain 'bivalent' marks
(both H3K4me3 and H3K27me3) in their promoter regions (although
the H3K4me3 levels at these inactive genes are typically lower, on average,
than they are at active genes, hinting at the possible importance of quantitative
effects of the two marks) (Tie, 2009).
A speculative model is proposed for the regulation of Polycomb silencing
that incorporates the activities of TRX, CBP, E(Z), RPD3 and UTX. Repressed genes are marked with
H3K27me3. H3K27 trimethylation by PRC2 (which can also control DNA methylation
in mammals) requires RPD3 (and possibly other histone deacetylases) to
deacetylate any pre-existing H3K27ac. H3K27me3 promotes binding of
PC-containing PRC1 complexes, which may inhibit H3K27 acetylation and maintain
silencing through 'downstream' events, including those promoted by the H2AK119
mono-ubiquitylation mediated by its RING subunit.
Conversely, active genes are marked with H3K4me3 and H3K27ac. H3K27
acetylation by CBP is dependent on TRX and possibly other TrxG proteins, as
suggested by the observation that H3K27me3 levels are significantly increased
on salivary gland polytene chromosomes from trx, ash1 and
kis mutants. The current results predict that this increase will be accompanied by a decrease in H3K27ac. Interestingly, ash1 encodes another HMTase that also interacts with CBP and antagonizes Polycomb silencing. Acetylation of H3K27 is likely to also require the K27-specific demethylase UTX when removal of pre-existing H3K27me3 is a prerequisite for acetylation, e.g. for
developmentally regulated reversal of Polycomb silencing at the onset of
differentiation. H3K27ac prevents H3K27 trimethylation and might also serve as
a signal for recruitment of other TrxG proteins with additional
chromatin-modifying activities that may protect the H3K27ac modification and
also alter local chromatin structure to promote transcription and further
inhibit Polycomb silencing (Tie, 2009).
The MOF (males absent on the first)-containing NSL (non-specific lethal) complex binds to a subset of active promoters in Drosophila melanogaster and is thought to contribute to proper gene expression. The determinants that target NSL to specific promoters and the circumstances in which the complex engages in regulating transcription are currently unknown. This study shows that the NSL complex primarily targets active promoters and in particular housekeeping genes, at which it colocalizes with the chromatin remodeler NURF (nucleosome remodeling factor) and the histone methyltransferase Trithorax. However, only a subset of housekeeping genes associated with NSL are actually activated by it. These analyses reveal that these NSL-activated promoters are depleted of certain insulator binding proteins and are enriched for the core promoter motif 'Ohler 5'. Based on these results, it is possible to predict whether the NSL complex is likely to regulate a particular promoter. It is conclude that the regulatory capacity of the NSL complex is highly context-dependent. Activation by the NSL complex requires a particular promoter architecture defined by combinations of chromatin regulators and core promoter motifs (Feller, 2012).
Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. This study devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. It is suggested that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks (Petruk, 2012).
The key logical problem that has prevented identification of epigenetic marks is the difficulty of distinguishing effects on transcriptional regulation from heritable effects. Any change to transcriptional regulation will affect inheritance, and vice versa. Reasoning that any protein or posttranslational modification (PTM) that is not stable to replication is unlikely to be the epigenetic mark, this study focused on identification of proteins or PTMs that are stable to DNA replication. To do so, it was asked what proteins or PTMs are closely associated with the RF based on association with proteins found near the RF, using proximity ligation assay (PLA) to assess in vivo protein-protein interactions and sequential chromatin immunoprecipitation (re-ChIP) to examine protein associations on specific nascent DNA sequences. To examine events at longer distances from the RF, protein and PTM association with nascent DNA labeled with EdU or BrdU was investigated using 'Chromatin Assembly Assay' (CAA or reverse re-ChIP to ensure that protein-DNA interactions were being examined rather than transient interactions with the replication machinery. Together, these approaches give consistent, repeatable results, allowing events to be assayed at different distances from the RF (Petruk, 2012).
The results of PLA assays show that unmodified H3 and H4K5Ac are in close proximity to PCNA and CAF-1, but H3K4me3 and H3K27me3 are not), agreeing with the results from re-ChIP with PCNA. Electron micrographs of replication bubbles from cleavage-stage Drosophila embryos show less than 200 bp of nucleosome-free DNA adjacent to the RF on one strand and on the other strand show partial nucleosome assembly very close to the RF. These micrographs, together with the current observation that 70% of DNA fragments after sonication are less than 200 bp, suggest that the majority of the histones detected in re-ChIP assays with PCNA are associated with DNA. However, it cannot be ruled out that protein-protein and protein-DNA interactions are being detected in the PLA assays, since small amounts of histones may be detected that are associated with CAF-1 prior to deposition rather than histones bound to nascent DNA. It is possible that parental-modified H3 forms are recruited to nascent DNA with some delay. This would not be detected in re-ChIP experiments because the fragment sizes are short (Petruk, 2012).
Therefore, the CAA assay was developed to detect direct association of modified histones on much larger fragments of nascent DNA in vivo. Surprisingly, no H3K4me3 and H4K27me3 PTMs were detected for at least 30 min after passage of the RF, and the first clear signal was detected at 1 hr. This agrees with the observation that H3K4me3 and H4K27me3 are present at low amounts at the cellular blastoderm or are undetectable in S phase during gastrulation. Taken together, the results of CAA, PLA, and re-ChIP suggest that methylated H3 forms are not associated with nascent DNA from the time of the passage of the RF to the end of the S phase. This is in contrast to unmodified H3, which is easily detected by re-ChIP and CAA on nascent DNA of any size (Petruk, 2012).
The data suggest that in embryos, CAF-1 may deposit acetylated histones and unmodified H3 but does not transfer parental H3K4me3 and H3K4me27 to nascent DNA soon after replication as proposed. In yeast, experiments in which parental histones can be distinguished from de novosynthesized histones show that parental histones are deposited within 400 bp of their original locations (Radman-Livaja, 2011). These experiments did not consider PTMs or timing of deposition but make the important point that if parental histones retaining PTMs are deposited at a different location on nascent DNA, then these would be 'epi-mutations' because the same mark would be at a different location. Considering the yeast and the current data, it is proposed that parental histones are dissociated from parental DNA, lose their trimethyl PTMs, and are then
transferred to nascent DNA along with the newly synthesized histones. This suggestion is consistent with earlier results showing that methylation of lysines 9 and 27 of histone H3 is lost during DNA synthesis), and that most histone methylation occurs after deposition (Petruk, 2012).
Together these results suggest that in embryos, trimethylated parental histones are not transferred to nascent DNA and that trimethylation of H3K4 and H3K27 occurs after deposition, and they support the previous suggestion that trimethylation of bound, unmodified H3 is regulated (Scharf, 2009). These data suggest that H3K4me3 and H3K27me3 are unlikely to be epigenetic marks in Drosophila embryos (Petruk, 2012).
In contrast to methylated histones, Trx, Pc, and E(z) associated with PCNA was detected in PLA and re-ChIP assays and stably bound to labeled nascent DNA in CAA assays and re-ChIP assays with BrdU. The results show that Trx, Pc, and E(z) are stable to DNA replication and are constitutively associated with nascent DNA through the S phase, consistent with previous observations that Psc and Pc are stable to replication in an SV40 in vitro replication system (Francis, 2009). Stability to DNA replication and the ability to restore the structure of chromatin required for transcriptional regulation are prerequisites for any putative epigenetic mark. Thus Trx and E(z) fulfill these criteria for epigenetic marks, although functional analysis will be needed to confirm this suggestion. MEs have been proposed to be 'cellular memory modules' because they are sufficient to retain gene-expression pattern. The observation that Trx, E(z), and Pc are retained at the ME after DNA replication supports the model that MEs are cellular memory modules and is consistent with the possibility that these proteins could be epigenetic marks. However, the current observations do not rule out the possibility that other proteins not tested in these experiments are epigenetic marks (Petruk, 2012).
The data imply that Trx, Pc, and E(z) remain bound or rapidly rebind to nascent DNA in the absence of trimethylated histones. There are many reports of trimethylated histone-independent binding of PcG proteins and of MLL1. The current data do not preclude a role for modified histones in binding of PcG and TrxG proteins during transcriptional regulation but suggest that their retention on nascent DNA does not require trimethylation in Drosophila embryos. A recent report shows that in mammalian cell lines, EZH2 associates with PCNA and DNA labeled with BrdU for 5 min, that H3K27me3 is needed to propagate transcriptional repression at a reporter locus, and that H3K27me3 is required for recruitment of PRC2 in interphase in mammalian cells. However that paper did not directly assay the role of H3K27me3 for recruitment of EZH2 in S phase. It is possible that chromatin assembly in Drosophila embryos differs from that in mammalian cells (Petruk, 2012).
The mechanism of retention of Trx and Pc during DNA replication requires association with the ssDNA following unwinding by DNA helicase and transfer from the ssDNA to the nascent dsDNA following passage of the DNA polymerase. The preSET domains of Trx and E(z) bind very tightly to ssDNA, thus providing a plausible explanation for retention of these proteins on the ssDNA found immediately downstream of helicase. It is not known how TrxG and PcG proteins are transferred to nascent DNA. Stable association of the core components of the PRC1 PcG complex, including Pc, with replicating SV40 DNA in vitro may occur by mass-action. Alternatively, Trx, Pc, and E(z) may be retained on replicating DNA by transient interaction with components of the DNA replication complex (Petruk, 2012).
The results suggest that the appropriate amounts of Trx and Pc are replenished late in the S phase or in the interphase, but the mechanism remains unknown. One possibility is that this may occur through interactions between these proteins themselves, for example through dimerization of the SET domain of Trx that has been demonstrated previously. Interestingly, Trx binding can withstand assembly of nucleosomes and interferes with the formation of regular nucleosomal arrays. Thus, Trx may specify a particular nucleosome structure in the MEs. This is a likely possibility given the recent discovery of a specific nucleosome that associates with Gal4 at its binding sites. Importantly, this complex works as a barrier that is essential for establishing specific chromatin architecture of the region surrounding Gal4-binding sites (Petruk, 2012).
PRC2 activity is cell cycle regulated. Several authors have examined retention of PcG and TrxG proteins in mitosis with varying results. The current results suggest that in future it will be interesting to monitor DNA binding and enzymatic activity of PcG and TrxG proteins to determine how epigenetic marks are propagated in G2 and M phases of the cell cycle (Petruk, 2012).
A model is proposed for the reconstitution of chromatin structure during DNA replication in Drosophila embryos. It is suggested that Trx, Pc, and E(z), and likely other TrxG and PcG proteins, are stably associated with their response elements during the progression of the RF, potentially through direct interactions with components of the DNA polymerase complex. Importantly, the stability of the TrxG and PcG proteins during replication ensures sequence specificity in association of these proteins with their response elements after replication. During replication, methylated histones are rapidly replaced by unmethylated histones. The continuous presence of histone-modifying TrxG and PcG proteins may result in histone modification, leading to restoration of the specific chromatin structure that allows either activation or repression of the target gene in the corresponding cells (Petruk, 2012).
The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. This study reports that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btb) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btb is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btb restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity (Komori, 2014).
Maintaining functionally distinct stem cell populations allows higher organisms to generate the requisite number of diverse cell types required for organogenesis. For example, neural stem cells in the subventricular zone and in the outer subventricular zone collectively contribute to the generation of all the cell types required for the development of a human brain. Similarly, heterogeneous stem cell pools have also been reported in other organs including blood and intestine. Although the mechanisms that specify the identity of distinct stem cell types within a given organ have been proposed, the mechanisms that maintain the functional heterogeneity of stem cells have never been reported. This study used the two well-defined yet functionally distinct types of neuroblasts in the fly larval brain to investigate the mechanisms that maintain stem cell functional heterogeneity during neurogenesis. It was discovered that Trx functions uniquely to maintain a type II neuroblast identity through the H3K4 methylation activity of the SET1/MLL complex, thereby contributing to neuroblast heterogeneity during larval brain neurogenesis. The homeodomain transcription factor Btd was identified as a direct downstream target of Trx in the maintenance of a type II neuroblast identity. This Trx-Btd-dependent mechanism provides the first mechanistic insight into the maintenance of stem cell functional heterogeneity within an organ. The homologs of Trx and Btd have been shown to play critical roles in regulating vertebrate neural stem cell functions. The current findings lead to a speculation that the SET1/MLL histone methyltransferase complex might also contribute to the maintenance of stem cell heterogeneity in other higher eukaryotes (Komori, 2014).
The SET1/MLL complex elicits biological responses by maintaining its target genes in an active state through the methylation of H3K4. The core components of the SET1/MLL complex is required for the maintenance of the H3K4 methylation in a type II neuroblast and the maintenance of a type II neuroblast functional identity. Most importantly, over-expression of rbbp5FL, but not rbbp5SG, which encodes a mutant Rbbp5 protein that partially compromises the H3K4 methylation activity of the SET1/MLL complex (Cao, 2010), restored both H3K4 methylation and a type II neuroblast functional identity in rbbp5 null type II neuroblasts. These results indicate that the H3K4 methylation activity of the SET1/MLL complex is required for maintaining the functional identity of a type II neuroblast. In the fly genome, Trx, Trr and dSet1 can each bind to the core components of the SET1/MLL complex. Although the methylation activity of Trx was required for maintaining the type II neuroblast functional identity, removing trx function did not alter the global H3K4 methylation. In contrast, knocking down the function of trr or dset1 did not affect the maintenance of a type II neuroblast functional identity despite resulting in the global loss of H3K4 mono- or tri-methylation. These data strongly suggest that Trx maintains a type II neuroblast functional dentity by regulating H3K4 methylation in specific downstream target loci (Komori, 2014).
The functional identity of a type II neuroblast is defined by the competence of a neuroblast to generate INPs. The data indicate Trx plays a central role in maintaining the functional identity of a type II neuroblast by promoting the expression of a small number of genes. This study identified the btb gene as a critical downstream target of Trx that is both necessary and sufficient for the regulation of the type II neuroblast functional identity. btb encodes a C2H2 zinc finger transcription factor required for required for proper patterning of the head segment during fly embryogenesis and likely functions as a transcription activator. However, the role of Btd in regulating neuroblasts has never been established, and the mechanisms by which Btd elicits biological responses remain unclear. Several possible reasons exist to explain the relatively inefficient nature of eliciting the type II neuroblast functional identity in a type I neuroblast by the mis-expression of btb. First, certain co-factors might be required for Btd to efficiently activate its target gene transcription, and a lower abundance of these co-factors in type I neuroblasts hinders the functional output of mis-expressed Btd. Second, the epigenetic landscape might be vastly different between the two types of neuroblasts such that mis-expressed Btd may not have access to all of its target genes required to elicit the type II neuroblast functional identity in a type I neuroblast. Lastly, additional transcription factors might function in parallel with Btd to regulate the functional identity of a type II neuroblast. Btd is a highly conserved transcription factor. Future studies to elucidate the mechanisms by which Btd regulates the functional identity of a type II neuroblast will provide critical insight in the regulation of neural stem cell heterogeneity during both invertebrate as well as vertebrate neurogenesis (Komori, 2014).
This study has identified the pnt gene as another direct downstream target of Trx. It was initially hypothesized that Pnt might function in parallel with Btd to maintain the functional identity of a type II neuroblast. This hypothesis was extremely appealing in light of a previous study demonstrating mis-expression of PntP1 can transform a type I neuroblast into a type II neuroblast. Unexpectedly, knocking down the function of the pnt gene, which encodes at least three alternatively spliced transcripts, had no effect on the maintenance of the type II neuroblast functional identity, and instead, resulted in the formation of supernumerary type II neuroblasts. This result to a proposal that Pnt functions in the immature INP to specify an INP identity. Consistently, heterozygosity of the
pnt locus dominantly enhanced the supernumerary neuroblast in the brat or erm hypomorphic genetic background. These two genetic backgrounds have been used extensively for elucidating the mechanisms that regulate the specification of an INP identity in the immature INP. Furthermore, over-expression of pntP1 failed to restore the functional identity of a type II neuroblast in trx mutant type II neuroblasts. Together, these data strongly suggest that pnt mainly functions to specify an INP identity rather than to maintain the type II neuroblast functional identity. Thus, it is proposed that in addition to maintaining the type II neuroblast functional identity, Trx also functions to promote INP identity specification through pnt (Komori, 2014).
Strategies that uniquely target the functional properties of cancer stem cells will revolutionize cancer treatments. Cancer stem cells generate a hierarchy of progeny that include cell types directly contributing to the exponential expansion of cancer stem cells. Thus,
reprogramming their functional identity to bypass the cell types that directly contribute to the exponential expansion of cancer stem cells should halt further tumor growth. In this study, removing trx function efficiently reduced the number of supernumerary type II neuroblasts, which are proposed to serve as cancer stem cells in several Drosophila brain tumor models increased the number of differentiated cells in the brat or erm mutant brain. Similarly, attenuating the competence of type II neuroblasts to generate INPs by removing btb function also efficiently halted the expansion of brat or erm mutant brain tumors/ The results strongly support the hypothesis that reprogramming the functional identity of putative cancer stem cells can significantly alter the course of tumorigenesis. As such, understanding the mechanisms that maintain stem cell heterogeneity during normal development might provide novel insight into designing rational therapies to promote switching of cancer stem cells to an alternative, non-cancerous stem cell type (Komori, 2014).
The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. This study analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, the trans-factors that regulate these CRMs were genetically and molecularly analyzed. The results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs (Bieli, 2015).
Genetic and cis-regulatory analysis has provided information about the logic of ap expression during wing development. It is proposed that ap expression is controlled by at least three CRMs that act in combination. The first element, apE is the earliest to be activated in proximal wing disc cells via the EGFR pathway; its expression subsequently weakens in the wing pouch. Deletion of this early enhancer (e.g., apDG12 or apC1345) completely abolishes wing formation. The asymmetry of ap expression to the proximal domain of the wing disc is probably due to the localized activation of the EGFR pathway by its ligand Vn and a distal repression by Wg signaling. The initial activation of the apE by the EGFR pathway was genetically and molecularly confirmed; however, other inputs are required for the continuous activation of this CRM in later wing discs (Bieli, 2015).
A few hours after apE activation, a second CRM, apDV, is activated in a subset of apE positive cells. In contrast to apE, apDV is restricted to the dorsal-distal domain of the wing pouch by direct positive inputs from Ap and Vg/Sd. The direct Ap autoregulatory input defines the time window when the apDV element is activated; apDV can only be active after the induction of Ap by the early enhancer (apE). It has been shown that Ap induces vg expression by triggering Notch signaling at the D/V boundary. Thus, the (direct) input of Vg/Sd on apDV can be regarded as an indirect positive autoregulation, which delimits the spatial domain where apDV can be actived. Consequently, the interface of Ap and Vg expression defines the region of apDV activity via positive autoregulation (Bieli, 2015).
The third ap CRM is the ap PRE/TRE region (apP), that, when deleted, leads to a strong hypomorphic wing phenotype (apc1.2b). The apP requires Trx input and maintains ap expression when placed in cis with the apDV and apE CRMs. Only the combination of the three CRMs faithfully reproduces ap expression in the wing disc. Moreover, the regulatory in locus deletion and in situ rescue analysis provide strong functional relevance for these CRMs (Bieli, 2015).
Ultimately, this cascade of ap CRMs provides a mechanism to initiate, refine and maintain ap expression during wing imaginal disc development, in which the later CRMs depend on the activity of the early ones. A similar mechanism has been described for Distal-less (Dll) regulation in the leg primordia where separate CRMs trigger and maintain Dll expression in part by an autoregulatory mechanism (Bieli, 2015).
It has been proposed that positive autoregulation may help to maintain the epigenetic memory of differentiation. In the case of ap, this study demonstrates that autoregulation works in conjunction with a PRE/TRE system; this might make the system very robust and refractory to perturbations (Bieli, 2015).
ChIP experiments have shown that many developmentally important genes are associated with a promoter proximal PRE as found at ap. The role of such a PRE has been studied at the engrailed (en) locus. It has been demonstrated that in imaginal discs, the promoter as well as the promoter proximal PRE are important for the long-range action of en enhancers. It has been proposed that this PRE brings chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments (Bieli, 2015).
The current results indicate that sequences around the transcription start of ap (apP) may serve a similar function. First, this element, when placed in cis with the ap CRMs (apE and apDV), maintains the ap expression pattern and keeps reporter gene expression off in cells where low or no activity of apDV and apE has been observed. Second, in the absence of trx, the expression of ap and apDV+E+P-lacZ is strongly reduced. All these data suggest that sequences within the apP integrate Trx input, thereby maintaining ap expression in a highly proliferative tissue such as the wing disc. Interestingly, trx mutant clones were not round and did not show ectopic wg activation, which is a hallmark of ap loss-of-function clones. This suggests that in trx mutant clones enough Ap protein is still present to maintain wg expression off. However, derepression of the ventral-specific integrin αPS2 was found in trx mutant clones in the wing pouch as previously described for ap mutant clones (Bieli, 2015).
It has been suggested that TrxG proteins could act passively antagonizing PcG silencing, rather than playing an active role as co-activators of gene transcription. For example, Ubx expression in the leg and haltere does not require Trx in the absence of Polycomb repression. These possibilities were tested and trx mutant clones were generated that were also mutant for the PcG member Sex combs on midlegs (Scm). Dorsally-located Scm- trx- double mutant clones still downregulate ap-lacZ expression while ventral-induced ones are unable to derepress ap-lacZ as was observed for Scm- single mutant clones. Therefore, the results suggest that TrxG maintains ap expression in dorsal cells, while ap expression is repressed in the ventral compartment by PcG proteins. Moreover, it has been shown that the sequences around the ap transcription start, including the PRE, are occupied by PcG complexes PRC1 and PRC2, as well as Trx (Bieli, 2015).
Enhancers-promoter interactions initiate transcription but their dynamics during development have remained poorly understood. A Chromosome conformation capture (3C) experiment provides evidence for the direct interaction between the ap CRMs apE and apDV with the maintenance element encoded by the apP. Beyond this, it was also found that these elements cooperate continuously during wing development. Flip-out experiments, in which the apDV and apE CRMs were removed at different time points, suggest that these elements need to be present continuously to ensure correct ap expression. Additionally, flies carrying apE only on one chromosome and apDV only on the homologue were unable to fully rescue wing development suggesting that these CRMs need to be in cis. It is conceivable that in cis configuration of the three ap CRMs facilitates and stabilizes enhancer-promoter looping. It could also help to rapidly establish relevant chromatin contacts after each cell division. These results are in accordance with previous observations, in which constant interactions between ap enhancers and promoter during embryogenesis have been described. The current results extend these observations to the wing disc, a highly proliferative tissue, where the expression of the trans-factors that regulate the activity of the apE and apDV is very dynamic. This raises the question on how this contact is re-assembled over many cell generations. It is possible that some epigenetic modifications are laid down in the activated apE and apDV CRMs, which are then inherited during cell divisions to ensure contact with apP. Studies of the chromatin status of these elements will be required to fully understand this process (Bieli, 2015).
A key question in developmental biology is how transcriptional regulation is coupled to tissue growth to precisely regulate gene expression in a spatio-temporal manner. For example, during Drosophila leg development, initial activation of the ventral appendage gene Dll by high levels of Wg and Dpp initiates a cascade of cross-regulation between Dll and Dachshund (Dac) and positive feedback loops that patterns the proximo-distal axis. Other mechanisms to expand gene expression patterns depend on memory modules such as PREs, as it is the case for the Hox genes or other developmental genes like hh. To direct wing formation, expression of ap in the highly proliferative tissue of the wing disc must be precisely induced to generate and maintain the D/V border. These in-depth analyses at the ap locus provide a functional and molecular explanation of how expression of this dorsal selector gene is initiated, refined at the D/V border, and maintained during wing disc development. It is proposed that this three-step mechanism may be common for developmental patterning genes to make the developmental program robust to perturbations (Bieli, 2015).
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