Abdominal-B
The transvection mediating region of the Abdominal-B locus Genetic studies have identified an unusual transvection process in the Abdominal-B (Abd-B) locus of Drosophila. In some cases distal infraabdominal (iab) regulatory domains
continue to activate the Abd-B promoter even when translocated onto different chromosomes. Transvection
depends on an approx. 10 kb genomic DNA sequence, termed the transvection mediating region (tmr), located
immediately downstream of the Abd-B transcription unit. A detailed analysis of this region is reported. Different
DNA fragments from the tmr were inserted into a variety of P-transformation vectors. Analyses of reporter gene
expression in transgenic embryos and adults have identified at least three cis-regulatory elements, including two enhancers (IAB7 and IAB8) and a new insulator DNA between IAB7 and IAB8 (Frontabdominal-8, Fab-8). Evidence is also presented for a Polycomb response element ( PRE) linked to the IAB8
enhancer, and an internal promoter in the iab-8 domain that transcribes the iab-7 and iab-8 cis-regulatory DNA, including the Fab-8 insulator. The significance of
these findings is discussed with regard to Abd-B transvection and long-range
enhancer-promoter interactions in mammalian globin loci (Zhou, 1999a).
The approx. 10 kb tmr is located just downstream of the Abd-B
transcription unit. Chromosomal breakpoints within
the tmr disrupt long-range interactions between iab-5, iab-6
and iab-7 enhancers and the Abd-B promoter. Such disruptions appear to cause a reduction of Abd-B
function. Cis-regulatory elements were identified within the
tmr by inserting different DNA fragments into P-element
transformation vectors containing lacZ, white and yellow
reporter genes. The regulatory activities of these DNA
fragments were determined by analyzing reporter gene
expression in transgenic embryos and adult flies. In situ
hybridization was used to analyze reporter gene expression in
embryos, while eye color and body color were used to examine
reporter gene activity in adults (Zhou, 1999a).
The distal approx. 4 kb region of the tmr extends into the iab-7
domain, which controls the
morphogenesis of the tissues that comprise the seventh
abdominal segment. Thus far, no enhancers, either embryonic
or larval, have been identified in this region. A 1.9 kb PCR
fragment, which includes nearly half of the iab-7 sequences
contained within the tmr, was inserted into a P-transformation
vector containing divergently transcribed white and lacZ
reporter genes. This iab-7 DNA directs
the expression of the white reporter gene within the limits of a
posterior stripe that resolves into two stripes
in late stage 5 embryos. Reporter gene
expression was visualized after hybridizing transgenic
embryos with a digoxigenin-labeled antisense RNA probe. A
similar staining pattern is observed when the lacZ reporter
gene is monitored via in situ hybridization.
The early stripes of white and lacZ expression appear to fall
within the limits of parasegments 12-14.
Different pieces of the 1.9 kb iab-7 DNA fragment were
analyzed in an effort to identify the minimal IAB7 enhancer. A 700 bp fragment retains full activity.
The 5' portion of the 1.9 kb fragment lacks regulatory activity, and larger fragments containing the minimal 700 bp
enhancer direct the same pattern of white expression as that
observed with the minimal fragment. The IAB7 enhancer directs somewhat stronger expression
in dorsal versus ventral regions; a similar asymmetry is
observed for the IAB5 enhancer in precellular embryos (Zhou, 1999a).
Transcriptional repressors encoded by the gap genes have
been shown to be essential for establishing the initial limits of
homeotic gene expression. Direct
regulatory interactions have been identified for the Krüppel
repressor and iab-2 cis-regulatory elements within the abd-A
locus. Mutations in critical Krüppel
binding sites result in an anterior expansion of the normal abd-A
expression pattern. Genetic studies suggest that Krüppel
might also regulate Abd-B expression. Krüppel minus mutants exhibit an ectopic stripe of
Abd-B expression in central regions of developing
embryos, within the presumptive thorax. A
computational search of Krüppel recognition sequences
throughout the BX-C reveals two closely linked sites
within an approx. 130 bp region of the minimal 700 bp
IAB7 enhancer. Nucleotide substitutions in
both core sequences (consensus: AACGGGTTAA) alter
the activity of the IAB7 fusion gene in transgenic
embryos. The white reporter gene exhibits ectopic
expression in anterior regions as compared
with the normal pattern directed by the wild-type enhancer.
These results suggest that Krüppel functions as a
direct repressor of Abd-B expression (Zhou, 1999a).
The tmr includes approx. 5 kb from the iab-8 cis-regulatory
domain. A 5.3 kb BamHI-HindIII
DNA fragment spanning this region was inserted into
a P-transformation vector containing divergently transcribed
white and lacZ reporter genes. This
DNA directs the expression of both reporter genes within a
posterior stripe in transgenic embryos. This stripe
appears to span parasegments 13 and 14. This expression pattern suggests that the 5.3 kb region
belongs to the iab-8 regulatory domain. Smaller DNA
fragments have also been examined to identify the minimal IAB8
enhancer; a distal 1.6 kb fragment fails to direct lacZ
expression, while the proximal 2.7 kb fragment retains full
activity (Zhou, 1999a).
Both the 5.3 kb, and to a lesser extent, the 2.7 kb DNA
fragments continue to direct reporter gene expression in the
posterior germ band of stage 10 to stage 13
embryos. In contrast, the iab-7 cis-regulatory
DNAs direct transient stripes of expression only during early
periods of embryogenesis. It is therefore
conceivable that the iab-8 region includes a maintenance
element, perhaps a PRE, which sustains expression during
development. Additional evidence for a PRE stems from the analysis of eye
color in the transgenic strains. Adults carrying the 5.3 kb iab-8
DNA exhibit strong position effect variegation of mini
white expression, which has been
shown to be diagnostic for other PREs. The 2.7 kb DNA fragment causes substantially
weaker variegation. It is interesting to note that a
conserved sequence motif (CRGCCATYDTRG)
found in other PREs is present in the 5.3 kb, but not
the 2.7 kb, iab-8 DNA (Zhou, 1999a).
It has been proposed that the different iab regulatory
regions are organized into separate chromatin loop
domains by a series of insulator DNAs. If
so, the most likely location of an insulator within the
tmr is between the IAB7 and IAB8 enhancers
identified in the preceding analyses. Three different
DNA fragments spanning this interval, each about 2
kb in length, were tested for insulator activity. The assay involves the use
of a P-transposon that contains two different
enhancers, 2xPE and IAB5, positioned 5' to the
divergently transcribed white and lacZ reporter genes. The
2xPE enhancer corresponds to two tandem copies of
the 180 bp proximal enhancer (PE) from the twist
promoter region that directs expression in the
ventral mesoderm. The IAB5 enhancer directs a
broad band of expression in the presumptive
abdomen. When a neutral
spacer DNA is inserted between the two enhancers,
both white and lacZ exhibit composite patterns of
expression in the mesoderm and abdomen. Only one of the DNA fragments that was
tested in this assay exhibits an insulator activity. Insertion of this fragment, hereafter called
Fab-8, in place of the spacer sequence alters both the
white and lacZ staining patterns. The white reporter
gene is now expressed exclusively in the mesoderm;
staining is essentially eliminated in the presumptive
abdomen. The opposite staining pattern is
observed for the lacZ reporter gene. In this
case, expression is detected primarily in the
presumptive abdomen, while staining in the
mesoderm is markedly reduced. These results suggest that the
Fab-8 DNA functions as an insulator, and selectively blocks
the interaction of a distal enhancer with a target promoter. It
blocks interactions between IAB5 and white, as well as 2xPE
and lacZ. Fab-8 is located approximately 2.5 kb 3' of the IAB7
enhancer and 2 kb 5' of IAB8 within the tmr (Zhou, 1999a).
Fab-8 and the previously identified Fab-7 insulator were
tested for their ability to block enhancer-promoter interactions
in heterologous tissues. The P-transformation vector
used for these experiments contains the white and yellow
genes; the latter reporter gene is normally expressed in the
body cuticle, wings and bristles of adult flies. The yellow promoter region contains separate
enhancers that regulate expression in the wings and body
cuticle. Previous studies have
identified the y2 mutation as an insertion of the gypsy
retrotransposon. The gypsy insulator
specifically blocks the activities of the distal wing and body
enhancers, but not the intronic bristle enhancer.
P-transposons were prepared that contain either a Fab-7 or
Fab-8 cassette. Each cassette contains five copies of
an eye-specific enhancer from the glass gene, and an insulator
DNA (Fab-7 or Fab-8) flanked by binding sites (frt) for the
yeast Flip recombinase. Transgenic flies carrying either
insulator cassette possess only yellow to light orange eyes, presumably due to a block in glass-white
interactions. These transgenic strains were mated with flies
containing the Flip recombinase under the control of the hsp70
promoter. Heat-induction of the recombinase in first to second
instar larvae results in red patches, due to the mosaic removal
of the insulator cassette. These results suggest that
the block in glass-white interactions is due to the Fab-7
and Fab-8 insulators, and not the result of position effects
resulting from differing sites of transgene integration. When
the insulators are removed by the Flip recombinase, the glass
enhancer leads to robust expression of the white gene. The
insulator cassettes also block the activation of the yellow gene
by the wing and body enhancers but not the intronic bristle
enhancer. Heat-induced expression of the Flip
recombinase results in patches of pigmented body cuticle, due to the mosaic activation of yellow by
the distal body enhancer (Zhou, 1999a).
The Fab-7 insulator DNA that was used in the preceding
is distinct from Fab-7
sequences examined in other studies, although all three Fab-7
DNAs contain a cluster of six linked GAGA sites. GAGA
binds to a zinc finger protein, Trithorax-like (Trl), and Trl-GAGA
interactions have been shown to be important for the
enhancer blocking activity of the eve promoter. It is
conceivable that GAGA is also important for the insulator
activity of Fab-7 (Zhou, 1999a).
Previous studies provide evidence that the Abd-B 3' cis-regulatory
DNA is transcribed. To investigate this
issue, different DNA fragments from the tmr were used for in
situ hybridization assays. All three cis-regulatory
elements, IAB7, Fab-8 and IAB8, detect specific RNAs. Staining is observed in a posterior stripe, similar to the
pattern directed by the IAB8 enhancer when attached to a
reporter gene. The use of small DNA fragments
spanning the iab-8 regulatory region suggests that there may
be one major internal promoter, located just downstream of the
Abd-B transcription unit. This
promoter can respond to heterologous enhancers. A
P-transposon was prepared that contains the 9.5 kb tmr and the
heterologous 200 bp hairy stripe 1 (H1) enhancer.
Transgenic embryos were then hybridized with the IAB7
enhancer as a probe. Normally, the enhancer is expressed in a
posterior stripe. However, the enhancer is expressed
in both a posterior stripe and an anterior band in transgenic
embryos. The latter pattern is presumably due to
activation of the iab-8 promoter by the H1 enhancer (Zhou, 1999a).
In the case of the Abd-B
3' cis DNA, it would appear that there is a major internal
promoter located just downstream of the Abd-B transcription
unit. The identification of an iab-8
promoter is reminiscent of the situation seen in mammalian
immunoglobulin genes, whereby unrearranged, germline
genes contain internal promoters that are thought to maintain
neighboring intronic enhancers in an open conformation for
interactions with appropriate transcriptional regulatory
proteins. It is conceivable that
the iab-8 promoter plays a similar role in Abd-B regulation.
The maintenance of an open chromatin conformation might
help account for Abd-B transvection. Perhaps proteins bound
to the 3' region serve as 'signposts' for the distal Abd-B
enhancers.
The iab-8 promoter might also play a more specific role in
ensuring peak expression of Abd-B in parasegment 13 (and
perhaps ps 14). Transcription of the 3' region might inactivate
the Fab-7 and Fab-8 insulators within parasegment 13 so that
all distal regulatory elements can activate the Abd-B promoter
in this posterior region of the embryo. It is also conceivable
that the iab-8 promoter, as well as other internal promoters in
the 3' region, might direct long-range enhancer-promoter
interactions. Perhaps such promoters help reel-in distal
enhancers to the Abd-B transcription unit.
There are parallels between the organization of the Abd-B
locus in Drosophila and mammalian globin loci. Both contain
insulator DNAs and exhibit extensive transcription of the cis-regulatory
DNA. It has
been shown that exogenous DNAs from the globin locus, either
the globin transcription units or the neighboring cis DNAs,
physically associate with the endogenous globin locus in
transfected tissue culture cells. This physical interaction of
exogenous DNA with the endogenous gene leads to the
activation of the internal promoters in the endogenous locus.
There may be a similar long-range physical interaction
between distal cis-regulatory elements and the Abd-B locus in
the transvection phenomenon. Future studies will determine
whether the cis-elements identified in this study are sufficient
to account for transvection, or whether the tmr contains
additional elements that facilitate long-range enhancer-promoter
interactions (Zhou, 1999a).
The Abd-B Hox gene contains an extended 3' cis-regulatory region that is subdivided into a series of separate iab domains. The iab-7 domain activates Abd-B in parasegment 12 (ps12), whereas iab-8 controls expression in ps13. iab-7 is flanked by two insulators, Fab-7 and Fab-8, which are thought to prevent regulatory factors, such as Polycomb silencers, from influencing neighboring iab domains. This organization poses a potential paradox, since insulator DNAs can work in a dominant fashion to block enhancer-promoter interactions over long distances. Evidence exists for a novel cis-regulatory sequence located within iab-7, the promoter targeting sequence (PTS), which permits distal enhancers to overcome the blocking effects of Fab-8 and the heterologous su(Hw) insulator. It is proposed that the PTS converts dominant, long-range insulators into local regulatory elements that separate neighboring iab domains (Zhou, 1999b).
Previous genetic studies have identified a ~10 kb region 3' of Abd-B that is essential for long-range enhancer-promoter interactions. This regulatory region, the transvection mediating region (tmr), contains a dense assortment of cis elements, including an insulator DNA (Fab-8), two enhancers (IAB7 and IAB8), a PRE, and an internal promoter. The present study provides evidence that the 1.7 kb Fab-8 insulator region contains two closely linked regulatory elements: the minimal 590 bp Fab-8 insulator, and a promoter targeting sequence (PTS), which abrogates the enhancer blocking activities of Fab-8 and the heterologous su(Hw) insulator. It is suggested that the newly identified PTS modifies the regulatory properties of insulator DNAs and facilitates long-range enhancer-promoter interactions (Zhou, 1999b).
The full-length, 9.5 kb tmr and the 1.7 kb Fab-8 DNA mediate two unusual regulatory activities. They can target 3' enhancers to a distal white reporter gene and suppress the enhancer blocking activity of the minimal Fab-8 insulator. Neither activity is detected when the 1.7 kb Fab-8 DNA is placed 5' of test promoters. In the 5' position, the DNA fragment exhibits a simple insulator activity, whereby distal, not proximal, enhancers are selectively prohibited from activating white and lacZ . Additional regulatory activities were uncovered only when the tmr and 1.7 kb Fab-8 DNAs were placed far (>5 kb) from the target promoters (Zhou, 1999b).
The PTS suppresses the enhancer blocking activities of Fab-8 when positioned in either orientation relative to the insulator or target promoter. There does not seem to be a requirement for tight linkage of the PTS and distal enhancer. For example, the rho NEE overcomes Fab-8 even when the PTS and NEE are separated by ~5 kb. The PTS abrogates enhancer blocking when located either upstream or downstream of the insulator. It is currently unclear whether anti-insulator activity depends on close proximity of the PTS and insulator (Zhou, 1999b).
It is proposed that proteins bound to the PTS somehow stabilize enhancer-promoter complexes and thereby help distal enhancers overcome the blocking activity of an intervening insulator. Moreover, a stable enhancer-promoter complex might prevent the enhancer from interacting with additional promoters once a target promoter is selected. PTS elements might play a role in the selection of a single receptor gene in mammalian odorant complexes. An alternative model is that the PTS works in a local fashion to inhibit linked insulators. For example, proteins bound to the PTS might physically interact with proteins bound to neighboring insulators (Zhou, 1999b).
Genetic studies suggest that the PTS can facilitate enhancer-promoter interactions even when the distal enhancer is located far from both the PTS and target promoter. The R73 (iab-7R73) chromosome contains a deletion in the Fab-7 insulator as well as in the PTS. The loss of Fab-7 causes a dominant tranformation of A6 into A7, suggesting that iab-7 activators 'spread' into the iab-6 domain and result in the overexpression of Abd-B in A6 (ps11). The R73 deletion suppresses this transformation, possibly due to a reduction in iab-6-Abd-B interactions resulting from the loss of the PTS. Moreover, R73/S10 transheterozygotes exhibit a relatively robust homeotic transformation of A5 into A4. This observation suggests that the loss of the PTS also reduces IAB5-Abd-B interactions; the IAB5 enhancer is located ~40 kb away from the PTS (Zhou, 1999b).
It is proposed that the PTS is responsible for converting dominant, long-range insulator DNAs into local, short-range regulatory elements. The Fab-7 and Fab-8 insulators prevent iab-7 and iab-8 cis-regulatory elements from disrupting the activities of neighboring iab domains. For example, the IAB8 enhancer directs Abd-B expression in ps13 and may be essential for the morphogenesis of the eighth abdominal segment. IAB8 activity is inhibited in all anterior segments, extending through ps12, and the iab-8 PRE maintains this pattern of repression throughout development. It is thought that Pc-G repressors bound to PREs work over long distances, possibly by propagating changes in chromatin structure. The Fab-8 insulator may be essential for preventing these repressors from spreading into iab-7 and inactivating the IAB7 enhancer in ps12. Similarly, Fab-8 might also prevent iab-7 activators from inducing ectopic expression of IAB8 in ps12. However, like other insulators, Fab-7 and Fab-8 can block distal enhancers over long distances. The newly identified PTS appears to permit distal enhancers to overcome the blocking effects of Fab-7 and Fab-8, but might not interfere with the local isolation of neighboring iab domains (Zhou, 1999b).
Recent genetic studies suggest that transvection depends on both the tmr and regulatory sequences in the Abd-B 5' promoter region. It
has been suggested that these 5' regulatory elements help tether distal iab enhancers to the Abd-B promoter. Thus, the PTS might
work in concert with dedicated enhancer-promoter interactions to ensure that distant iab elements can overcome the blocking effects of intervening
insulators. Specific enhancer-promoter interactions have been observed in a number of Drosophila loci, including the Antennapedia complex, gooseberry, and dpp (Zhou, 1999b and references therein).
Transcriptional enhancers in large gene complexes activate promoters over huge distances, little is known about the mechanism of these long-range interactions. The promoter targeting sequence (PTS) from the Abdominal-B locus of the Drosophila bithorax complex facilitates the activity of a distantly located enhancer in transgenic embryos and the PTS restricts the enhancer to a single promoter. These functions are heritable in all successive generations. The PTS functions only when itself and an insulator are located between the enhancer and the promoter. These findings suggest that the PTS may facilitate long-range enhancer-promoter interactions in the endogenous Abdominal-B locus. It is proposed that the PTS establishes a stable chromatin structure between an enhancer and a promoter, which facilitates yet restricts an enhancer to a single promoter (Lin, 2003).
The Fab-8 insulator can block
enhancers (including IAB8) from activating a promoter when it is interposed
between the enhancer and its promoter. The 625 bp PTS element is able to overcome
the enhancer blocking effect of the Fab-8 insulator
(Zhou, 1999). Tests were made to determine whether the minimal 290 bp 5' DNA from the 625 bp PTS can overcome the Fab-8 insulator, and, in addition, facilitate the IAB8 enhancer activity. A 5.3 kb BamHI-HindIII
fragment from the tmr that contains the minimal 290 bp of the PTS,
the 580 bp Fab-8 insulator and the 1.6 kb IAB8 enhancer were tested (Lin, 2003).
The IAB8 enhancer directs a narrow band of transcription in the posterior
region of the embryo. Similar to other early Drosophila enhancers that
have been tested, its activity attenuates as its distance from the promoter
increases. This 5.3 kb tmr was placed in both the forward (5'->3' when IAB8 is between the promoters and Fab-8/PTS) and the reverse
orientation. To monitor transcriptional activity of the IAB8 enhancer, embryos from individual transgenic strains were collected and subjected to whole-mount RNA in situ hybridization for the white (w) or lacZ genes (Lin, 2003).
In the forward orientation, IAB8 weakly stimulates both the divergently
transcribed w and Tp-lacZ genes. Its activity on the
Tp promoter is very similar to that of the 1.6 kb IAB8 alone at the
same location, suggesting that the distally located PTS and Fab-8 do not affect the communication between IAB8 and the transgenic promoters. In the reverse
orientation, however, IAB8 activates the transgenic promoters despite the
intervening Fab-8 insulator. In any particular strain, IAB8 exhibits
the selective activation of either the Tp-lacZ or the w gene. This effect is seen in about 50% of the transgenic lines. In the remaining
strains, IAB8 does not activate any of the transgenic promoters due to the
enhancer blocking effect of the Fab-8 insulator.
These results are similar to those of the previous study
(Zhou, 1999),
suggesting that the 290 bp PTS exhibits the anti-insulator and promoter
targeting activities. This is confirmed by transgene W14, where PTS could
overcome a heterologous suHw insulator and target IAB8 to the
w or Tp promoters. It should be noted that in the forward
orientation, IAB8 is
located 5.5 kb away from the w promoter and 4.9 kb away from the
lacZ promoter, whereas in the reverse orientation, it is 8.2 kb away
from w and 7.5 kb away from lacZ. Rather than reducing the
activity, the greater distance between IAB8 and the promoters caused by
inserting Fab-8 and PTS results in an increase of IAB8 enhancer
activity. The enhancer activity is much stronger when
both PTS and Fab-8 are located between IAB8 and the promoters. These
results indicate that PTS may facilitate the long distance interactions
between IAB8 and the w or the Tp promoter (Lin, 2003).
To confirm the enhancer facilitating activity and to eliminate position
effects due to differential chromosomal insertion sites of the transgene, the Flp-FRT system was used; this causes the removal of FRT-flanked DNA sequences
from the transgene after introducing the Flp recombinase by genetic cross.
This technique permits the analysis of the transgene in the same chromosomal
context before and after the test DNA is removed. A 2.0 kb
PstI-HindIII fragment from the tmr region that contains both the
minimal 290 bp PTS and the 580 bp Fab-8 insulator was flanked with the FRT sites
and this group of elements was placed between the 3' end of lacZ
and 2.7 kb EcoRI fragment from the same region that contains the 1.6
kb IAB8 enhancer. When transgenic embryos were analyzed, results similar to
those without FRT sites were obtained. In about half of the transgenic strains
IAB8 selectively activates one of the two divergently transcribed w
and the Tp-lacZ genes. In the remaining strains, IAB8 does not activate any of the transgenic promoters. These results strongly suggest
that the PTS, in combination with the Fab-8 insulator, facilitates
enhancer-promoter interaction, and that it restricts the enhancer activity to
a single promoter (Lin, 2003).
These studies have shown that PTS facilitates long-range enhancer-promoter
interactions in transgenic embryos. The enhancer facilitating activity depends
on the anti-insulator and promoter targeting functions in that it only
facilitates an enhancer when it is targeted to a promoter. Evidence is provided that the promoter targeting function is due to
restricting the access of an enhancer to a single promoter, and not due to,
for example, positional effects that might inactivate the other promoter
present in the transgene. The IAB5 or the IAB8 enhancer alone is capable of activating
both the w and Tp promoters after the PTS/insulator fragment
is removed by recombination. It is possible that the anti-insulator, promoter
targeting, and enhancer-facilitating activities are inseparable and are
possibly different aspects of the same activity. For example,
enhancer-facilitating effect could result, at least in part, from
restricting the enhancer to a single promoter, which would prevent the
enhancer from activating other promoters and consequently increase the
probability of activating the 'target' promoter (Lin, 2003).
In the Abd-B locus, the enhancer-facilitating property of the PTS
could help distal enhancers such as IAB7 overcome the long distances and
direct robust transcription activation of Abd-B. This notion is
consistent with the genetic functions of the tmr and the
loss-of-function phenotype of PTS mutants. The
single promoter-activating function could ensure that enhancers in the BX-C
activate the cognate Abd-B promoter only. In this study, it has also been
shown that promoter targeting occurs only when PTS/insulator is placed
between an enhancer and a promoter. This location-dependent characteristic of
PTS suggests that strategic placement of PTS within Abd-B can
facilitate specific enhancers. For example, in the Abd-B locus, PTS
and Fab-8 are located between IAB7 (but not IAB8) and Abd-B
promoter. In this
arrangement, PTS may facilitate only the distal IAB7 but not the proximal IAB8
to the Abd-B promoter. In fact, this appears to be the case, as
deletion of PTS causes a loss-of-function transformation of the seventh but
not the eighth abdominal segment. On
this note, it is possible that multiple PTS-like elements exist in the BX-C to
mediate long-distance regulatory interactions (Lin, 2003).
The PTS-facilitated single enhancer-promoter interaction is stronger than
the sum of enhancer-w and enhancer-Tp interactions when the
IAB5 or IAB8 enhancers are placed alone at the 3' of lacZ.
These results suggest that the PTS-mediated enhancer facilitation is not just
the consequence of restricting an enhancer to a single promoter -- it must also
actively promote long distance enhancer-promoter communications. It is
possible that the PTS functions by establishing an insulator-insensitive,
stable chromatin structure between the enhancer and a promoter, e.g., forming a
'stable loop' and bringing the enhancer closer to the promoter. Similar 'loop'
hypothesis has been proposed based on genetic analysis of the
enhancer-promoter interactions in the Abd-B locus. This
model can not only explain the anti-insulator activity but can also account
for the enhancer-facilitating and the single promoter activating activities.
Such a stable association would ensure that enhancer-interacting activators
are constantly present at the promoter, which would result in efficient
promoter activation and, at the same time, prevent the enhancer from
activating other promoters (Lin, 2003).
This study also suggests that PTS functions as a generic element in
transgenic embryos since it can target and facilitate a heterologous
neuroectoderm enhancer NEE. It is possible that in other large genetic loci such as the
odorant receptor gene complex and the neural cadherin-like adhesion gene complex, where
only one among several dozen promoters is activated in any given cell,
PTS-like elements may contribute to the promoter-selective transcriptional
activation (Lin, 2003).
In transgenic embryos, the PTS does not appear to exhibit promoter-specific
activity since it can target either the w or the Tp promoter
present in the transgene. It is not known what determines which of the two
promoters to select. One possibility is that the decision is made by the
interaction between the PTS and local chromatin structure. Alternatively, the
selection could be a stochastic process. In the endogenous BX-C, however, the
PTS must target the Abd-B promoter. Additional mechanism(s),
therefore, must be in place to ensure enhancer-promoter specificity in the
Abd-B locus (Lin, 2003).
Large gene complexes frequently use 'specialized' DNA elements to ensure proper regulation of gene activities. The Promoter Targeting Sequence (PTS) from the Abdominal-B locus of the Drosophila Bithorax complex overcomes an insulator, and facilitates, yet restricts, distant enhancers to a single promoter. This promoter-targeting activity is independent of an enhancer's tissue or temporal specificity, and can be remembered in all somatic cells in the absence of promoter activation. It requires an insulator for its establishment, but can be maintained by the PTS in the absence of an insulator. More important, the promoter-targeting activity can be remembered after the transgene is translocated to new chromosomal locations. These results suggest that promoter targeting is established independent of enhancer activity, and is maintained epigenetically throughout development and subsequent generations (Lin, 2004).
Surveys of a region 15 kb downstream from the Abd-B promoter have led to the identification of a novel cis-regulatory element, the Promoter Targeting Sequence (PTS). The minimal 290-bp PTS exhibits an anti-insulator activity, facilitates long-range enhancer-promoter interactions, and usually restricts the enhancer activity to a single promoter when more than one is present in the same transgene. These unique properties of the PTS suggest that it may normally regulate highly specific, long-range enhancer-promoter communications in the Abd-B locus by overcoming the enhancer-blocking activity of the Fab-8 element (Lin, 2004 and references therein).
Several lines of evidence are presented suggesting that the PTS-mediated promoter-targeting function is epigenetically heritable in both somatic and germ-line cells. (1) It was shown that promoter targeting is independent of tissue or temporal specificity of enhancers, and is memorized in the absence of transcription activity throughout development. (2) It was demonstrated that promoter targeting requires an insulator for establishment but can be maintained by the PTS alone in all successive generations. (3) It was observed that promoter-targeting memory is stable even after a transgene is translocated to new chromosomal locations (Lin, 2004).
When given the choice of two promoters in the transgene, the PTS usually targets just one, resulting in exclusive transcription activation of the targeted promoter. What determines which promoter is targeted in a specific transgenic strain is not known, but two models could be proposed regarding the role of the enhancer in promoter selection: (1) promoter targeting is determined partially by the enhancer or transcription factors bound to the enhancer; (2) it is epigenetically determined and maintained independent of the identity of the enhancer, and the enhancer-binding proteins. The first model predicts that if multiple enhancers exist in the transgene, the PTS could target different enhancers independently. For example, one enhancer could be targeted to the proximal Tp promoter, whereas the other could be targeted to the distal w promoter in the same transgenic strain. This is shown not to be the case. In all transgenic strains examined, different enhancers are cotargeted to the same promoter. These results strongly support the epigenetic inheritance model. Consistent with this model, both adult eye enhancer glass and embryonic enhancers IAB5 were observed to be cotargeted to the same promoter. Because the adult glass enhancer and the embryonic enhancer IAB5 are active in different tissues and at different times during the fly life cycle, cotargeting of these two enhancers suggests that in every somatic cell during the life span of the fly, the PTS maintains a memory of the target promoter should an enhancer become active during development (Lin, 2004).
Several models can be proposed to account for the role of the insulator in PTS function: an insulator may be necessary to direct the transgene to 'special' nuclear compartments that are compatible with PTS function. The suHw insulator is known to cause the transgene to associate with the nuclear envelope, whereas the vertebrate ß-globin HS4 insulator has been shown to associate with the nucleolus. In addition, artificially attaching transgenes to nuclear pores creates a chromatin boundary in the transgene. Although it provides an interesting mechanistic explanation, this model could not explain why the insulator is dispensable once promoter targeting is established, because the loss of insulator would dislodge the transgene from the nuclear compartment required for PTS function. The second possibility is that insulator DNA may cause the P-element to integrate into special regions of the chromosome during P-mediated transformation where the PTS can interact with the appropriate local chromatin structure favorable for promoter targeting. Although specific regulatory DNA sequences have been observed that cause preferential insertions into specific chromosomal locations, such a function has not been reported for insulators. It has been demonstrated that an insulator is not necessary to maintain the promoter-targeting memory during transgene mobilization to new locations; thus, it is unlikely that insulator functions by directing the P-element to the right chromosomal location for integration. A third potential mechanism is that insulator may help recruit proteins that are necessary for PTS activity. This is also unlikely because it was observed that promoter targeting only occurs when an insulator is inserted between an enhancer and its promoter. Because the insulator is a constitutive element that presumably recruits the same proteins regardless of insulator locations relative to the enhancer, or the promoter. The differential results on promoter targeting as a result of insulator location in the transgene is more likely due to which enhancer is blocked rather than what proteins the insulator helps recruit for the PTS. The model is favored that the insulator is necessary for promoter targeting because of its domain boundary or enhancer-blocking function. For example, the PTS could recognize a special chromatin structure established by an insulator, such as specific histone or DNA modification, and then establish enhancer-promoter interactions over the insulator (Lin, 2004).
Two important implications can be gleaned from the role of an insulator in PTS function. First, the Fab insulator elements may play a role in Abd-B that has not been previously realized. These elements are known to function as boundaries that keep individual regulatory domains functionally independent. The current study suggests that they are also necessary for long-range enhancer-promoter interactions in the Abd-B locus, because without an insulator the PTS would not be able to target the distant, Abd-B downstream enhancers such as IAB5 and IAB7 to their promoter, over long distances. Similar roles for insulators in long-range enhancer-promoter interactions have also been suggested by recent studies of the suHw insulator, where paired suHw could facilitate rather than block distantly located enhancers. The second implication for the initial requirement of an insulator in promoter targeting is epigenetic inheritance. An insulator is required for promoter targeting, but only when the transgene is first generated. In established promoter-targeted lines, the insulator could be deleted without any adverse effect on promoter targeting. These results suggest that the PTS could epigenetically maintain promoter targeting through multiple generations (Lin, 2004).
The most important implication of this study is the heritable nature of PTS function throughout the fly life cycle that is transmittable into the next generations. This is supported by three sets of experiments presented here: enhancer cotargeting, the initial requirement of an insulator, and P-element transposition. Enhancer cotargeting strongly suggests that promoter targeting is a constitutive chromatin effect that is not determined at the onset of transcription, that it is independent of the function of an enhancer, and that it is epigenetically stable throughout the life cycle of a fly. Because promoter targeting is stable over all successive generations, the memory of the initial targeting event is likely epigenetically maintained by the PTS in the germ line. This idea is further supported by the differential requirement of insulators in the initiation and maintenance of promoter targeting. An insulator must be present to obtain transgenic strains that exhibit promoter targeting, but it could be deleted from the targeted strains, with no loss of promoter targeting. There are two alternative explanations for this result: either the germ-line cells have the memory of promoter targeting, or the insulator must have helped the transgene to integrate into a chromosomal location where promoter targeting could occur (Lin, 2004).
More direct evidence for the epigenetic inheritance model comes from the subsequent P-element transposition experiment, which demonstrates that the promoter-targeting effect is remarkably stable even when the transgene is translocated to new locations. In one of the experiments, it was found that when a w-targeted, W59-1 strain is transposed to various locations, including two that moved to a different chromosome, enhancers from all new insertions appear to target the same w promoter, suggesting that a memory is present within the transgene during its transposition to different locations in the germ-line cells. In a separate experiment, new insertions were generated from an original promoter-targeted strain, from which the suHw insulator has been removed by FLP-FRT recombination. Most of the new insertion strains retain promoter targeting to the same Tp promoter. Although there are strain variations to the extent of how stable this memory is, these results suggest that an insulator is not required to maintain the promoter-targeting memory during transposition, and that an insulator probably does not function by directing the transgene to chromosome locations favorable for PTS function, since it is not present in the transgene when being transposed. Thus, in the original promoter-targeted strains, the consistent targeting to the same promoter generation after generation is largely due to epigenetic inheritance (Lin, 2004).
It could still be argued, however, that most P-element transpositions are relatively local where chromatin structures are similar, and hence the same type of promoter targeting can be generated without epigenetic memory. Considering that transpositions of nontargeted strains are also local hops, yet produce a normal distribution of Type I, II, and III strains, indistinguishable from generating promoter-targeted strains by DNA injection, the local chromatin near the original promoter-targeted transgene is less likely to exert a consistent effect on which promoter should be targeted when the P-element is excised and reinserted nearby. Presently, the possibility that a promoter-targeted transgene may associate with other chromatin structures or nuclear sites where similar function is processed cannot be ruled out. Consequently, such structure may dictate where the transgene could insert after being transposed. This possibility, however, is hinged on an epigenetic memory in the germ line, because some of the proteins associated with promoter targeting must be present to direct targeting after P-element reinsertion at a new site. It is concluded that promoter targeting can be memorized in both somatic and germ-line cells and can be transmitted to successive generations (Lin, 2004).
Heritable transcription memory has been reported in the Drosophila Abd-B locus. The 3.7-kb Fab-7 boundary region also contains a cellular memory module (CMM) that can transmit a Polycomb-dependent heritable transcription memory to the offspring that remains stable for a few generations. Similarly, the PTS-mediated promoter targeting appears to be stable in a majority of new insertions. However, once a newly targeting strain is obtained, it is stable in all successive generations, and can be transmitted through both the male and the female germ lines. It is not known what mechanism the PTS uses to transmit promoter targeting memory to subsequent generations. However, it is clearly different from what is used by the CMM, because PTS function is independent of Polycomb. Considering that certain chromatin loop structures remain intact through spermatogenesis, it is likely that the PTS function is transmitted by a special loop structure (Lin, 2004).
An important prediction of this study is that the inheritance of promoter targeting is not due to the transmission of an enhancer activity in the somatic cell into the germ line. It is a process that has to be determined in the germ-line lineage independent of enhancer functions (also in the absence of a history of enhancer activity) by a combination of the PTS, the promoter, the insulator, and, possibly, the local chromatin structure. The promoter-targeting activity detected in the embryo is not a de novo process, but it is built upon what has been determined in the germ-line cells. This prediction is supported by the FLP-FRT analysis and P-element transposition experiments because only germ-line recombination and transposition events could be recovered by the analysis. It is proposed that promoter-targeting memory is maintained by the PTS through either a stable chromatin loop or a special chromatin modification. Such structure or modification could be maintained in somatic tissues and germ-line cells in the absence of enhancer activity. When an enhancer becomes active, this structure or modification could automatically guide the enhancer to the targeted promoter. This unique property of the PTS element underscores the possibility that certain aspects of the mechanism controlling long-range enhancer-promoter interactions in the Drosophila Hox cluster may be programmed in the germ-line chromatin (Lin, 2004).
Polycomb response elements (PREs) are cis-acting DNA elements that mediate
epigenetic gene silencing by Polycomb group (PcG) proteins.
Pleiohomeotic (Pho) and a multiprotein Polycomb core complex (PCC) bind highly
cooperatively to PREs. A conserved sequence motif, named
PCC-binding element (PBE), has been identified
that is required for PcG silencing in vivo. Pho
sites and PBEs function as an integrated DNA platform for the synergistic
assembly of a repressive Pho/PCC complex. This nucleoprotein complex is termed the
silenceosome to reflect that the molecular principles underpinning its
assemblage are surprisingly similar to those that make an enhanceosome (Mohd-Sarip, 2005 ).
Because Pho can directly bind two subunits of the PCC complex, Pc and PH
(Mohd-Sarip, 2002), it was of interest to test whether Pho could
recruit PCC (PRC1 core complex comprising Pc, Ph, Psc, and dRING1) to DNA.
As representative PREs the bxd PRE, located, ~25 kb upstream of the Ubx transcription start site, and the iab-7 PRE, located ~60
kb downstream of the Abd-B promoter, were used. For initial binding studies, focus was placed on Pho sites 4 and 5
within the bxd PRE (Pho4/5-PRE), which are required for PcG silencing in
vivo. Pho, Pho lacking the 22-amino acid Pc- and PH-binding domain (DeltaPBD), Pc,
and PCC were expressed in Sf9 cells using the baculovirus
expression system and were immunopurified to near homogeneity from cell
extracts (Mohd-Sarip, 2005).
To test DNA binding by Pho and PCC, DNA mobility shift assays were performed.
Whereas Pho alone binds weakly to the Pho4/5-PRE, together with PCC, a Pho/PCC/DNA complex was forms very efficiently, resulting in complete saturation of the probe.
In contrast, PCC alone is unable to bind DNA sequence-specifically. Deletion of
the PBD of Pho impairs the synergistic formation of a higher-order Pho/PCC/DNA
complex, revealing the importance of
direct protein-protein interactions between Pho and PCC (Mohd-Sarip, 2005).
To identify the DNA sequences contacted by the Pho/PCC complex,
primer extension DNaseI footprinting assays were carried out. After
addition of PCC to a subsaturating amount of Pho, which by itself does not yield
a footprint, DNA binding is readily detected. The Pho/PCC footprinted area
is very large, comprising ~120 bp, indicative of extensive protein-DNA contacts. As expected, PCC alone is unable to bind DNA sequence-specifically. In
contrast to Pho/PCC, a saturating amount of Pho generates a small footprinted
area of ~40 bp, encompassing the two Pho sites. Next, tests were performed to see whether the cooperation between Pho and PCC also occurred on chromatin templates.
The Drosophila embryo-derived S190 assembly system was used to package the template into a nucleosomal array. Pho alone failed to bind its chromatinized sites. However, DNA binding was greatly facilitated by the addition of PCC,
which by itself is unable to target the PRE sequence. It is noted that no Pho binding to chromatin was detected even at the highest amounts add.
Thus, Pho binding to chromatin appears dependent upon PCC. Because
nucleosomes are not positioned on these templates, the DNaseI digestion ladder
resembles that of naked DNA. Chromatin footprinting requires the use of high
amounts of DNaseI, which completely digests any residual naked DNA in the
reaction (Mohd-Sarip, 2005).
To identify specific PCC subunits that directly contact the DNA, a
DNA cross-linking strategy was used. A radiolabeled Pho4/5-PRE fragment
substituted with bromodeoxyuridine (BrdU) was generated. After binding of Pho and PCC, the resulting protein-DNA complexes were subjected to ultraviolet (UV)
cross-linking. SDS-PAGE analysis, followed by autoradiography, revealed very
strong labeling of Pho and Pc and weaker labeling of Psc or Ph.
The cross-linked PSC and PH could not be resolved well. Because on low percentage gels PSC and PH form a radiolabeled doublet, it is assumed that both proteins bind DNA. No labeling of dRING1 was detected, suggesting that it does not directly contact DNA. Because Pc was strongly cross-linked to DNA and can directly bind Pho (Mohd-Sarip, 2002), tests were performed to see
whether Pc can bind DNA together with Pho. After addition of Pc to a
subsaturating amount of Pho, DNA binding was readily detected.
Pc alone is unable to bind DNA sequence-specifically. Also when Pc
was added to a saturating amount of Pho, the footprinting pattern changed and
was extended, suggesting additional
protein-DNA contacts. Although Pc can cooperate with Pho, the level of
cooperation and DNA area contacted is modest compared with Pho-PCC,
emphasizing the contribution of other PCC subunits (Mohd-Sarip, 2005).
What are the precise DNA sequence requirements for cooperative PRE binding by
Pho and PCC? Within many PREs, the Pho core recognition sequence forms part of a
larger conserved motif (Mihaly, 1998). To determine the
functional significance of these sequence constraints, the effect of
mutations on Pho binding by DNase were examined by footprinting and
bandshift analysis. Whereas the downstream motif
(D.mt) has no effect on Pho binding, mutation of the upstream motif (U.mt)
reduced Pho affinity. As expected, mutation of the core Pho site (C.mt)
abrogates Pho binding. These results suggested that the sequence constraints
directly upstream of the Pho core site reflect an extension of the Pho
recognition site. The sequence downstream of the Pho site, however, appeared to
play no role in Pho binding. Therefore, an attractive possibility was that this
motif might mediate docking of PCC and function as a PCC-binding element (PBE).
To determine whether synergistic Pho/PCC complex assembly is dependent on each
Pho site or the downstream sequence motifs, each Pho
site and putative PBEs was mutated individually. Strikingly, each mutation aborted formation of the
Pho-PCC-DNA complex. Likewise, synergistic binding
of Pho and Pc was also abrogated by PBE mutations. It is concluded
that cooperative DNA binding of Pho and PCC is strictly dependent on the
presence of at least two Pho sites and their juxtaposed PBEs (Mohd-Sarip, 2005).
The conservation of the PBE (Mihaly, 1998) and its
requirement for cooperative DNA binding by Pho and PCC led to a test if it is
also critical for PRE-directed silencing in vivo. The minimal
260-bp iab-7 PRE, for which an extensive collection of control lines has
already been established, was examined. The
iab-7 PRE harbors three Pho/PBE elements, but their spacing and phasing
is very different from that in the bxd PRE. Whether Pho and PCC
bind cooperatively to the iab-7 PRE was tested. In
agreement with the results on the bxd PRE, Pho and PCC synergistically
recognized the iab-7 PRE, resulting in a very large DNaseI footprint,
including all three Pho and PBE elements. Cooperative binding of Pho and PCC was completely abolished by mutations in the three PBEs juxtaposing the Pho sites. Thus, the PBEs are required for Pho/PCC complex formation on both the bxd and the iab-7 PRE (Mohd-Sarip, 2005).
A central problem in understanding epigenetic gene regulation is how specialized
DNA elements recruit silencing complexes to a linked gene. This study has identified the PBE, a small conserved sequence element required for PcG silencing in vivo. These results suggest that Pho sites and their juxtaposed PBEs function as an integrated DNA platform for the assembly of a repressive Pho/PCC complex. In a
previous study, the failure of Pho sequences fused to a heterologous DNA-binding
domain to nucleate the assembly of a silencing complex was interpreted as an
argument against its role as a tether of other PcG proteins.
However, in light of the critical role of the PBE in PcG
silencing, it is not to be expected that artificially tethered Pho can support
PcG complex assembly (Mohd-Sarip, 2005).
Synergistic Pho/PCC/PRE nucleocomplex formation is strictly dependent on the
presence of at least two Pho sites, their accompanying PBEs and protein-protein
interactions between Pho and PCC. The observations revealed a striking
similarity in the design of PREs and enhancers. The cooperative assembly of
unique transcription factor-enhancer complexes, termed enhanceosomes, is also
dependent upon a stereospecific arrangement of binding sites and a reciprocal
network of protein-protein interactions. Thus, the
basic principles governing the assembly of distinct higher-order nucleoprotein
assemblages with opposing activities are surprisingly similar. To reflect the
generality of these rules, it is proposed that PRE-bound PcG silencing
complexes be called silenceosomes (Mohd-Sarip, 2005).
Like enhancers, PREs are complex and their activity involves the combined
activity of distinct recognition elements and their cognate factors. In addition
to Pho/PBE sites, these modules include the (GA)n-element, recognized by GAGA or
Pipsqueak; Zeste sites; and the
recently identified GAAA motif bound by DSP1, a fly HMGB2 homolog (Dejardin, 2005). Finally, histone modifications, including H2A and
H2B (de)ubiquitylation, and H3-K27 or H3-K9 methylation, play a critical role in
PRE functioning. One scenario is that silenceosome formation is
nucleated by direct DNA binding and contextual protein-protein and protein-DNA
interactions. Next, the silenceosome could be stabilized further through
multivalent interactions with the histones guided by selective covalent
modifications. The available evidence strongly suggests that a cooperative
network of individually weak protein-DNA and protein-protein interactions drive
the formation of a PcG silencing complex. It is proposed that the molecular
principles governing silenceosome or enhanceosome formation are very similar (Mohd-Sarip, 2005).
Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer-blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking: Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF (CCCTC-binding factor), with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. This study characterized an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, this study demonstrates that one of the insulators (Fab-8) in the Drosophila Abdominal-B locus mediates enhancer blocking by CTCF. Therefore, the enhancer-blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans (Moon, 2005).
Expression of the eukaryotic genome is controlled by enhancer and silencer elements, both of which can mediate their function from a distance. Insulator elements with enhancer-blocking activity curb enhancer activity, such that only appropriate promoters are activated. The proteins that mediate insulator function have been identified for only a few Drosophila insulator sequences. These are Zw5, BEAF-32, GAGA factor and Su(Hw) (Moon, 2005).
Another perspective on the requirement of insulators comes from the fact that many genes are controlled by several regulatory elements needed for tissue- and cell-specific expression. For example, the Drosophila gene Abdominal-B (Abd-B) contains an extended 3' regulatory region that is functionally subdivided into distinct enhancer domains. Functional separation of the enhancer sequences is achieved by intervening insulators such as Frontabdominal (Fab)-7 and Fab-8. Although both elements have been shown to mediate enhancer-blocking function, the protein involved in this activity has not been described (Moon, 2005).
In sharp contrast to Drosophila, the genome of vertebrates is much more expanded, due primarily to larger distances between genes. Therefore, the need for insulators to separate genes may not seem as pronounced as it is in Drosophila. Indeed, until now, only a single protein, CTCF, has been identified to mediate enhancer-blocking activity. Binding sites for CTCF have been shown to be involved in gene activation, gene repression and enhancer blocking. Furthermore, vertebrate- and mammalian-specific functions, such as X-chromosome inactivation and control of the epigenetic DNA methylation state, seem to involve CTCF (Moon, 2005 and references therein).
Obviously, the function of enhancer blocking has developed during evolution such that Drosophila uses several proteins and mechanisms for enhancer blocking and insulation . However, none of the known Drosophila insulator proteins has a counterpart found to be conserved in vertebrates. Rather, vertebrates use CTCF, which has not previously been found in Drosophila. This study characterizes a Drosophila orthologue of CTCF with similarities to many of the features identified for vertebrate CTCF. Furthermore, a previously characterized Drosophila insulator, Fab-8, mediates enhancer blocking by CTCF in Drosophila as well as in vertebrate cells. Thus, the enhancer-blocking protein CTCF and, probably, the mechanisms of CTCF-driven enhancer blocking are both conserved from Drosophila to humans (Moon, 2005).
FlyBase data entries and cDNA sequence analysis revealed an open reading frame (ORF) coding for a protein similar to vertebrate CTCF with respect to the overall structure. dCTCF contains all of the expected 11 zinc fingers (Zn-fingers), separated by both standard and noncanonical inter-finger linkers. Furthermore, most of the crucial DNA base recognition residues at positions −1, 2, 3 and 6 are identical. Variation in position 6 for fingers #6 and #9 generates a change from alanine or serine to methionine; this is of no consequence for the DNA-binding specificity, as the recognition code is not changed (Moon, 2005).
Similarities in Zn-fingers do not necessarily imply similarities in function. Therefore, whether dCTCF can act as a transcriptional repressor, as has been demonstrated previously for vertebrate CTCF, has been examined. The strongest repressive function has been shown to reside within the combined carboxy-terminal plus Zn-finger domains. Equivalent regions of Drosophila and chicken CTCF (chCTCF) were fused to the yeast GAL4 transcription factor DNA-binding domain. Both Drosophila and chicken GAL4-CTCF fusions repressed reporter gene activity to a similar extent in two different cell lines and in a way comparable with the previously characterized strong repressor GAL4-v-erbA362. These results clearly indicate that dCTCF, like its vertebrate counterpart, has transcriptional repressor activity (Moon, 2005).
In vertebrates, CTCF is ubiquitously expressed (Burke, 2002), apparently functioning as a global transcriptional regulator in all cell types (Ohlsson, 2001). In comparison, dCTCF RNA expression levels were monitered at various stages of fly development. Using in situ hybridization, it was found that dCTCF RNA is present in the cytoplasm of the nurse cells within the fly egg chamber, transported into and distributed uniformly in the developing oocyte and in 0-24 h embryos as a maternal. Later stages show expression in all tissues and stages, revealing that dCTCF is a ubiquitous factor as in vertebrates. Location of dCTCF protein is clearly nuclear, exemplified by the nuclear staining of syncytial blastoderm embryos with dCTCF-specific antibodies (Moon, 2005).
To extend the comparison of vertebrate and Drosophila CTCF, in vitro-translated Drosophila and vertebrate CTCF were tested for binding to several previously characterized vertebrate CTCF targets (CTS). The sequences tested included the CTS of the FII insulator element of the β-globin gene, the APP gene, the myc genes and the mouse ARF promoter. With the exception of the two myc FPV and A sites, all the other sequences bound chicken and Drosophila CTCF similarly (Moon, 2005).
A methylation-interference assay was used to determine whether both proteins contact the same guanidine nucleotides on a given target DNA site. Both Drosophila and human CTCF were found to contact the same nucleotides on the β-globin FII insulator fragment. These results indicate that, despite considerable overall sequence divergence, fly and human CTCF show a striking degree of functional conservation with respect to DNA binding (Moon, 2005).
To identify potential Drosophila CTCF regulatory targets, an in vitro screen was performed for CTCF-binding sites, and the Fab-8 element, for which enhancer-blocking and boundary function have been shown, was found. This sequence is situated in the Abd-B locus, separating and insulating enhancer domains infraabdominal-7 (iab-7) from iab-8. Since the protein involved in this mechanism was unknown, and since vertebrate CTCF mediates enhancer-blocking activity, whether dCTCF might have a similar role in the context of the Fab-8 element was tested. In vitro binding of dCTCF to Fab-8, as determined by methylation interference, suggested two binding sites for CTCF. Binding site mutations resulting in single-site mutations (mut1 or mut2) and in a double-site mutation (mut1+2) were used for electrophoretic mobility shift assay (EMSA). The wild-type Fab-8 element generates two retarded bands corresponding to a different mobility of the same DNA molecule occupied by CTCF at one of the two closely spaced CTS sequences. These different mobilities are probably caused by a site-specific DNA bending, which has also been observed on other dual binding sites, such as the H19 locus. Excess protein generated a slow mobility complex only resolved after a long run of the gel, reflecting binding of CTCF to both sites (Moon, 2005).
To test in vivo dCTCF binding to this important element, crosslinked chromatin was prepared from Drosophila embryos and CTCF-occupied sites were precipitated with the anti-dCTCF-C antibody. PCR primers for the Fab-8 sequence identified specifically precipitated chromatin, whereas primers against a different non-dCTCF-binding site and mock-precipitated chromatin resulted in no signal (Moon, 2005).
To test the functional similarity between dCTCF and vertebrate CTCF, enhancer blocking of Fab-8 was analyzed in vertebrate K562 cells. In comparison to the known enhancer-blocking effect mediated by the FII sequence, a similar reduction in colony numbers mediated by Fab-8 was seen. More importantly, specific abrogation of CTCF binding by the double mutation, mut1+2, resulted in loss of enhancer blocking (Moon, 2005).
The crucial test for enhancer-blocking activity of dCTCF had to be carried out in flies. Therefore, a vector was used with two regulatory regions containing the iab-5 enhancer from the Abd-B locus and two copies of the minimal twist enhancer, PE, directing an additive pattern of expression when placed between divergently transcribed white and lacZ reporter genes. The iab-5 enhancer directs expression in the posterior one-third of the blastoderm stage embryo, whereas the 2 × PE enhancer activates transcription in the ventral-most region where twist is normally expressed. Enhancer elements are enhancing both the white gene as well as the lacZ gene. Altered patterns of transcription were observed when the 1 kb spacer sequence was replaced by the 680 bp Fab-8 element. On the white promoter, the iab-5 activity was completely abolished (shown as the lack of staining in the iab-5 activity region), while the 2 × PE enhancer was still activating the white gene. The lacZ promoter, conversely, could be activated only by the proximal iab-5 but not by the distal 2 × PE. This result suggests that the Fab-8 fragment blocks the respective distal enhancer for both the white and the lacZ promoters. When the CTCF sites were mutagenized (mut1+2), the iab-5 activity on white was partly restored. Similarly, the 2 × PE element again directed the transcription of the lacZ gene. Chromatin/CTCF immunoprecipitation revealed specific CTCF binding to the Fab-8 element of the enhancer-blocking vector, whereas binding to the Fab-8 mut element was clearly reduced. This correlation between strong CTCF binding and full enhancer-blocking function indicates that the activity of Fab-8 is at least partly mediated by CTCF and that dCTCF, similar to vertebrate CTCF, confers enhancer blocking (Moon, 2005).
Thus, at least one enhancer-blocking protein (CTCF) in Drosophila and vertebrates is conserved with a similar enhancer-blocking function. In addition to enhancer blocking, mammalian CTCF has gained functions involving the control of epigenetic states in the context of imprinted genes and X-chromosome inactivation (Moon, 2005 and references therein).
Insulator or enhancer-blocking elements are proposed to play an important role in the regulation of transcription by preventing inappropriate enhancer/promoter interaction. The zinc-finger protein CTCF is well studied in vertebrates as an enhancer blocking factor, but Drosophila CTCF has only been characterised recently. To date only one endogenous binding location for CTCF has been identified in the Drosophila genome, the Fab-8 insulator in the Abdominal-B locus in the Bithorax complex (BX-C). This study carried out chromatin immunopurification coupled with genomic microarray analysis to identify CTCF binding sites within representative regions of the Drosophila genome, including the 3-Mb Adh region, the BX-C, and the Antennapedia complex. Location of in vivo CTCF binding within these regions enabled construction of a robust CTCF binding-site consensus sequence (AGGNGGC, the same ase mammalian CTCF). CTCF binding sites identified in the BX-C map precisely to the known insulator elements Mcp, Fab-6, and Fab-8. Other CTCF binding sites correlate with boundaries of regulatory domains allowing localization of three additional presumptive insulator elements; 'Fab-2', 'Fab-3', and 'Fab-4'. With the exception of Fab-7, these data indicate that CTCF is directly associated with all known or predicted insulators in the BX-C, suggesting that the functioning of these insulators involves a common CTCF-dependent mechanism. Comparison of the locations of the CTCF sites with characterised Polycomb target sites and histone modification provides support for the domain model of BX-C regulation (Holohan, 2007).
The multiple zinc-finger DNA-binding protein CTCF is known to be required for the enhancer blocking action of vertebrate insulators, and a clear role for CTCF in the regulation of endogenous gene expression has been demonstrated at the imprinted Igf2. The mode of action of CTCF is, however, still unclear, although several studies have implicated CTCF in the formation of higher-order chromatin structure. CTCF molecules can interact to form clusters and thereby may mediate the formation of chromatin loop domains. Partitioning of regulatory elements into independent chromatin loop domains is postulated to play a key role in the interactions between enhancers and promoters. The CTCF homolog of Drosophila is required for the insulator function of the Fab-8 element in the BX-C. This observation has opened up the prospect of utilising the wealth of genetic and molecular characterisation of BX-C transcriptional regulation for the analysis of CTCF function. This study used ChIP-array to investigate CTCF binding sites in regions of the Drosophila genome with a particular focus on the BX-C. CTCF not only associates with the Fab-8 insulator, but also with other mapped boundary elements, Fab-6 and Mcp. In addition, CTCF sites are located at other postulated boundaries within the BX-C; 'Fab-2', 'Fab-3', and 'Fab-4'. This provides a precise mapping of regulatory domain boundaries and a specific molecular foundation for the domain model of BX-C regulation (Holohan, 2007).
It is noted that the Fab-7 boundary may differ from the other characterised boundaries in the BX-C since no strong Patser match was found to the CTCF consensus in the functionally mapped Fab-7 boundary element. Although Fab-7 was not demonstrably enriched in the ChIP-array, significant CTCF association with Fab-7 was found in the more sensitive PCR-base ChIP assay. Given the lack of a strong Patser match (ChIP enrichment) this may suggest an indirect association. No CTCF site was seen between the abx/bx and the bxd/pbx regulatory elements. However, these elements are separated by a long distance, and it is not clear whether they require insulation (Holohan, 2007).
According to the domain model, the parasegment-specific regulatory domains that control the expression patterns of the Ubx, abd-A, and Abd-B genes of the BX-C are initially activated in appropriate parasegments by the early pattern-forming genes acting on initiator elements. Each regulatory domain is predicted to contain a particular initiator element, tuned to respond to a specific combination of gap and pair-rule gene products, thus activating the regulatory domain in the appropriate set of parasegments. This activation would be read by maintenance elements consisting of PREs that thereafter autonomously maintain each regulatory domain in either the OFF (silenced) or ON (active) state. Within a domain in the ON state, enhancers present in that domain would be able to engage with the relevant gene promoter and regulate expression of the gene. Boundary elements that flank each domain are proposed to restrict the effects of the initiator and maintenance elements to a single domain (Holohan, 2007).
Although boundary elements are postulated to have the common property of insulating the regulatory domains, no sequence similarity between the mapped boundary elements has been reported until now. This study shows that a set of these boundary elements contain CTCF binding sites and bind CTCF in vivo. CTCF has been shown to be required for the insulator activity of Fab-8, and it seems likely that CTCF will also be a required component at the other boundary elements. In support of this suggestion, it was found that the CTCF sites are well conserved within the sequenced insect genomes. The observation that CTCF sites flank a set of regulatory domains in the BX-C, together with the vertebrate studies that suggest that CTCF can mediate the formation of chromatin loops (Splinter, 2006; Yusufzai, 2004) supports the idea that interaction between CTCF sites may organise these domains into chromatin loops. However, how such a looping mechanism enables the autonomy of the individual regulatory domains and facilitates appropriate enhancer/promoter interactions is still unclear (Holohan, 2007).
A key feature of the domain model is the relationship between the boundary and maintenance elements. For the domains to be capable of independently being set to the ON or OFF state, the range of influence of PREs needs to be restricted by the domain boundaries. Each domain would require at least one PRE. Precise mapping of in vivo CTCF binding sites has enabled examination of their relationship with Polycomb target sites. In strong support of the domain model, it was found that the domains demarcated by CTCF sites contain Polycomb target sites. Indeed, an intimate relationship was found between CTCF and Polycomb binding sites for 'Fab-4', Mcp, Fab-6, and CTCF site 'C'. This fits with previous functional mapping indicating that boundary elements and PREs are closely associated at Fab-7, Fab-8, and Mcp. This arrangement would impose a polarity on the spread of chromatin modification from the PRE, such that modification may start at the PRE abutting one boundary and spread across the domain in one direction towards the next boundary. At the boundaries, CTCF may play many possible roles. It could participate in boundary element function allowing the independence of chromatin domains by acting as a chromatin insulator blocking the spread of chromatin modification. However, at the chicken ß-globin locus, the chromatin boundary appears to be separable from the CTCF binding site. Another possibility is suggested by that fact that CTCF has been demonstrated to block the progression of RNA polymerase. This could potentially play an important role at boundaries in the BX-C to enable the independent function of PREs in neighbouring domains. There is considerable evidence that transcription through PREs may control their state, and many noncoding RNAs have been detected in the regulatory regions of the BX-C. One role for CTCF could be to act as a barrier to such noncoding transcription, preventing transcripts arising in one regulatory domain from crossing into the neighbouring domain and affecting the PRE state. Such a role would be consistent with the observed location of CTCF sites in this region, as a CTCF site closely abuts one side of each PRE (Holohan, 2007).
The individual regulatory domains must not only be able to act autonomously to set and maintain their activity state, but they must also be able to interact appropriately with the relevant gene promoters. Boundaries may play a role in this, and recently a long-range interaction has been demonstrated between Fab-7 and the Abd-B-RB promoter. This interaction was associated with lack of Abd-B expression, but similar interactions, bringing in appropriate enhancers, may also activate expression. The ability of CTCF to form clusters may facilitate such interactions, and it is intriguing that there are CTCF sites not only at the boundaries but also close to Abd-B promoters; the CTCF site 'B' is 300 bp upstream of the Adb-B-RB promoter. Clustering of boundaries together with Abd-B promoter sequences may enable interaction between the promoter and enhancers in domains in the ON state. The clustering may also be more selective; in S2 cells, which specifically express Abd-B-RB, several boundaries are embedded in chromatin bearing the repressive H3K27me3 modification, whereas Fab-8, CTCF site 'B', and the Abd-B-RB promoter are in the unmodified, presumably 'open', chromatin domain. It could be speculated that the expression of Abd-B-RB in these cells might be facilitated by interaction of the CTCF sites in the 'open' domain, Fab-8 and site 'B', enabling Fab-8 to bring appropriate enhancers to the Abd-B-RB promoter (Holohan, 2007).
ChIP-array analysis of CTCF genomic sites can be compared with ChIP-array analysis of binding sites for another Drosophila insulator-binding protein, Su(Hw). CTCF and Su(Hw) are both multi-zinc- finger DNA-binding proteins, and in both cases relatively long (~20 bp) consensus binding sites have been identified. In contrast to most DNA-binding proteins, it was found that strength of match to the consensus binding sites is a good predictor of in vivo occupancy. It was also investigated whether the data indicate any collaboration between CTCF and Su(Hw). This seemed an attractive possibility since removing Su(Hw) function in vivo has little effect; su(Hw) null mutant flies are female-sterile but viable. Also, the insulating activity of Fab-8 was significantly reduced when the CTCF sites were mutated but not completely abolished. However this study found no evidence for general colocalisation between CTCF and Su(Hw). A total of 60 Su(Hw) sites were identified in the Adh region, and only one of the fragments covering this region contained both CTCF and Su(Hw) sites. The single CTCF site identified in the achaete-scute complex was also some distance from the two Su(Hw) sites found. Subsequent ChIP-array analysis in the BX-C led to the identification of only one Su(Hw) site within the entire BX-C region, in a location devoid of CTCF binding sites. Indeed while the BX-C appears relatively enriched in CTCF sites compared to the Adh region, the converse is true for Su(Hw). For CTCF there are 4.7 sites/100 kb in the BX-C and 1.7 sites/100 kb in the Adh region, whereas for Su(Hw) the BX-C is depleted in sites with only 0.29/100 kb in comparison to 2.7/100 kb in the Adh region. Clearly, although CTCF and Su(Hw) both possess insulating ability, their sites of action do not correlate and there is no evidence from this analysis, covering approximately 3% of the Drosophila genome, for cooperative activity (Holohan, 2007).
By comparing the sequences of ChIP-enriched fragments a strong Drosophila consensus CTCF binding site was identified. Analysis of vertebrate CTCF target sequences leads to a proposal that vertebrate CTCF also binds to a similar consensus sequence. These findings do not support the current view that CTCF binds to divergent DNA sequences by engaging different subsets of the zinc fingers. Indeed, the binding site revealed here has been previously noted. A CTCF binding site has been identified in the chicken β-globin insulator, and sequence comparisons between this site and other known CTCF sites identified a conserved 3' region, the mutation of which completely abolished CTCF binding and enhancer blocking. This comparison was extended to include the Dm1 sites, mouse H19 DMD4 and DMD7 and human MYC A, and again identified a conserved region within the larger approximately 50-bp DNase footprint for each site. It is this conserved region that corresponds to the vertebrate CTCF site found here. Very recently, an analysis of CTCF binding in the human genome has generated a vertebrate CTCF consensus site (Kim, 2007), and a CTCF consensus has also been derived from analysis of conserved regions in the human genome. Both these sites are very similar to the consensus identified in this study; in particular they share the strong features of the CC at positions 1 and 2, the AG at positions 6 and 7, and the GGC at positions 10, 11, and 12. Overall, these findings indicate that CTCF in both Drosophila and vertebrates binds to a single core consensus sequence (Holohan, 2007).
In summary, ChIP-array analysis has enabled construction of a CTCF binding site consensus. Mapping of genomic binding sites leads to a proposal that all known or predicted insulators in the BX-C (with the possible exception of Fab-7) function in a CTCF dependent manner (Holohan, 2007).
Insulator sequences guide the function of distantly located enhancer elements to the appropriate target genes by blocking inappropriate interactions. In Drosophila, five different insulator binding proteins have been identified, Zw5, BEAF-32, GAGA factor, Su(Hw) and dCTCF. Only dCTCF has a known conserved counterpart in vertebrates. This study found that the structurally related factors dCTCF and Su(Hw) have distinct binding targets. In contrast, the Su(Hw) interacting factor CP190 largely overlaps with dCTCF binding sites and interacts with dCTCF. Binding of dCTCF to targets requires CP190 in many cases, whereas others are independent of CP190. Analysis of the bithorax complex revealed that six of the borders between the parasegment specific regulatory domains are bound by dCTCF and by CP190 in vivo. dCTCF null mutations affect expression of Abdominal-B, cause pharate lethality and a homeotic phenotype. A short pulse of dCTCF expression during larval development rescues the dCTCF loss of function phenotype. Overall, this study demonstrates the importance of dCTCF in fly development and in the regulation of abdominal segmentation (Mohan, 2007).
The CP190 protein contains three classical C2H2 zinc-finger motifs and an N-terminal BTB/POZ domain. Both domains could potentially be involved in chromatin binding. In contrast, chromatin binding might be achieved by interaction with other factors, such as dCTCF. A possible interaction of dCTCF with CP190 was tested using co-immunoprecipitation. Precipitation of CP190 from Schneider cell extracts resulted in the detection of dCTCF. To confirm the interaction a FLAG-dCTCF fusion protein was expressed in Schneider cells and precipitated with either an antibody against CP190 or an antibody against FLAG. The CP190 precipitate contained endogenous dCTCF as well as FLAG-dCTCF in the same ratio as the input, suggesting that both dCTCF proteins are similarly associated with CP190. Furthermore, the reverse experiment using FLAG precipitation demonstrated that dCTCF and CP190 interact in vivo (Mohan, 2007).
Because CP190 and dCTCF colocalize on polytene chromosomes and interact in vivo, it was asked whether the overall amount of dCTCF protein might be changed in CP190-deficient third instar larvae. A Western blot analysis of both Cp1901 homozygotes (deficient in CP190) and wild-type larval extracts showed that the amount of dCTCF is reduced in Cp1901 homozygotes (Mohan, 2007).
Next it was of interest to know whether the reduced amount of dCTCF caused by the loss of CP190 affects dCTCF binding on the polytene chromosomes. It was found that the total number of dCTCF labeled sites is reduced in the Cp1901 mutant, whereas the number of CP190 sites was not affected by dCTCF mutants. The analysis of dCTCF binding in the two hypomorphic mutants CTCFEY15833/CTCFEY15833 and GE24185/GE24185 revealed that that the number of bound sites is reduced to about 50% and 25%, respectively. By close inspection of the chromosomes it was found that the set of dCTCF sites missing in the CP190 or in the dCTCF mutants overlap but are not identical. Thus, different sites vary in their requirement for CP190/dCTCF cooperation (Mohan, 2007).
Insulator elements with enhancer blocking activity establish independent regulatory domains. An analysis of binding sites (CTS) for the enhancer blocking factor dCTCF on salivary gland polytene chromosomes resulted in the identification of several hundred sites bound by dCTCF. All of these sites are found in interbands, and when inspected more precisely are often at the borders of interbands next to bands. Interbands harbor active housekeeping genes or regulatory regions of inactive genes, whereas bands contain the bodies of inactive genes. Interbands and bands differ in chromatin composition and modification. Thus, there is a clear border between interbands and bands. Any factors generating functional chromatin boundaries would be expected to be localized to the interband/band transition. This is not only the case for dCTCF, as a similar location has been found for Su(Hw). Also, BEAF-32 and Zw5 are located in interbands at hundreds of binding sites throughout the genome (Mohan, 2007).
The obvious question was whether dCTCF has a redundant function and therefore similar targets as the other Drosophila enhancer blocking factors. No significant colocalization of dCTCF with either BEAF-32 or with Su(Hw) on polytene chromosomes was detected. This may provide an explanation of how an organism with a small genome, such as Drosophila, can prevent promiscuous enhancer interaction with any nearby gene. Apparently, an elaborate system of different enhancer blockers and barrier factors fulfills the insulation of regulatory units (Mohan, 2007).
The biochemical composition and function of insulator complexes involving Su(Hw) have been studied in detail. The best studied binding site is the gypsy transposon with a 350-bp sequence containing 12 binding sites for Su(Hw). A functional complex of Su(Hw), Mod(mdg4)67.2, CP190, and possibly other factors has been documented. Although there is no colocalization of Su(Hw) with dCTCF on polytene chromosomes, and only partial colocalization with Mod(mdg4), it was of interest to examine whether CP190 plays a role in dCTCF function. Vertebrate CTCF is a centrosomal factor during mitosis and a nuclear protein during interphase, and that CP190 (centrosome binding protein) is associated with centrosomes as well. CP190 is essential for viability, but is not required for cell division (Butcher, 2004). CP190 knockdown in Schneider cells has no effect, whereas a null mutation in flies leads to pharate lethality. A similar phenotype is seen after dCTCF depletion in Schneider cells and in the pharate lethality in flies. The centrosomal function of CP190 is not required for the insulator activity in the context of Su(Hw) bound to gypsy. The localization of CP190 on polytene chromosomes overlaps with sites bound by Su(Hw) or by Mod(mdg4)67.2. In addition, CP190 is found at loci devoid of Su(Hw) or Mod(mdg4)67.2, suggesting that other factors might recruit CP190 to these sites. There is a significant overlap in dCTCF with CP190 binding sites. A functional dependence is seen, because at many sites binding of dCTCF depends on CP190. Although there is an overall reduction in the dCTCF amount observed in the CP190 mutant, differences in dCTCF occupancy in dCTCF and CP190 mutants indicate a discrimination between CP190-independent and -dependent sites. Furthermore, the previously characterized insulator Fab-8 is impaired in the absence of dCTCF and by the reduction of CP190 (Mohan, 2007 adn references therein).
Another perspective on the requirement of insulators comes from the fact that many genes are controlled by several regulatory elements that are required for tissue and cell-specific expression. A prominent example is the Drosophila BX-C. This is one of two Hox gene clusters, which contain regulator genes controlling development. The BX-C is responsible for the correct specification of the posterior thorax segment (T3) and all of the abdominal segments. Within BX-C, only three protein coding genes, Ubx, abd-A and Abd-B, are responsible for the segment-specific development of organs and tissues. On the other hand, nine separate groups of many mutations are affecting segment-specific functions. The borders of some of these domains are genetically defined by elements Fab-6, Fab-7, Fab-8 and by Mcp. Proteins involved in such a functional separation are the GAGA factor in case of the Fab-7 element, and dCTCF for the Fab-8 sequence. Recently, it has been demonstrated that six of the BX-C domain junctions are bound by dCTCF (Holohan, 2007). Consequently, if these sites contribute to boundary function, gene activity within this locus should be changed. Indeed, a homeotic phenotype and a reduced expression of Abd-B was found in larval nerve cord. If dCTCF plays a central role in separating the different regulator domains in the BX-C and elsewhere in the genome, it is difficult to predict the dCTCF phenotype. The situation could be complicated as the three BX-C genes are controlling realizator genes as well as other regulators. Furthermore, individual BX-C genes repress others, for example Abd-B as well as the miRNA iab-4 and bxd expression repress Ubx. In addition, other factors, such as CP190 and perhaps additional unknown factors may contribute to the enhancer blocking function of dCTCF. For all of the CTS in the BX-C, dCTCF and CP190 binding was found. Although both factors clearly interact as seen by co-immunoprecipitation, CP190 may contact other DNA-bound factors as well, or may be directly targeted to chromatin (Mohan, 2007).
Thus, dCTCF shares several biochemical and functional features with Su(Hw), but is clearly targeted to dCTCF-specific sites. Overall, this study has shown that dCTCF is important for fly development, and has important functions in the regulation of abdominal segmentation (Mohan, 2007).
Specific targeting of the protein complexes formed by the Polycomb group of
proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. The grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, it is proposed that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity (Blastyak, 2006).
The iab-7 PRE lies next to the Fab-7 boundary, a chromatin domain insulator element between the neighboring iab-6 and iab-7 cis-regulatory domains of BX-C. Fab-7 ensures the functional autonomy of these cis-regulatory domains; iab-7 is inactive in the sixth abdominal segment (A6), where iab-6 is active, while iab-7 is activated in segment A7. A large set of internal BX-C deficiencies is available, making this region ideal for genetic studies (Blastyak, 2006).
Class II deletions, which remove only the boundary region, fuse the otherwise intact cis-regulatory elements iab-6 and iab-7. The consequence of this fusion is that in some A6 cells iab-6 is inactivated by iab-7, while in some other A6 cells iab-6 ectopically activates iab-7. As a result, A6 will become a mixture of cell clones with either A5 or A7 identity. Due to the fact that the Abd-B gene, the expression of which is controlled by these cis regulators, is haploinsufficient, such transformations are evident even under heterozygous conditions. Class I deletions, which remove both the Fab-7 boundary and the adjacent iab-7 PRE, transform A6 into a perfect copy of A7, suggesting that in the case of class II deletions it is the iab-7 PRE that mediates the inactivation of iab-6 in A6; thus, the inactivation may depend on Pc-G-mediated silencing. Indeed, if a class II deletion is combined with some, but not all, Pc-G mutations, the resulting phenotype is indistinguishable from that of class I deletions. Based on this result, it should be possible to identify mutations in factors that specifically interact with the iab-7 PRE as enhancers of the phenotype of class II deletions (Blastyak, 2006).
Accordingly, several X-ray mutagenesis screenings were performed with the class II allele Fab-72. Among the enhancer mutants, one complementation group, represented by five alleles in the collection, is described here. Two alleles are associated with a cytologically visible breakpoint in 54F, and deficiency mapping placed the locus between the proximal breakpoints of the Pcl11b and Pcl7b deletions. Previously, four complementation groups were isolated within this interval. Noncomplementation with alleles of one of the four complementation groups showed that new mutant alleles were isolated of the previously described gene grainyhead (grh). The previously isolated grh alleles, including the molecularly characterized amorphic allele B37, are also strong Fab-72 enhancers, indicating that loss-of-function grh mutations affect the function of the iab-7 PRE (Blastyak, 2006). Genome-wide prediction has indicated that the occurrence of the same limited set of consensus motifs can fairly accurately predict the PRE function of a DNA sequence (Ringrose, 2003). This observation suggests that many, if not all, PREs use the same set of DNA binding proteins. One of the frequently occurring consensus sequences within PREs is a poly-T motif. Many, although not all, GRH binding sites are T rich, and the current studies indicate that at least in some cases the poly-T consensus sequence may be a binding site for this protein. However, like other DNA binding proteins involved in PRE function, GRH alone cannot explain the specificity of targeting, since its function is not limited to PREs. In other contexts, GRH acts as a transcriptional activator. The fact that an array of distinct sequence motifs is required to accurately predict PREs probably means that there is no single major targeting activity. Indeed, in the case of the engrailed PRE it was demonstrated that all binding sites of DNA binding proteins are equally important for silencing activity. Identification of GRH as a PRE-related DNA binding protein and, in particular, its
cooperative interaction with another member of this group both in vivo
and in vitro may help in understanding the targeting of PC-G to PREs
during development (Blastyak, 2006).
A cooperative interaction between GAF (Trithorax-like) and PHO has been demonstrated (Mahmoudi, 2003). In contrast to the case of GRH and PHO, cooperation between GAF and PHO is independent of the physical interaction between the two proteins and requires a nucleosomal context. Although the physical basis of this cooperative interaction is not understood, it also suggests that cooperativity may be an important principle in the organization of nucleoprotein assembly at PREs (Blastyak, 2006). What could be the impact of cooperativity on PC-G targeting? Theoretically, one of the most significant problems encountered by a DNA binding protein is the huge excess of potential binding sites in the genome, including both functional sites and pseudosites. It can be assumed that if any of the DNA binding proteins involved in targeting are present in limited amounts in the nucleus, then their binding occurs only at the highest-affinity sites, where a combination of certain binding sites facilitates their cooperative binding. Several observations contradict this simple model. First, if the amount of these DNA binding proteins were limited, their mutations would be expected to result in strong haploinsufficient phenotypes, which is not the case. Second, studies on the DNA binding proteins EVE, FTZ, and GAF demonstrated that in vivo they also bind to genes that are not controlled by them. These functionally irrelevant sequences may represent pseudosites, and the relatively low level of binding at these sites may indicate a low binding affinity. Thus, it appears that restricted binding site occupancy of DNA binding proteins is not necessary for specificity in gene regulation. Likewise, even though the DNA binding proteins present on PREs may bind to nonfunctional sites, it is likely that the functionally relevant high-affinity sites are distinguished from pseudosites in vivo by the unique arrangement of distinct, stably bound cooperative partners. However, although in this model of targeting of PRC1 to the iab-7 PRE, cooperativity at the level of the DNA binding proteins is critically required for binding stability, by itself it is insufficient to provide the required specificity of the targeting process (Blastyak, 2006).
In contrast to the DNA binding components, other constituents of the silencing complex appear to be limiting factors. This is suggested by the fact that most Pc-G genes were identified either on the basis of their characteristic haploinsufficient phenotypes or on the basis of their dominant genetic interaction with other known Pc-G members. The number of potential PRE sequences is also relatively small, as a genome-wide survey estimated it to be not more than a few hundred in Drosophila. This brings us to the question of how the abundant DNA binding proteins link the limited amount of PC-G complexes to the low-frequency target sites with high specificity (Blastyak, 2006). The first clue comes from studies showing that all of the PRE DNA binding proteins have the ability to interact with various PC-G proteins that are all subunits of the same preformed protein complex, PRC1. These interactions appear to be weak by themselves, as illustrated by the fact that although the occurrence of these interactions can be demonstrated by using short protocols like immunoprecipitation, the resulting complexes do not survive nonequilibrium methods used for traditional biochemical purification of protein complexes. The consequence of the cooperativity at the level of DNA binding proteins is that the otherwise weak interaction surfaces are integrated into a stable composite surface that can serve as a high-affinity docking site for the limited amount of PRC1 complex. In the model, this second level of cooperativity would provide targeting specificity (Blastyak, 2006).
Notably, the same DNA binding proteins involved in PC-G targeting can separately participate in weak interactions with various other protein complexes involved in processes unrelated to, or the opposite of, Pc-G-dependent silencing, such as TFIID-dependent transcription or chromatin remodeling by SWI/SNF. Based on the available data, interaction surfaces of any such complex are not shared by these DNA binding proteins, and according to this model, their concerted recruitment to PREs is unlikely. Also, in agreement with the experimental data, this model predicts that in the absence of DNA none of the DNA binding proteins will be able to interact stably with the complex to be recruited. The integration of several weak protein-protein interaction modules into a single entity is a prerequisite for the complex to dock on chromatin (Blastyak, 2006).
It has been shown that transcription through the iab-7 PRE
displaces PC-G proteins and results in concomitant recruitment of the
TRX and BRM proteins. Thus, iab-7 PRE appears to be a switchable element and the potential, for example, of PHO to interact with protein partners having a function that is the opposite of PC-G silencing might be realized under certain circumstances. There is insufficient data to explain the mechanism underlying this switch. One possibility is that binding of some DNA binding proteins to DNA or to their interacting partners is modified by posttranslational modifications, as it was shown in the case of the human homologue of Grh. According to the model, even the modification of a single actor (e.g., GRH) can radically influence the overall assembly configuration of the targeting complex and might be responsible for the dynamic nature of the iab-7 PRE (Blastyak, 2006).
This model shows remarkable similarity to the functional and structural organization of enhanceosomes. For example,
multimerization of the binding sites of any of the DNA binding proteins involved in beta interferon (IFN-ß) enhanceosome formation does not reproduce faithfully the virus inducibility of the intact enhancer. Instead, these synthetic enhancers respond promiscuously to inducers that are normally not involved in regulation of the IFN-ß gene. The molecular basis of the selective inducer response of the enhanceosome is established by the following cooperative interactions. First, in their original context, the mutually cooperative interactions at the level of DNA binding proteins promote binding stability. Second, on the resulting spatially arranged protein surface, each DNA binding protein contributes to the recruitment of a protein complex through interactions with one of its subunits. It is concluded that the integration of different, hierarchical levels of cooperativity could be a general principle in the targeting of protein complexes to chromatin (Blastyak, 2006).
The validity of the enhanceosome model has already been demonstrated by in vitro reassembly of the IFN-ß enhanceosome with well-defined recombinant components. In vitro studies with a nucleosomal template have provided valuable insights into the role of PRC1 in regulation of the chromatin structure. However, in this experimental system the excess of PRC1 and nonspecific DNA binding of PRC1 complex members overcomes the problem of targeting. An initial attempt to reconstitute cooperativity at the level of DNA binding proteins failed, possibly because the simultaneous presence of several other DNA binding proteins is required for cooperative assembly. Until these components of PREs are identified, it is likely that PC-G targeting cannot be faithfully reconstituted in vitro. Hopefully, the identification of as-yet-unknown DNA binding protein components of PREs, together with the conceptual framework presented here, will facilitate these studies (Blastyak, 2006).
Recent results showed that in vivo stable recruitment of PC to the Ubx PRE critically depends on the presence of the E(Z) protein. E(Z) is a member of a PC-G complex, which is distinct from PRC1, and possesses histone methyltransferase activity. These findings led to a model wherein, upon binding of the EZ complex, its enzymatic activity could provide the mark for the specific targeting of PRC1. Hence, recruiting of PRC1 would only indirectly depend on sequence-specific DNA binding proteins, as they primarily act as recruiters of the E(Z) complex, but not PRC1. Contrary to the predictions of this model, it was found that although mutations in PRC1 complex members are similarly strong dominant enhancers of the Fab-72 phenotype as grh and pho, amorphic E(z) alleles in heterozygous condition are not. Thus, the current results indicate a rather intimate link between these DNA binding proteins and PRC1 complex members. However, it is still possible that in a nucleosomal context the histone mark could provide an additional constituent for binding whose presence can be critical in vivo in certain tissues. Certain PC-G group members have a tissue-specific phenotype, and GRH is also not ubiquitously expressed, which supports this notion (Blastyak, 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).
Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. Drosophila homeotic gene clusters have been examined by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. These results suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements (Mito, 2007).
Chromatin can be differentiated by the replication-independent replacement of one histone variant with another. For example, histone H3.3 is deposited throughout the cell cycle, replacing H3 that is deposited during replication. Unlike replication-coupled assembly of H3, which occurs in gaps between old nucleosomes on daughter helices, the insertion of H3.3 is preceded by disruption of preexisting histones during transcription and other active processes. H3.3 replacement profiles resemble those for RNA polymerase II, which suggests that gradual replacement of H3.3 occurs in the wake of transiting polymerase to repair disrupted chromatin. This study asked whether histone replacement and nucleosome occupancy are also distinctive at cis-regulatory elements (Mito, 2007).
Log-phase Drosophila S2 cells were induced to produce biotin-tagged H3.3 for two or three cell cycles. DNA was extracted from streptavidin pull-down assay and input material, labeled with Cy3 and Cy5 dyes, and cohybridized to microarrays. To provide a standard, biotin-tagged H3-containing chromatin was profiled in parallel. Analysis of H3.3/H3 levels over the entire 3R chromosome arm revealed that the 350-kb bithorax complex (BX-C) region displays the lowest H3.3/H3 ratio of any region of comparable size on 3R, and the Antennapedia homeotic gene complex (ANTP-C) also displays an unusually low H3.3/H3 ratio. Low H3.3/H3 ratios at the homeotic gene clusters are attributable to infrequent histone replacement, and not to low nucleosome occupancy, because H3.3 levels at the BX-C are far below the median for all of 3R, whereas H3 levels are slightly above the median overall. Even the heterochromatic chromosome 4 includes only shorter (100-kb) stretches that are as depleted in H3.3 as the BX-C (Mito, 2007).
A close-up view of the BX-C iab region reveals the presence of several prominent H3.3 peaks. Notably, the seven highest peaks correspond to the functional boundaries of the seven proximal-to-distal cis-regulatory domains that regulate the abd-A (iab2 to iab4) and Abd-B (iab5 to iab8) homeotic genes successively from anterior to posterior in the abdomen. Conspicuous peaks of H3.3 also correspond to the bxd Polycomb response element (PRE) and to promoters within the Abd-B gene, which is known to be active in S2 cells. Therefore, each of the most prominent H3.3 peaks in the region corresponds to a previously defined cis-regulatory element. These findings are likely to be general, because in budding yeast, promoters and boundaries are also sites of intense histone replacement (Mito, 2007).
A characteristic feature of both boundaries and PREs in the BX-C is that they span deoxyribonuclease I (DNaseI)-hypersensitive sites in a variety of cell types, including S2 cells. To better delineate histone replacement patterns in the vicinity of hypersensitive sites, the entire BX-C was tiled at 20-bp resolution. The bxd, Mcp, Fab-7, and Fab-8 PRE-boundaries each encompass conspicuous peaks of H3.3 abundance that closely correspond to all the known nuclease-hypersensitive sites within the region. Nuclease hypersensitivity identifies sites of relatively accessible DNA, so that their correspondences to peaks of histone replacement suggest that continuous disruption of nucleosomes exposes cis-regulatory DNA relative to surrounding regions (Mito, 2007).
PRE-boundary elements in the BX-C and other regions are binding sites for multiple Polycomb group (PcG) proteins, which have been mapped in an S2 cell line at high resolution. If the process that disrupts nucleosomes also facilitates PcG binding, then a correspondence would be expected between peaks of PcG binding and peaks of H3.3. Indeed, when H3.3 profiles were compared with those for Enhancer-of-zeste (EZ) and Posterior-sex-combs (PSC) PcG proteins, all 10 peaks of PcG binding in the abdominal region were found to be local peaks of H3.3. Likewise at the ANTP-C, all 13 peaks of PcG binding in the Scr-Antp region correspond to high levels of H3.3. H3.3 enrichment at PcG-binding sites is not attributable to higher nucleosome occupancy, because essentially identical results were obtained for H3.3/H3 profiles (Mito, 2007).
Not all PREs in the BX-C are found to be sites of PcG binding; for example, neither Fab-7 nor Fab-8 is detectably bound by EZ or PSC. The fact that all PcG sites are peaks of histone replacement, but not vice versa, suggests that histone replacement at PREs and boundaries is constitutive and independent of the expression of the homeotic genes that they regulate. For example, Abd-B is expressed at high levels in S2 cells and displays the typical H3.3 5' peak for an active gene, whereas Ubx and abd-A are nearly inactive, yet the PREs and boundaries regulating all three genes are sites of conspicuous histone replacement over a low background (Mito, 2007).
Histone replacement averaged over the 175 genomewide EZ+PSC peaks outside of the BX-C and ANTP-C was examined and an H3.3 peak was observed centered over the PcG maximum. Therefore, the strong association between PcG protein binding and histone replacement is not limited to homeotic gene clusters. The genomewide H3.3 peak is higher than that for the BX-C and ANTP-C, presumably because other PcG-binding sites are not superimposed over such deep H3.3 valleys (Mito, 2007).
The colocalization of PcG-binding sites and local peaks of H3.3 suggests that the process that disrupts nucleosomes locally maintains the accessibility of cis-regulatory DNA to PcG proteins. If so, then there should be a lower average occupancy of nucleosomes over sites of PcG protein binding than over their surrounding regions. To test this possibility, nucleosomal DNA and fragmented genomic DNA were hybridized on the same microarrays, and nucleosomal/genomic DNA log ratios were measured. Around peak regions of EZ+PSC binding, nucleosomal DNA was clearly depleted on average, similar to the depletion seen for active gene promoters, and essentially the same results were obtained with different methods for genomic DNA fragmentation. It is concluded that the correspondence between histone replacement and nucleosome depletion is a genomewide feature of PcG-binding sites (Mito, 2007).
In Drosophila, many cis-regulatory elements, including PREs and boundaries, are bound by the trxG proteins, Zeste and GAGA factor (GAF). To test the possibility that histone replacement is enhanced and nucleosome occupancy is reduced where Zeste protein preferentially binds, 390 Zeste-binding sites identified by high-resolution chromatin immunoprecipitation (ChIP) combined with tiling microarrays (ChIP-chip profiling) were aligned, and log ratios of H3.3/H3 and nucleosome occupancy were averaged. A prominent maximum of histone replacement and a sharp minimum of nucleosome occupancy was observed centered over the point of alignment. Similar results were obtained for predicted GAF sites, which suggests that nucleosome disruption is a general feature of trxG protein DNA-binding sites. H3.3 enrichment at PcG- and trxG protein-binding sites results from a replication-independent replacement process, because essentially identical profiles were obtained for H3.3core, which lacks the N-terminal tail and does not assemble during replication (Mito, 2007).
Like Fab-7 and Fab-8, heat shock gene promoters are prominent sites of GAF binding, nuclease hypersensitivity, and reduced nucleosome occupancy. Heat shock protein Hsp70 genes are constitutively 'poised' for rapid induction, but do not produce detectable mRNAs in the uninduced state. Hsp70 genes were aligned at their 5' ends and H3.3 and H3 profiles were averaged. For comparison, similarly aligned H3.3 and H3 profiles were averaged for all 2165 genes on 3R with known 5' and 3' ends, divided into quintiles based on expression levels. H3.3 patterns were similar to those of highly active genes, with histone replacement levels peaking on either side of heat shock promoters. As do transcriptionally active gene promoters, heat shock genes display prominent H3.3 and H3 dips in abundance that are attributable to partial nucleosome depletion. Constitutive histone replacement also appears to be a feature of poised promoters in vertebrates, because H3.3 is strongly enriched in the upstream region of the chicken folate receptor gene, regardless of whether the gene is active or inactive (Mito, 2007).
What process maintains the chromatin of cis-regulatory elements in a state of flux? Many DNA-binding and chromatin-binding proteins involved in gene regulation display short residence times on DNA, and some mouse transcription factors show dynamic behavior at their functional binding sites. A model for this process has been proposed, involving alternating cycles of nucleosome disruption by a Brahma-related SWI/SNF chromatin-remodeler and transcription factor binding. The binding of PcG and trxG proteins is also dynamic, and it is proposed that a similar cycle of nucleosome disruption and factor binding takes place at boundaries and PREs. Nucleosome disruption by SWI/SNF remodeling complexes would occasionally evict nucleosomes and transiently expose DNA, which would become available to other diffusible factors, including PcG proteins. The continued local presence of nucleosome remodelers would result in another cycle of remodeling, nucleosome depletion, nuclease hypersensitivity, and histone replacement at the site. This model could account for the diversity of trxG proteins, which include DNA-binding proteins (Zeste and GAF), nucleosome remodelers (Brahma and Kismet), and histone methyltransferases (Trithorax and Ash1) that are specific for H3K4, a modification that is highly enriched on H3.3. The resulting dynamic process would allow for proteins that promote opposite epigenetic outcomes to act at common cis-regulatory sites (Mito, 2007).
A key question in understanding of the cis-regulation of gene expression during embryonic development has been the molecular mechanism that
directs enhancers to specific promoters within a gene complex. Promoter
competition and insulators are thought to play a role in regulating these
interactions. In the bithorax complex of Drosophila, the IAB5
enhancer is located 55 kb 3' of the Abdominal-B
(Abd-B) promoter and 48 kb 5' of the abdominal-A
(abd-A) promoter. Although roughly equidistant from the two
promoters, IAB5 specifically interacts only with the Abdominal-B
promoter, even though the enhancer and promoter are separated by at least two
insulators. This study demonstrates that a 255 bp element, located 40 bp 5'
of the Abd-B transcriptional start site, has a novel cis-regulatory
activity as it is able to tether IAB5 to the Abd-B promoter in
transgenic embryos. The tethering element is sufficient to direct IAB5 to an
ectopic promoter in competition assays. Deletion of the promoter-tethering
element results in the redirection of enhancer-driven gene expression on
transgenes. Taken together, these results provide evidence that specific
long-range enhancer-promoter interactions in the bithorax complex are
regulated by a tethering element 5' of the Abd-B promoter. A bioinformatic analysis is described of the tethering element across different
Drosophila species and a possible molecular mechanism by which this
element functions. Existing evidence is examined that this novel class of
cis-regulatory elements might regulate enhancer-promoter specificity at other
gene complexes (Akbari, 2008).
A 1.5 kb Abd-B promoter specifically recruits the IAB5 enhancer on
reporter transgenes. The tethering activity contained in the Abd-B
promoter region is able to interact with IAB5 over a long distance (>5 kb)
and is capable of facilitating the bypass of an intervening promoter
(abd-A) from the BX-C. Deletion of a 255 bp
sequence located in the 5' region of the Abd-B promoter reveals
the existence of a novel cis-element responsible for tethering of the IAB5
enhancer to the promoter. Removal of this PTE sequence from the Abd-B
promoter is sufficient to redirect the IAB5 enhancer to the adjacent
abd-A promoter on transgenes. In addition, fusion of
the PTE sequence to an ectopic promoter results in complete recruitment of the
IAB5 enhancer to the promoter (Akbari, 2008).
The transgenic experiments presented in this study demonstrate that the 255 bp DNA
sequence located 5' of the Abd-B promoter contains a
promoter-tethering element (PTE) that serves a key regulatory function by
specifically recruiting the IAB5 enhancer to the Abd-B promoter. It
is conceivable that at the endogenous complex the PTE may be involved in
recruiting multiple intergenic enhancers to the Abd-B promoter, such
as IAB6 and IAB7. Although this has yet to be tested on transgenes, the
available genetic evidence supports this idea, since a relatively small deletion
in the Abd-B upstream region is sufficient to disrupt activation by
the IAB7 enhancer in trans. By contrast, the IAB8 enhancer may not require a tethering element to activate the Abd-B promoter, possibly due to the close
proximity of this enhancer to the Abd-B promoter and the fact that
there are no intervening insulator elements (Akbari, 2008).
Previous studies demonstrated that a deletion 5' of the
Abd-B promoter region, which included the PTE, resulted in reduced
IAB enhancer-Abd-B promoter interactions in trans
(Sipos, 1998). As larger deletions were made, the interactions between the IAB7 enhancer and the target Abd-B promoter became increasingly weaker. One explanation for this observation could be the existence of additional elements in the extended 5' promoter sequence which may play a role in the tethering of the IAB enhancers to the Abd-B gene at the endogenous BX-C. Bioinformatic
studies across different Drosophila species support this idea. The
neighboring sequence 5' of the PTE is highly conserved; suggesting that
part of this upstream region may also contain sequences that aid in the
tethering activity. In this case, the critical in vivo function of the PTE may be supported by, as yet unidentified, additional cis-regulatory sequences capable of facilitating promoter-enhancer tethering. Transgenic constructs containing a PTE sequence extended to include part of this 5' region will be important in determining the function of this region (Akbari, 2008).
The PTE may also function in conjunction with a different class of
anti-insulator elements at the BX-C, the promoter targeting sequences (PTS),
which are known to facilitate promoter-enhancer interactions. Future
transgenic and genetic experimental approaches will help to unravel the
combinatorial regulatory activities of these complex cis-elements. However, it
is clear that the promoter-enhancer interactions facilitated by the PTE are
relatively strong, since neither spacer DNA nor endogenous insulator elements
are capable of disrupting these interactions in transgenic assays. These
results provide insight into the regulatory requirements at the endogenous
locus. It seems likely that these strong interactions are necessary for the
IAB5 enhancer element to bypass the two known insulator elements to activate
the Abd-B promoter approximately 55 kb away in cis (Akbari, 2008).
The precise molecular mechanism by which the tethering element functions is
not clear. It is possible that common trans factors may bind to both the IAB5
enhancer and PTE and establish protein-protein interactions, although the
Abd-B PTE and IAB5 enhancer do not share any extensive sequence
homology. There is, however, a precedent for this type of interaction; Sp1
has been shown to form DNA loops between binding sites proximal to promoter
sequences and distant binding sites to mediate an increased concentration of
activator protein at the promoter. Bioinformatic analysis reveals two separate short sequences within the PTE that are highly conserved in different Drosophila species, when compared to the other sequences in the PTE. It is possible that
these sequences harbor binding sites critical for the recruitment of the trans
factors involved in the molecular function of the PTE. It is possible that a mechanism involving the IAB enhancers looping to
interact with the PTE and drive expression from the target Abd-B
promoter could be facilitated by specific chromatin structures in the BX-C. A
similar spatial nuclear organization has also been suggested as a global
regulator of developmental gene expression in higher eukaryotes.
Studies have demonstrated that there are indeed physical interactions between distant regulatory regions with the BX-C, although the details remain to be fully elucidated. This model would explain the necessity of a functional PTE in the BX-C, as the disruption of this element would prevent the formation of the chromatin loop structures essential for promoter-enhancer communication, leaving the Abd-B target promoter inactive (Akbari, 2008).
Promoter-tethering elements represent a precise mechanism for regulating
specific enhancer-promoter interactions in gene complexes. Other mechanisms of
cis-regulation may be less flexible. Recent studies have identified enhancers
in the Drosophila genome capable of interacting only with distinct
sub-sets of promoters. Some enhancers will only interact with DPE-containing
promoters, whereas others only interact with TATA-containing promoters. At
the BX-C this may not be a feasible method for regulating enhancer-promoter
interactions since the addition of a TATA box to one promoter, for example, may
result in recruitment of all the enhancers in the complex. However, the PTE
may function similarly to promoter competition in some respects, since the
specific IAB5-Abd-B interaction it mediates appears to predominantly
prevent the enhancer from activating other promoters (Akbari, 2008).
The existence of a tethering element capable of specifically recruiting the
distal T1 enhancer to the Scr gene promoter at the antennapedia
Hox gene complex in Drosophila suggests
that promoter-tethering elements may represent a common mechanism for
regulating enhancer-promoter interactions at complex loci. The ability of the
IAB5 enhancer to activate Abd-B across insulator DNAs provides an
intriguing model for tethering activities at other gene complexes. An example
is the well characterized insulator at the mouse H19 imprinting
control region which separates 5' enhancers from the H19
promoter. It is possible that a tethering element is required to
selectively recruit these 5' enhancers to the target promoter. The
identification of an enhancer-containing global control region at the mouse
Hoxd complex raises the possibility that promoter tethering over long
distances may also be required at mammalian Hox genes (Akbari, 2008).
Polycomb-group (PcG) and trithorax-group (trxG) genes encode important regulators of homeotic genes, repressors and activators, respectively. They act through epigenetic mechanisms that maintain chromatin structure. The corto gene of Drosophila encodes a co-factor of these regulators belonging to the Enhancer of Trithorax and Polycomb class. Corto maintains the silencing of the homeotic gene Abdominal-B in the embryo and it interacts with a cyclin, Cyclin G, suggesting that it could be a major actor in the connection between Polycomb/Trithorax function and the cell cycle. This study shows that inactivation of Cyclin G by RNA interference leads to rotated genitalia and cuticle defects in the posterior abdomen of pupae and that corto genetically interacts with Cyclin G for generating these phenotypes. Examination of these pupae shows that development of the dorsal histoblast nests that will give rise to the adult epithelium is impaired in the posterior segments which identity is specified by Abdominal-B. Using a line that expresses LacZ in the Abdominal-B domain, it was shown that corto maintains Abdominal-B repression in the pupal epithelium whereas Cyclin G maintains its activation. These results prompt a proposal that the interaction between the Enhancer of Trithorax and Polycomb Corto and Cyclin G is involved in regulating the balance between cell proliferation and cell differentiation during abdominal epithelium development (Salvaing, 2008b).
Ubiquitous downregulation of CycG by RNA interference (using da::Gal4 or Act::Gal4 drivers) led to a high percentage of lethality in late third instar larvae or pharates depending on the CycG line and on the sex (Salvaing, 2008a). Lethality was complete in Act::Gal4/+; UAS::dsCycG2/+ males which intriguingly never underwent pupariation and stopped their development as third instar larvae, dying after a few days. In contrast, most females died as late pharates. UAS::dsCycG2/+; da::Gal4/+ as well as Act::Gal4/+; UAS::dsCycG2/+ emerging animals presented defects in the abdominal cuticle restricted to the posterior tergites A4 to A6. Apart from disorientation of abdominal bristles, the tergites of these segments exhibit unsclerotized patches of variable size. Males were more strongly affected than females and also frequently exhibited rotated genitalia (Salvaing, 2008b).
Genetic interactions between CycG and the loss-of-function alleles corto420 and corto07128 were examined. Their combination with ubiquitous RNAi inactivation of CycG increased lethality, cuticle defects and rotated genitalia. These data suggest that CycG and corto interact genetically and corroborate the existence of a functional relationship between CycG and corto (Salvaing, 2008b).
To understand the underlying defects of the cuticular phenotypes observed in RNAi-inactivated CycG flies, the development of the abdominal epithelium in pupae was addressed. In Drosophila, the abdominal epithelium of adults is derived from a fixed number of diploid histoblast cells, nested within the polyploid larval epithelium. Each abdominal hemisegment contains four histoblast nests, anterior and posterior dorsal, ventral and spiracle nests, that contribute to tergite and sternite of each abdominal segment. Histoblasts start to proliferate at the beginning of metamorphosis, replacing the larval cells, to eventually build up the adult abdominal integument. In wild-type pupae, the anterior and posterior dorsal histoblast nests of each hemisegment begin to fuse between 15 and 18 h APF. Fusion is completed at 24 h APF and the histoblasts have replaced all the polyploid larval cells at 48 h APF. In Act::Gal4/+; UAS::dsCycG2/+ 48 h APF pupae, whereas the dorsal histoblast nests of segment A3 fuse, the histoblast nests of segments A4 to A6 still remained small and unfused. Nevertheless, the development of these flies was not notably delayed, with regard either to puparium formation or to emergence of adult escapers. Therefore, it is concluded that RNAi inactivation of CycG especially impedes abdominal epithelium development of segments A4 to A6 where histoblast proliferation seemed to have stopped completely (Salvaing, 2008b).
It has been shown that corto is involved in the regulation of Abd-B and that Corto and CycG bind to the iab-7 PRE and to the promoter of Abd-B in embryos (Salvaing, 2008a). Since the epithelium defects of RNAi-inactivated CycG individuals affect abdominal segments A4 to A6, and are enhanced in corto mutants, it was hypothesized that they might be associated with misregulation of Abd-B, which specifies posterior abdominal identity. To address the role of corto and CycG in Abd-B regulation in the abdominal epithelium, genetic interactions between Abd-B and corto or CycG mutants was studied. The Fab-71 allele was used, in which both the Fab-7 boundary and the iab-7 PRE of the Abd-B cis-regulatory sequences have been deleted. This mutation induces a higher level of Abd-B expression in A6 which leads to a shift of A6 cell identity toward A7. As there is no normal sclerotized A7 segment in wild-type males, Fab7 homeotic A6 to A7 transformation results in loss of cells. As a result, Fab-71/+ males thus present a half-reduced A6 segment. It was observed that corto alleles enhance the expressivity of this phenotype leading to complete disappearance of the A6 segment in 100% of the males. Next, the effect of inactivation of CycG was examined in a Fab-71/+ genetic context. The expressivity of the Fab-71 phenotype was slightly enhanced in most (86%) of the UAS::dsCycG2/+; da::Gal4/Fab-71 males but at least a thin A6 segment always persisted. Curiously, no cuticular defects were observed neither in A5 nor in the remaining A6 tergites of these males suggesting that they might partly result from altered Abd-B expression. Overexpression of CycG also led to enhancement of the Fab-71 phenotype expressivity but in this case complete disappearance of A6 was observed in 56% of UAS::CycG/+; da::Gal4/Fab-71 males and in 100% of Act::Gal4/+; UAS::CycG/+; Fab-71/+ males. Lastly, the Fab-71 phenotype was investigated in corto, RNAi-inactivated CycG males. Crosses of Act::Gal4; Fab-71 females with UAS::dsCycG2/CyO; corto07128/TM6b males gave only few Act::Gal4/+; UAS::dsCycG2/+; Fab-71/corto07128 male escapers that all exhibited complete disappearance of A6 segment. These results suggest that both corto and CycG participate in maintenance of A6 cell identity by regulating Abd-B expression. corto clearly acted as a repressor of Abd-B since it enhanced the gain-of-function phenotype of Fab-71. However, it is not possible to conclude about the precise role of CycG on Abd-B expression since overexpression as well as inactivation led to enhancement of the Fab-71 phenotype, although to a lesser extent in case of inactivation. To understand this issue, the expression of Abd-B was addressed in corto mutants and in RNAi-inactivated CycG or overexpressing CycG individuals (Salvaing, 2008b).
Thus, Abd-B expression was examined in the abdominal epithelium of pupae. Since monoclonal anti-Abd-B antibodies show unspecific ubiquitous staining in the pupal epithelium, the HCJ199 strain was used where a P{LacZ} element is inserted in the cis-regulatory sequences of Abd-B. In agreement with published reports, it was observed that LacZ expression mimics Abd-B expression forming a decreasing gradient from A7 (in females) to the posterior part of A4, the expression in this segment being very faint and only detectable at high magnification. This pattern was also observed in Act::Gal4/+; HCJ199/+ control female pupae showing that the Act::Gal4 driver has no effect on Abd-B expression per se. At 24 h APF, LacZ was expressed in polyploid larval cells as well as in proliferating diploid histoblasts. Later on (48 h APF), LacZ was still expressed in the proliferating diploid histoblasts of A7, A6 and A5, and a barely discernible staining could be seen in posterior . In HCJ199/corto420 and HCJ199/corto07128 48 h APF pupae, LacZ was also expressed from A7 to the posterior part of A4 but expression in posterior A4 was much stronger than in control pupae. Moreover, some cells in the posterior region of A3 also expressed LacZ. This suggests that, in the abdominal epithelium of pupae as in embryos, corto maintains repression of Abd-B expression. In RNAi-inactivated CycG female pupae (Act::Gal4/+; UAS::dsCycG2/+; HCJ199/+) at 48 h APF, almost complete loss of LacZ expression was observed in A5 and A6, whereas it was still expressed in A7. In contrast, 48 h APF Act::Gal4/+; UAS::CycG/+; HCJ199/+ female pupae that overexpressed ubiquitously CycG showed ectopic expression of LacZ in the whole abdomen. Taken together, these results suggest that CycG has the ability to activate Abd-B expression in the abdominal epithelium and contributes to Abd-B expression maintenance in A6 and A5. Thus, corto and CycG play opposite roles on the control of Abd-B expression in the abdominal epithelium, corto being a repressor and CycG an activator. The expression of Abd-B was addressed in pupae where corto and CycG expressions were simultaneously reduced. In Act::Gal4/+; UAS::dsCycG2/+; HCJ199/corto420 48 h APF female pupae, although it was not possible to precisely determine segment borders due to impaired histoblast nest development, rescue of LacZ pattern was obseved that extended more anteriorly than in Act::Gal4/+; UAS::dsCycG2/+; HCJ199/+ pupae. Then, Abd-B loss of expression in A5 and A6 induced by CycG inactivation was abrogated when the amount of Corto was simultaneously reduced (Salvaing, 2008b).
This study has show that ubiquitous downregulation of CycG in pupae results in failure of epithelium formation in the posterior abdomen. Abdominal epithelium of adults derives from imaginal histoblasts that are recruited during embryogenic stages and form small group of diploid cells nested in the polyploid larval epithelium. The anterior dorsal nest is composed of about 15 to 18 cells whereas the posterior dorsal nest is composed of about 5 to 6 cells. These cells stay quiescent being arrested in G2 during the larval stages. At the onset of metamorphosis, they first undergo a phase of rapid proliferation triggered by ecdysone signalling and consisting of three synchronous and fast divisions. Also set off by ecdysone signalling are the second phase of histoblast proliferation which is slow and asynchronous and the simultaneous death of the polyploid larval cells. At 24 h APF, the anterior and posterior histoblast nests have fused. It is shown here that inactivation of CycG impedes the proliferation of histoblasts in the posterior part of the abdomen, the dorsal anterior and posterior nests being still individualized at 48 h APF. This probably results in cuticle defects in the less severely affected individuals that will become adult. Similar cuticle defects have been described in some mutants (Arrowhead, escargot, cdc2, myb, torpedo, EcR) where they extend more often over the entire abdomen. Arrowhead has been shown to be involved in the establishment of abdominal histoblasts during embryogenesis. In RNAi-inactivated CycG larvae, the number of cells in the dorsal anterior and posterior histoblast nests is identical to that of wild-type larvae, suggesting that CycG inactivation does not affect histoblast recruitment during embryogenesis. esgargot and cdc2 are required to maintain diploidy of histoblast cells. In RNAi-inactivated CycG larvae, the size of histoblast nuclei in dorsal nests appears similar to the size of the corresponding wild-type nuclei thus suggesting that CycG inactivation does not affect ploidy. Lastly, the epithelium defects could be related to defects in cell proliferation. This is the case for the myb mutant, which proliferating histoblasts exhibit mitosis defects, or the torpedo mutant, which shows loss of mitotic figures in the histoblast nests at 25 h APF. In RNAi-inactivated CycG pupae at 48 h APF, approximately 100 and 40 cells were observed in the anterior and posterior nests of the A6 segment, respectively, suggesting that they might have undergone the 3 first rounds of division. Then, it could be that cells slow down during the second phase of proliferation. Intriguingly, like in RNAi-inactivated CycG pupae, slowdown of histoblast proliferation in segments A5 and A6 has been observed in the torpedo mutant; torpedo encodes the EGF receptor. This suggests that the role of CycG in the proliferation of the abdominal epithelium could be related to MAP kinase signalling. Furthermore, the use of a dominant-negative form of the Ecdysone-receptor that blocks death of the larval epidermal polyploid cells also induces cuticle defects. In this case, cell-autonomous inhibition of EcR activity leads to abortive delamination and persistence of larval polyploid cells in the pupal epithelium. A similar phenomenon, linked to disruption of ecdysone signal reception, could arise when CycG is inactivated. Interestingly, that Act::Gal4>UAS::dsCycG males never go through pupariation; this could reflect a defect in EcR signalling reception. Although the abdominal cuticle of corto mutants seems to be unaffected, the cuticle defects were enhanced by combining them with CycG inactivation. It suggests that corto and CycG together regulate the formation of the abdominal epithelium during metamorphosis (Salvaing, 2008b).
These data also show that corto and CycG oppositely regulate the expression of the Hox gene Abd-B in the growing pupal epithelium, corto behaving as a repressor whereas CycG behaves as an activator. Since Corto also represses Abd-B in embryos, it can be considered as a global repressor of Abd-B. Nevertheless, neither Corto nor CycG were detected on the BX-C locus in salivary glands suggesting that they regulate Abd-B in a tissue-specific manner (Salvaing, 2008a). In accordance with expression data, reduction of corto or overexpression of CycG leads to enhancement of the gain-of-function phenotype of Fab-71 heterozygotes. Surprisingly, whereas loss of Abd-B expression was observed upon inactivation of CycG, a mild enhancement of the gain-of-function phenotype of Fab-71 was seen in the same genetic background. This enhancement may result from perturbation of proliferation in the remaining tergite rather than from homeotic transformation of A6 cells to A7 cells. However, it may also reflect the intrinsic mechanism of action of CycG. Indeed, it has been shown that CycG binds both the iab-7 PRE and the promoter of Abd-B (Salvaing, 2008). It is well known that PREs have a stronger silencing activity when present in two copies in the genome, a phenomenon called pairing-sensitive repression. Then, if CycG activates Abd-B partly by working at the promoter and partly by limiting pairing-sensitive repression, loss of Abd-B activation at promoter could be overwhelmed by loss of pairing-sensitive repression when a single copy of the iab-7 PRE is present which is the case in the Fab-71/+ flies (Salvaing, 2008b).
Finally, in pupae combining RNAi-inactivated CycG and corto mutation, histoblast proliferation is still impeded whereas Abd-B expression seems to be restored. It suggests that the ratio between Corto and CycG activities must be preserved to insure appropriate regulation of Abd-B in the posterior abdomen. Altogether, these results suggest that a tripartite interaction between corto, CycG and Abd-B together regulates the balance between proliferation and differentiation during the formation of the abdominal epithelium at metamorphosis. Further experiments are now required to better understand how these processes are coordinated (Salvaing, 2008b).
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Abdominal-B:
Biological Overview
| Evolutionary Homologs
| Transcriptional Regulation
| Targets of activity
| Protein Interactions
| Developmental Biology
| Effects of Mutation
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