pleiohomeotic
Mutations in genes of the Polycomb (Pc) group cause abnormal segmental development due to ectopic expression of the
homeotic products of the Antennapedia and bithorax complexes. This paper describes the requirements for Pc group genes in controlling the
abdA and AbdB products of the bithorax complex. Examined were embryos containing mutations in the genes Polycomb (Pc),
extra sex combs (esc),Enhancer of zeste [E(z)], polyhomeotic (ph), Sex comb on midleg (Scm), Polycomb-like (Pcl), Sex
comb extra (Sce), Additional sex combs (Asx), Posterior sex combs (Psc) and pleiohomeotic (pho). In every
case, both abdA and AbdB are expressed outside of their normal domains along the anterior-posterior axis, consistent
with these Pc group products acting in a single pathway or molecular complex. The earliest detectable ectopic expression is
highest in the parasegments immediately adjacent to the normal expression boundary. Surprisingly, in the most severe Pc group
mutants, the earliest ectopic AbdB is distributed in a pair-rule pattern. At all stages, ectopic abdA in the epidermis is highest
along the anterior edges of the parasegments, in a pattern that mimics the normal abdA cell-specific pattern. These examples of
highly patterned mis-expression show that Pc group mutations do not cause indiscriminate activation of homeotic products. It is
suggested that the ectopic expression patterns result from factors that normally activate abdA and AbdB only in certain
parasegments, but that in Pc group mutants these factors gain access to regulatory DNA in all parasegments (Simon, 1992).
A 176 bp fragment located at -576 to -400 bp of the engrailed locus has been shown to act as a pairing-sensitive silencer in
transgenic Drosophila. An evolutionarily conserved 17 bp sequence from this fragment was used to search for DNA binding proteins that might be
required for silencing. Pleohomeotic is shown to bind to this 17 bp sequence and mediate a pairing-sensitive silencer function (Brown, 1998)
Polycomb response elements (PREs) in several genes contain conserved sequence motifs. One of these motifs is the binding site for the protein coded for by the recently cloned gene polyhomeotic (pho), the Drosophila homolog of mammalian YY1. The conserved sequence extends beyond the YY1 core consensus sequence suggesting that parts of Pho may impose additional DNA sequence requirements. In this respect and unlike YY1, PHO has an additional 45 amino acids following the fourth zinc finger. It is also possible that Pho may bind to PREs together with another protein in order to fully exploit the conserved sequence. The conserved sequence motif CNGCCATNDNND, includes the YY1 core consensus CCATNWY. Eight consensus sites have been identified in 6 PREs of the bithorax complex (BX-C): bxd, iab-2, Mcp, iab-6, iab-7 and iab-8. The bxd PRE harbors all three characteristics used to define PREs (maintenance of expression of a lacZ reporter assay throughout development; pairing-sensitive repression of a mini-white reporter, and creation of an additional chromosomal binding site of the PcG-repressing complex in a salivary gland assay). The iab-2 PRE contains two homology boxes (a and b) and has been identifed in the maintenance and pairing-sensitive assays. The Mcp and iab-6 PREs have been characterized in the pairing-sensitive assay. The iab-7 PRE contains two homology motifs, a and b. This PRE has been characterized in all three assays. The iab-8 PRE has been identified in the maintenance assay. The conserved sequence motif is found in three PREs from Sexcombs reduced regulatory regions, and has been identified in the pairing-sensitive assay. The sequence motif found in two PREs from the engrailed regulatory region has been characterized in the pairing-sensitive assay. The sequence motif is also found in polyhomeotic, and has been identified in the pairing-sensitive and salivary gland assays (Mihaly, 1998)
In Drosophila, two classes of genes, the
trithorax group and the Polycomb group, are
required in concert to maintain gene expression by regulating chromatin
structure. Trithorax protein (Trx) binding elements have been identified
within the bithorax complex. Within the
bxd/pbx regulatory region these elements are functionally
relevant for normal expression patterns in embryos and they confer Trx
binding in vivo. Trx binding elements have been localized to three closely situated sites
within a 3-kb chromatin maintenance unit with a modular structure.
Results of an in vivo analysis have shown that these DNA fragments (each
~400 bp) contain both Trx- and Polycomb-group response elements (TREs
and PREs) and that in the context of the endogenous
Ultrabithorax gene, all of these elements are essential for
proper maintenance of expression in embryos. Dissection of one of these
maintenance modules has shown that Trx- and Polycomb-group responsiveness
is conferred by neighboring but separable DNA sequences, suggesting
that independent protein complexes are formed at their respective
response elements. The activity of this
TRE requires a sequence (~90 bp) that maps to within several tens of
base pairs from the closest neighboring PRE and the PRE activity
in one of the elements may require a binding site for Pho, the protein
product of the Polycomb-group gene
pleiohomeotic. These results show that long-range maintenance
of Ultrabithorax expression requires a complex element
composed of cooperating modules, each capable of interacting with both
positive and negative chromatin regulators (Tillib, 1999).
Polycomb group (PcG) proteins repress homeotic genes in
cells where these genes must remain inactive during
development. This repression requires cis-acting silencers,
also called PcG response elements (PREs). The
Drosophila PcG protein Pleiohomeotic has been shown to bind to specific
sites in a silencer of the homeotic gene Ultrabithorax. In an
Ultrabithorax reporter gene, point mutations in these
Pleiohomeotic binding sites abolish PcG repression in vivo.
Hence, DNA-bound Pleiohomeotic protein may function in
the recruitment of other non-DNA-binding PcG proteins to
homeotic gene silencers (Fritsch, 1999).
To dissect the 1.6 kb Ubx PRE, a Ubx-lacZ reporter
gene was used to monitor silencing capacity of PRE subfragments. PBX is an embryonic enhancer and
IDE is an imaginal disc enhancer: Both are located
about 30 kb upstream of the Ubx transcription start site. PBX directs
expression in early embryos in a pattern similar to Ubx with a
sharp anterior boundary in parasegment 6 (ps 6). In contrast, if IDE is linked to a reporter gene it
activates transcription not only in haltere discs where
endogenous Ubx is expressed but also in wing discs where Ubx
is not expressed. A PBX-IDE reporter gene is thus active within Ubx
expression boundaries in early embryos but is later expressed
also outside of the Ubx domain, i.e. in the wing disc. A test was therefore performed to see whether PRE or subfragments thereof would
silence this misexpression if inserted into the PBX-IDE
reporter gene. The 1.6 kb PRE was inserted between
the PBX and IDE enhancers and this reporter gene
(PRE1.6) was introduced into flies. Whereas PBX-IDE transformants
without the PRE fragment show nearly uniform beta-galactosidase
(beta-gal) expression in wing and haltere discs, beta-gal
expression in PRE1.6 transformants is confined to the
posterior compartment of haltere discs. The
boundary between beta-gal-positive and beta-gal-negative cells runs
through the middle of the haltere disc and apparently coincides
with the ps 6 compartment boundary. Thus, IDE activity is
completely suppressed anterior to ps 6 but is unaffected in ps
6 itself. This suggests that PRE1.6 silences the reporter gene
anterior to ps 6 and thereby preserves the anterior expression
boundary delimited by PBX in the embryo. The
expression pattern directed by PBX in the embryo is not
silenced by PRE1.6 (Fritsch, 1999).
Subfragments of the 1.6 kb PRE have been tested for silencing
function. PRE silencer is contained in the central 567
bp PRED fragment.
It was asked whether the silencing mediated by the PRE
fragments depends on PcG gene function. A
reduction in Pc gene dosage leads to a partial loss of silencing;
the extent of the observed misexpression is comparable to the
misexpression of the endogenous Ubx gene in Pc
heterozygotes. The patterns of PRED
lines were examined in larvae homozygous for a pho mutation. In
each case pho mutant wing and haltere discs show an
extensive loss of silencing. These results demonstrate
that silencing by PRED requires PcG gene function.
It was next examined whether Pho protein binds directly to
PRED. Pho contains a DNA-binding domain with very high
similarity to the DNA-binding domain of YY1, which is known
to bind to the sequence G/t C/t/a CATN T/a T/g/c. The PRED fragment contains several
motifs that match versions of this YY1 protein binding site.
Oligos spanning each of these motifs were tested for Pho
binding in gel-shift assays. Pho protein
forms a specific complex with six of the ten tested oligos. These and additional binding tests with other oligos
suggest GCCATTAC as an optimal binding site for Pho. To test
whether Pho protein binds to the PRED construct in vivo, antibodies were generated against the Pho protein. On polytene
chromosomes from salivary glands, Pho antibodies bind to
approximately 35 different loci. The strongest signal was found
at the location of the Bithorax-Complex (BXC), suggesting
that Pho protein is bound to the BXC genes.
On polytene chromosomes of a PRED
transformant line, a strong additional signal was found at the
transposon insertion site. These data suggest that
Pho protein binds directly to PRED in vitro and in vivo (Fritsch, 1999).
Are Pho protein binding sites
needed for silencing in imaginal discs? All six Pho
binding sites in the PRED fragment were mutated by altering two or three
nucleotides in each CCAT core motif. The introduced base changes abolish binding of
Pho protein in vitro. The mutated PRED fragment was
inserted into the PBX-IDE reporter gene to obtain PREDphomut. These
PREDphomut transformants show uniform beta-gal staining in
wing and haltere discs that is comparable to transformants carrying
the reporter gene without PRE. Thus, mutations in the
Pho binding sites abolish PRE function. Taken together, these experiments provide strong evidence
that Pho protein binds directly to PRE and is required for
silencing (Fritsch, 1999).
Expression of the endogenous Ubx gene was examined
in imaginal discs of pho mutants. Animals that are
homozygous for pho null mutations develop into pharate adults
with only relatively mild homeotic transformations.
Consistent with this, it was found that pho1 and phob homozygotes
show only slight misexpression of Ubx in wing and antennal
discs. The observed misexpression
is comparable to the misexpression of Ubx in Pc heterozygotes. In pho mutants, the PRED reporter gene
shows substantially more misexpression than the endogenous
Ubx gene. Thus, silencing of the reporter gene is more
sensitive to the lack of pho product than the native Ubx gene.
Animals that are mutant for two different PcG mutations often
show more severe misexpression of homeotic genes and
consequently enhanced homeotic transformations, when compared
to the single mutants by themselves. pho
homozygotes that are also heterozygous for Pc show very
dramatic misexpression of Ubx in wing and other discs. Thus, in this genetically sensitized background due to only
one rather than two copies of Pc, pho is required to repress Ubx
in all imaginal disc cells (Fritsch, 1999).
What is the role of maternal Pho? Most pho mutant embryos, which lack maternal wild-type pho
product, fail to develop altogether and the rare putatively
paternally rescued embryos that do develop die with
segmentation defects and homeotic transformations. In contrast, if maternal pho product is present,
pho homozygotes survive to pharate adults. This suggests that
pho function is particularly important in the very early embryo.
Mutation of the Pho binding sites in the
PREDphomut reporter gene abolish silencing in all disc cells.
Thus, it appears that if Pho protein is prevented from binding
to PRE, i.e. in the PREDphomut reporter gene, silencing is
probably never established. Conversely, silencing of the PRED
reporter gene is only partially lost in larvae homozygous for
a pho null mutation. Thus, in pho
homozygous embryos (which contain maternal Pho protein)
silencing of the PRED reporter is probably established but is
subsequently lost in imaginal discs. In summary, these
observations strongly suggest that maternally deposited Pho
protein is crucial for the establishment of silencing but that
zygotic Pho protein is required for complete silencing (Fritsch, 1999).
The Mcp element from the Drosophila melanogaster bithorax complex (BX-C) was initially identified because deletions of the element cause a dominant gain-of-function transformation of PS9 into PS10. This transformation in parasegmental identity is due to the inappropriate activation of the iab-5 cis-regulatory domain (which specifies PS10 identity) of the Abd-B gene in PS9 (a parasegment in which Abd-B is normally turned off). Two models have been proposed to explain the gain-of-function phenotypes associated with Mcp deletions. In the first, the Mcp deletions remove a PS10 silencer that functions to keep the iab-5 cis-regulatory domain off in PS9. When this silencer is removed, iab-5 is activated in PS9, turning on Abd-B. In the second model, Mcp corresponds to a boundary element that functions to preserve the functional autonomy of the iab-4 and iab-5 cis-regulatory domains. While the question of whether Mcp corresponds to a silencer, a boundary, or both (as is the case for the element deleted by another BX-C gain-of-function
mutation, Fab-71, a novel activity has been uncovered. Sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and they silence adjacent reporters by means of a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a 'pairing-sensitive' silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression (Muller, 1999).
The Mcp element in BX-C is defined by three overlapping deletions. Although these deletions differ in size and location, all three have indistinguishable, dominant gain-of-function phenotypes: they transform PS9 into PS10. This transformation in parasegmental identity is due to the ectopic activation of Abd-B in PS9, a parasegment in which Abd-B is normally off. The three deletions remove a common region of ~450 bp in length. This common region spans a major ~400-bp chromatin-specific, nuclease-hypersensitive site that is present throughout embryogenesis and in tissue culture cells. The smallest deletion, McpB116, is slightly larger than this common region, and it removes an additional ~350 bp proximal to the major nuclease-hypersensitive region. DNA fragments extending to either side of the small Mcp deletion have silencing activity when linked to either a mini-white or y reporter. Like other silencers in the PRE class, silencing activity depends on Pc-G proteins. Included in the Pc-G group is pleiohomeotic, a gene that encodes a DNA-binding protein that appears to be closely related to the mammalian YY1 transcription factor. As has been found for several other PREs in BX-C, the major Mcp nuclease-hypersensitive region has a consensus Pho/YY1-binding sequence. The presence of this sequence, together with the fact that silencing activity depends on the pho gene, argue that this DNA-binding protein may play a key role in the assembly of Pc-G-silencing complexes by the Mcp element (Muller, 1999).
The most unusual feature of the Mcp element is its ability to promote long-distance interactions. Regulatory interactions are observed between Mcp transgenes
inserted at different sites on the same chromosomal arm, on different chromosomal arms, and even between transgenes inserted on different chromosomes. The
long-distance regulatory activity of the Mcp element is unusual. Among the previously characterized PREs from BX-C and other genetic loci, pairing-sensitive
silencing is generally observed only between interacting partners inserted at the same site. Only in a few instances have
interactions been observed between partners inserted at different sites, and these usually involved PRE transgenes located in quite close proximity. How does Mcp promote regulatory interactions over long distances? One model suggests that Mcp might function by dragging paired DNA into a heterchromatic nuclear compartment. Contrary to the expectations of the compartmentalization model, the Mcp element can mediate not only long-distance silencing, but also long distance activation. An alternative, more plausible model suggests that the Mcp element facilitates long-distance regulatory interactions because it is able to locate and then pair with Mcp elements at other sites. After this locating process the formation and spread of a function silencing complex around each element would occur. Specificity is likely to be generated by a combination of proteins, some that are found in most PREs and some that are unique to the 800-bp MCP element (Muller, 1999).
Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the
gap gene products and the Polycomb group proteins. DNA elements and
trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein have been investigated. Analysis of a 1.7-kb
enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that both Hb
and Kruppel (Kr) are required to set the Abd-A anterior boundary in parasegment 7. DNase I footprinting and site-directed
mutagenesis show that Hb and Kr are direct regulators of this iab-2 enhancer. The single Kr site can be moved to a new
location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results
suggest that Kr repression occurs through a local quenching mechanism. The gap repressor Giant (Gt)
initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. This iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (Pho). These iab-2
Pho sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the Pho
sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late
embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer (Shimell, 2000).
After Hb and Kr decay during early gastrulation, the
repressed state is propagated through later stages of development
by the PcG proteins. How the transition from early
gap repressors to long-term PcG repressors occurs at the
molecular level is not known. Two basic models have been
proposed: (1) direct recruitment, and (2) chromatin recognition. Model (1): The gap gene products, especially Hb, have
been proposed to help recruit PcG proteins directly to
specific DNA sites. Based upon its
early time of action, a role for the PcG protein Extra sex
combs (Esc) as a molecular bridge between the two sets of
repressors has been suggested. However, direct interactions
between Esc and gap repressors have not been reported.
A better candidate for such a molecular link is
dMi-2, which binds directly to Hb and behaves genetically
as an enhancer of PcG repression. In its
simplest form the direct recruitment model is unlikely
because the iab-2, bx, and pbx enhancers all contain Hb
sites but do not effectively recruit PcG proteins. These
elements fail to maintain A-P boundaries of expression and
are unable to attract PcG proteins to sites on chromosomes. Furthermore,
the continuous requirement for PRE sequences during
development shows that DNA
site recognition by PcG proteins can occur long after Hb
and Kr have decayed. Model (2):The second model proposes that PcG
proteins recognize some feature of silenced chromatin,
rather than particular gap repressors.
This model is supported by patterns of PcG-dependent
silencing that reflect patterns of early gene activity rather
than the distributions of gap proteins. In
this view, PcG proteins sense the transcriptional off state
and then assemble locally to imprint this state through
later stages. These two models are not mutually exclusive. Both the
Hb-interacting protein dMi-2 and the
Kr-interacting protein dCtBP have
mammalian homologs that interact with histone deacetylases. Perhaps the gap repressors work by targeting these
deacetylases, whose action alters the local acetylation state
of the histone tails. This could provide a feature of silenced
chromatin that is recognized by PcG proteins and that
promotes their association at nearby PREs (Shimell, 2000 and references therein).
In addition to sites for the gap repressors, the role of iab-2 binding sites was characterized for the recently identified
PcG protein, Plieohomeotic. Pho
sites on the iab-2(1.7) fragment are required for pairing-sensitive repression (PSR) of a mini-white reporter. Thus, Pho can mediate this
type of gene repression in the context of a homeotic
regulatory fragment, analogous to its activity with an
engrailed regulatory fragment. Similarly, Pho
binding sites are required for function of a different PRE
located in the bxd region.
Are the iab-2(1.7) Pho sites sufficient for full PcG
repression? The results suggest that they are not, since lacZ
maintenance in the embryo, as opposed to PSR function
during late stages, requires more distally located iab-2
sequences in combination with the iab-2(1.7) fragment. Thus, assays for PSR and for lacZ maintenance are not
measuring precisely the same activity. In molecular terms,
this could reflect association of distinct complexes at PSR
sites as opposed to sites that supply full PRE function.
Alternatively, a larger critical number of Pho sites might
be needed for lacZ maintenance and fewer sites might
suffice for PSR. The iab-2(534) fragment,
which enables lacZ maintenance, contains two additional
Pho consensus core sites. However, three lines of evidence
indicate that Pho is not likely the sole factor that
recruits PcG proteins either to PREs or to PSR sites. (1) A
multimerized Pho site is insufficient to mediate PSR; (2) in vivo crosslinking studies show heterogeneity among PcG proteins assembled onto
DNA from different regions of the engrailed locus, and (3) the DNA-binding GAGA protein has also been implicated in PRE function and has been found associated with PRE sequences in chromatin binding assays. These observations strongly suggest that multiple DNA-binding factors form the landing pad for association of distinct types of PcG complexes.
What might be the in vivo role of PSR sites, such as the
one on the iab-2(1.7) fragment, which by themselves cannot
provide full PRE activity? One possibility is that, in
their normal context, they act as secondary recruitment
sites to extend and/or stabilize chromatin changes that are
nucleated at strong PREs. In agreement with this, is has been found that PC protein first assembles onto
core PREs at the blastoderm stage and that high levels of PC
association with fragments outside of these core regions
do not occur until later in embryogenesis. The scattering
of PSR sites throughout large regulatory domains, such as
those within the BX-C, might assist assembly and propagation
of repressive chromatin complexes over large DNA
distances (Shimell, 2000 and references therein).
A functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila bithorax complex (BX-C) has been undertaken. Previous studies mapped the iab-7 PRE to
an 860-bp fragment located just distal to the Fab-7 boundary. Located within this fragment is an ~230-bp chromatin-specific
nuclease-hypersensitive region called HS3. HS3 has been shown to be capable of functioning as a Polycomb-dependent silencer
in vivo, inducing pairing-dependent silencing of a mini-white reporter. The HS3 sequence contains consensus binding sites for
the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is
related to the mammalian transcription factor YY1. GAGA and Pho interact with these sequences in vitro, and the consensus binding sites
for the two proteins are critical for the silencing activity of the iab-7 PRE in vivo (Mishra, 2001).
Like the GAGA factor, Pho appears to function by directly interacting with target sequences in HS3. Several
lines of evidence support this conclusion: (1) the silencing
activity of the iab-7 PRE in vivo depends on pho
function and is eliminated by mutations in the pho gene;
(2) the Pho protein binds to two conserved target sequences in the
iab-7 PRE; (3) mutations in these two sites not only
eliminate binding in vitro but also compromise silencing activity in
vivo. Pho has also been directly implicated in the silencing activity
of three other PREs, one from the en gene and two from BX-C. The Pho protein has been shown to bind to these PREs in vitro, while mutations in either the Pho binding sites or in the pho gene itself reduce or eliminate silencing (Mishra, 2001).
Unlike that of Trl, the phenotypes of pho mutants
are similar to those seen for other Pc-G genes. Animals homozygous for loss-of-function alleles die at the
pupal stage and exhibit homeotic transformations of legs and abdomen.
The late lethal phase is due to a substantial maternal contribution,
and mutant embryos lacking a maternal source of wild-type Pho die with
severe homeotic transformations and other developmental defects. The homeotic transformations evident in mutant animals indicate that pho is likely to have a direct role in Pc-G
silencing. For the iab-7 PRE, the results argue that
silencing activity depends on the binding of the Pho protein to the two
target sites in HS3. Both sites seem to be important, since silencing activity
is compromised when one site is deleted. Whereas it is supposed that the major
function of the GAGA factor is to ensure that sequences in HS3 are
accessible to other proteins, the phenotypic effects of pho
mutations suggest that it plays a more active role in silencing. A
plausible hypothesis is that it functions (perhaps together with as yet
unidentified factors) to recruit components of the silencing machinery
to the PRE, such as Polycomb or Sex Combs Midleg, which do not appear to interact directly with DNA. Supporting the possibility that other
factors besides Pho play a critical role in recruiting Polycomb group
complexes, a PRE
fragment from iab-2, which contains Pho binding sites and
which is able to silence mini-white, has been shown to be insufficient to
confer full Pc-G maintenance activity. Moreover, mutations in the two Pho binding sites have only a minor
effect on the maintenance activity of the 860-bp iab-7 PRE
fragment in an iab-7 Ubx-LacZ assay system. Clearly it will be of interest to identify these other factors (Mishra, 2001).
Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response
Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to
behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene
assays, a 138 bp sequence has been defined within the MCP silencer that is sufficient for silencing of a reporter gene in the
imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (Pho) and
two binding sites for the GAGA factor, encoded by the Trithorax-like gene. PHO and the Trl proteins bind to these sites in vitro. Mutational
analysis of Pho and Trl binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends
on the function of Trl, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that,
individually, either Pho or Trl binding sites retain only weak silencing activity. However, when both Pho and Trl binding sites are present, they
achieve strong silencing. A model is presented in which robust silencing is achieved by sequential and facilitated binding of Pho and Trl (Busturia, 2001).
How does Trl or perhaps another GAGA binding protein contribute to the silencing by MCP, and what is its relationship to the Pho protein function? Two models to explain their relationship which leads to strong silencing are suggested. These models are based on the following observations. (1) Pho binding sites by themselves show little silencing activity (MCP1 and MCP7* constructs). (2)Trl or some other protein that binds to MCP can weakly recruit silencing complexes in the absence of Pho binding (5MPho construct). (3) When present together, Trl and Pho binding sites exhibit robust silencing activity (MCP7 construct). In the first model, Trl and Pho bind to the MCP silencer in a sequential order. One version would be that Trl binding is absolutely required for binding or activity of Pho. Trl may open up chromatin at MCP, allowing binding of Pho. Upon binding, Pho may recruit PcG silencing complexes, although there is still little evidence that this happens. Trl has been shown to induce DNase I hypersensitive sites, or nucleosome-free regions, and this may create a prerequisite condition for Pho to bind to its recognition sites. There is indeed a DNase hypersensitive region associated with MCP that includes the location of the Trl binding site (Busturia, 2001).
In a second version of the model, Pho acts as a facilitator of Trl binding by creating some pre-condition, perhaps by bending DNA as YY1 does. Since Pho binding sites are not absolutely required for MCP silencing activity, Trl presumably can bind weakly to MCP in the absence of Pho.
Enhanced binding of Trl leads to increased recruitment of silencing complexes. Trl bound to MCP may recruit PcG silencing complexes by directly interacting
with PC or other members of PcG complexes. Alternatively, Trl could first recruit SIN3 histone deacetylation complexes through its
interaction with SAP18, which then might generate a chromatin state favorable for PcG complex binding. Whichever version of the model is
correct, the important feature of the model is the sequential recruitment of DNA binding proteins, Trl and Pho, to MCP. Binding of one protein creates a
condition favorable to the binding of a second protein, eventually leading to the recruitment of PcG complexes. Note that the requirement of Trl and Pho proteins
applies to MCP silencing, but not necessarily to all PREs. Other PREs may use other combinations of proteins. This model is analogous to Swi5 protein binding to the yeast HO promoter and recruiting the chromatin remodeling complex Swi/Snf. Swi/Snf in turn recruits the histone acetylase complex SAGA, eventually leading to the binding of the transcription factor SBF to the HO promoter. In such a sequential recruitment model, compromising one step in the sequence
may become rate limitating so that combining two mutations that disable two different steps may not necessarily lead to synergistic effects. This may explain why no synergistic effects are observed when Trl and PcG mutations are combined. In the second model, Trl and Pho bind to MCP independently of one another. Each protein may induce a unique chromatin modification that, together, can have a
positive synergistic effect on the recruitment of PcG silencing complexes (Busturia, 2001).
Regulatory DNA from engrailed causes silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called 'pairing-sensitive silencing.' The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), the activities of each of these engrailed fragments were tested in a construct designed to detect PRE activity in embryos. Both fragments behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment shows additional silencing capabilities. Using the mini-white reporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggest that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function (Americo, 2002).
Polycomb group (PcG) proteins function as high molecular weight complexes that maintain transcriptional repression patterns during embryogenesis. The vertebrate DNA binding protein and transcriptional repressor, YY1, shows sequence homology with the Drosophila PcG protein, Pleiohomeotic. YY1 might therefore be a vertebrate PcG protein. Drosophila embryo and larval/imaginal disc transcriptional repression systems were used to determine whether YY1 represses transcription in a manner consistent with PcG function in vivo. YY1 represses transcription in Drosophila, and this repression is stable on a PcG-responsive promoter, but not on a PcG-non-responsive promoter. PcG mutants ablate YY1 repression, and YY1 can substitute for Pho in repressing transcription in wing imaginal discs. YY1 functionally compensates for loss of PHO in pho mutant flies and partially corrects mutant phenotypes. Taken together, these results indicate that YY1 functions as a PcG protein. Finally, YY1, as well as Polycomb, was found to require the co-repressor protein CtBP for repression in vivo. These results provide a mechanism for recruitment of vertebrate PcG complexes to DNA and demonstrate new functions for YY1 (Atchison, 2003).
The YY1 repression patterns are the same as those obtained previously with a known PcG protein. Therefore, it was asked whether YY1 repression required PcG function. To determine this, an hbGALYY1 BXDGALUbxLacZ (BGUZ) recombinant chromosome line was prepared and this chomosome was crossed into various homozygous PcG mutant backgrounds. Since PcG proteins function as complexes, mutation of a single PcG gene often abrogates PcG-dependent repression. Strikingly, homozygous mutant Polycomb (Pc), Polycomb-like (Pcl) or Sex combs on midleg (Scm) backgrounds led to complete derepression of YY1 function. Even heterozygous Pc and Pcl mutants abolished YY1 repression. Homozygous mutant Sex combs extra (Sce), Additional sex combs (Asx) or Suppressor of zeste [Su(Z)2] plus Posterior sex combs (Psc) backgrounds yield partial derepression of YY1 activity, perhaps due to maternal effects. Therefore, YY1 repression in vivo required PcG function (Atchison, 2003).
Two distinct PcG complexes have been identified. The first complex, termed the PRC1 complex, contains Pc, Scm, Polyhomeotic (Ph) and Psc proteins. This complex is clearly necessary for YY1 repression since Pc and Scm mutants abolish YY1 function. The second complex contains Esc and E(z). YY1 physically interacts with EED, the vertebrate homolog of Drosophila Esc. Therefore, the necessity of Esc for YY1 repression was tested in vivo. Homozygous mutation of the esc gene causes partial loss of YY1 repression. Thus, both complexes are needed for maximal YY1 repression, although mutations of proteins in the PRC1 complex cause more dramatic loss of YY1 repression (Atchison, 2003).
Most biochemical studies have not revealed a physical association of YY1 with the known PcG complexes, although substoichiometric levels are observed in human Pc complexes, and some associations have been documented for Drosophila Pho. The transient nature of the Drosophila associations suggest that an intermediary protein exists. This study demonstrates genetic and physical associations between YY1 and CtBP, which link YY1 to PcG function and provide a mechanism for the recruitment of vertebrate PcG complexes to DNA. Since CtBP is able to homodimerize, it may interact with Pc by one dimer partner and with YY1 by the other dimer partner. These interactions could define the mechanism by which YY1 functions to repress transcription in both a PcG- and CtBP-dependent fashion. In addition, the CtBP and Pc experiments indicate that CtBP plays a more direct role in PcG repression. Thus, CtBP may perform more than one function in the repression mechanism (Atchison, 2003).
The PcG function of YY1 that was identified in this study extends a list of YY1 functions including transcriptional activation and repression via apparently non-PcG pathways. YY1 binds to numerous promoters and can mediate repression by a variety of mechanisms including binding site competition, DNA bending and interference with activator interactions with the basal transcription machinery. YY1 repression can be influenced by interactions with proteins such as adenoviral E1A and the co-activator p300. YY1 can also interact with histone deacetylase proteins and is speculated to play a role in chromatin remodeling. Thus, the PcG function of YY1 identified in this study may be one of numerous functions mediated by this complex transcription factor. It may not be surprising that YY1 carries out multiple functions, because diverse functions of other PcG proteins are now being elucidated. For example, the PcG proteins Bmi-1 and Mel-18 play roles in controlling the cell cycle and their mutation leads to proliferative defects that impact the hematopoietic system. Therefore, PcG proteins play roles in multiple processes in addition to body axis formation (Atchison, 2003).
Stable transcriptional repression by YY1 is observed, but it was also found that YY1 appears to repress expression of a previously active gene. Generally, PcG proteins are believed to be maintenance repressors that do not initiate de novo repression. However, YY1 has the feature that it can repress de novo and may be able to repress transcription by multiple mechanisms that include PcG-dependent and -independent mechanisms. This is in agreement with the multiple YY1 repression mechanisms that have already been identified (Atchison, 2003).
The peri-implantation lethal phenotype of YY1 knock-out mice is similar to the phenotype of eed–/– mice. In contrast, pho mutant Drosophila show a phenotype much later in development, potentially indicating some differences between YY1 and Pho. Phenotypic rescue experiments demonstrate considerable functional similarity between these proteins, but 75% of vertebrate YY1 and Drosophila Pho protein sequences contain no discernable homology, suggesting some distinct functions. Pho appears insufficient for repression at early embryonic stages in Drosophila, since a LexA-Pho chimeric protein is incapable of repressing transcription of a LexA-Ubx-LacZ reporter, and a GAL-Pho chimeric protein is incapable of repressing the BGUZ construct. Thus, unlike YY1, Pho does not repress transcription in early embryos. However, Pho is necessary for repression at later stages of development, since mutating Pho binding sites in the Ubx PRE results in loss of silencing in wing imaginal discs. YY1 can clearly repress transcription at both early embryonic stages, as well as at later larval stages in wing imaginal discs. The early function of YY1 is consistent with its early lethal phenotype in YY1 mutant mice. This repression indicates that YY1 can mediate embryonic functions lacking in the Pho protein. Specifically, the association of YY1 with CtBP may provide a bridging function not mediated by Pho. Most proteins that bind to CtBP contain a canonical PXDLS motif. While YY1 contains a similar sequence, this motif is absent from Pho (Atchison, 2003).
The precise role of CtBP in PcG repression is unclear. CtBP mutants in flies show segmentation defects, but homeotic derepression has not been observed. Similarly, mouse ctbp1 and ctbp2 null mutants show a variety of defects including skeletal abnormalities, but these defects do not precisely match the skeletal posterior transformations seen with mammalian PcG mutants. It is quite possible that YY1 and CtBP are necessary for a subset of PcG functions. Similarly, it has been proposed that multiple distinct PcG complexes exist to regulate distinct genes. An additional potential link between YY1 and the PcG complex is the protein RYBP. Similar to CtBP, RYBP can physically interact with both YY1 and PcG proteins. The absence of a corresponding mutant in Drosophila precluded testing of the necessity of RYBP for YY1 repression (Atchison, 2003).
The demonstration that YY1 functions as a PcG protein predicts that vertebrate PREs should contain YY1 binding sites. YY1/Pho binding sites (CGCCATNTT) are indeed present within many Drosophila PRE sequences, and are required for function. Since the YY1 binding motif is well characterized, these results should facilitate the identification of vertebrate PRE regions, which thus far have proved elusive. The experiments linking YY1 to PcG function reveal mechanistic features of YY1-mediated transcriptional repression, with implications for PcG activity in mammals. It will be very interesting in the future to determine whether YY1 heterozygous mice augment mutant phenotypes in PcG mutant heterozyotes (Atchison, 2003).
Polycomb/Trithorax response elements (PRE/TREs) maintain transcriptional decisions to ensure correct cell identity during development and differentiation. There are thought to be over 100 PRE/TREs in the Drosophila genome, but only very few have been identified due to the lack of a defining consensus sequence. The definition of sequence criteria that distinguish PRE/TREs from non-PRE/TREs is reported in this study. Using this approach for genome-wide PRE/TRE prediction, 167 candidate PRE/TREs are reported, that map to genes involved in development and cell proliferation. Candidate PRE/TREs are shown to be bound and regulated by Polycomb proteins in vivo, thus demonstrating the validity of PRE/TRE prediction. Using the larger data set thus generated, three sequence motifs that are conserved in PRE/TRE sequences have been identified (Ringrose, 2003).
The detection of PRE/TREs by prediction generates a large data set that can be used to search for further common sequence features. To this end, the 30 highest scoring PRE/TRE hits were scanned for motifs that occur significantly more often in PRE/TREs than in randomly generated sequence. Five significant motifs were found. Not surprisingly, but reassuringly, two known motifs, the GAF and PHO binding sites were found. The Zeste binding motif was not found by this analysis, although it occurs as frequently as GAGA factor in the 30 sequences analyzed. This is probably due to the shortness and degeneracy of the Zeste motif, and suggests that other such short motifs will also be missed by this approach (Ringrose, 2003).
Nevertheless, three additional motifs were found. The first, called GTGT, is found several times in 14 of the sequences. The second motif, poly T, is found several times in almost all 30 PRE/TRE sequences analyzed. Some variants of this site match the binding consensus for the Hunchback protein, which has been shown to be an early regulator at some PRE/TREs. The third motif, TGC triplets, occurs several times in 13 of the PRE/TRE sequences. No binding factor for this sequence has yet been identified (Ringrose, 2003).
To further examine these three motifs, motif occurrence was evaluated in all 167 predicted PRE/TREs and in the promoter peaks described above. In contrast to the known GAF, Z, and PHO motifs, the three motifs each occur in only a subset of predicted and known PRE/TREs, and do not occur significantly together. These motifs may thus each define a subclass of PRE/TREs. Consistent with this idea, some of the lowest scoring known PRE/TRE sequences indeed contain one or more of the three motifs (Ringrose, 2003).
Although no correlation between particular sites and high scores was found, a negative correlation was found between numbers of GAF/Z and PHO sites (a correlation coefficient of -0.78, indicating that when many GAF/Z sites are present, there are few PHO sites, and vice versa). This suggests that each PRE/TRE may have a preferred ground state, in which it is either predisposed to silencing (many PHO sites) or to activation (many GAF/Z sites) (Ringrose, 2003).
In summary, this analysis identifies three motifs that occur significantly in association with known PRE/TRE motifs. Further functional characterization of these motifs and the proteins that bind them may contribute to a more complete definition of the sequence requirement for PRE/TRE function, and of subclasses of PRE/TREs (Ringrose, 2003).
This study offers four main contributions to the understanding of PRE/TRE function. First, a larger set of sequences have been defined that will facilitate the more complete definition of PRE/TRE sequence requirements. Three motifs have been identified that may contribute to this goal. The definition of the minimal requirement for PRE/TRE function will not be a trivial task. Analysis of motif composition and order in the 167 predicted PRE/TREs reveals that there is a great diversity of patterns, with no preferred linear order. It is possible that each different pattern of motifs reflects a subtly different function. However, the concept of a linear order of motifs may well be irrelevant, because these elements operate in the three-dimensional context of chromatin. The fact that such a diversity of PRE/TRE designs exist indicates that the vast majority of them would defy detection by conventional pattern-finding algorithms, and underlines the advantages of the approach described in this study (Ringrose, 2003).
Although no linear constraints on motif order were found, the fact that only motif pairs, and not single motifs, are able to identify PRE/TREs strongly suggests that this close spacing of sites has functional significance. Multiple sites may work in concert, to promote cooperative binding of similar proteins (e.g., repeated PHO sites) or to provoke competition between dissimilar proteins (e.g., closely spaced GAGA factor and PHO sites). In addition, in chromatin, only a subset of sites will be exposed and optimally available for binding at any one time, while others will be occluded by nucleosomes. The trxG includes nucleosome remodeling machines, raising the intriguing possibility that remodeling of PRE/TREs in chromatin may contribute to epigenetic switching by exposing different sets of protein binding sites (Ringrose, 2003).
Second, a PRE/TRE peak is observed at the promoter of all the genes examined. This strongly suggests that promoter binding is a general principle of PRE/TRE function. It has been reported that PcG proteins can interact with general transcription factors. It has hitherto been unclear whether the observed PcG/trxG binding at promoters of the genes they regulate is mediated indirectly via such an interaction, or whether the PcG and trxG bind directly to PRE/TREs at the promoters. The high scores observed at promoters favor the latter interpretation (Ringrose, 2003).
Third, it has been shown that in most cases, PRE/TREs do not occur in isolation, but are accompanied by one or more other peaks nearby. These grouped PRE/TREs may create multiple attachment sites for PcG and trxG proteins, which come together to build a fully operational complex at the promoter. Alternatively, grouped PRE/TREs may be individually regulated by tissue-specific enhancers as in the BX-C. Thus, each of the many PRE/TREs of the homothorax gene may interact with the promoter PRE/TRE in different tissues. This idea is consistent with the fact that Homothorax has specific roles in diverse developmental processes (Ringrose, 2003).
Finally, the current list of about ten PcG/trxG target genes has been expanded to over 150 genes, identifying candidates for epigenetic regulation. The genes thus identified encompass every stage of development, suggesting that the PcG/trxG are global regulators of cellular memory. Experiments to further investigate and compare this regulation for individual genes are currently underway (Ringrose, 2003).
Polycomb group (PcG) proteins maintain the transcriptional silence of target genes through many cycles of cell division. This study provides evidence for the sequential binding of PcG proteins at a Polycomb response element (PRE) in proliferating cells in which the sequence-specific DNA binding Pho and Phol proteins directly recruit E(z)-containing complexes, which in turn methylate histone H3 at lysine 27 (H3mK27). This provides a tag that facilitates binding by a Pc-containing complex. In wing imaginal discs, these PcG proteins also are present at discrete locations at or downstream of the promoter of a silenced target gene, Ubx. E(z)-dependent H3mK27 is also present near the Ubx promoter and is needed for Pc binding. The location of E(z)- and Pc-containing complexes downstream of the Ubx transcription start site suggests that they may inhibit transcription by interfering with assembly of the preinitiation complex or by blocking transcription initiation or elongation (L Wang, 2004; full text of article).
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).
Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Using DNaseI footprinting analysis, a high-resolution map is provided of sites for the sequence-specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. While GAGA can efficiently bind to a chromatinized PRE, PHO alone is incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, is strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins (Mahmoudi, 2003).
This study has determined the precise distribution within the Ubx
PRE of the recognition elements for four sequence-specific DNA-binding proteins that have all been implicated in Ubx regulation in vivo: PcG protein PHO, gap protein HB and trxG proteins GAGA and Zeste. The results indicate that, rather than a random collection, the binding site distribution within the Ubx PRE reflects a functional arrangement, allowing cooperation between distinct PRE binding proteins. Of particular interest is the observation that chromatin binding by the PcG protein PHO is strongly facilitated by the trxG protein GAGA. This finding provides a molecular mechanism for the requirement for both factors during PRE-directed silencing in vivo, and suggests that PHO and GAGA elements together may form a functional module (Mahmoudi, 2003).
Several independent genetic studies have pointed to a concurrent requirement for GAGA and PHO during gene silencing directed by distinct PREs. The PcG-dependent silencing conferred by a 230 bp fragment of the iab-7 PRE is dependent on both GAGA and PHO binding. Similarly, a 138 bp fragment of the MCP silencer, which was found to be sufficient for maintenance of embryonic silencing, contains PHO and GAGA sites. Mutations in either PHO or GAGA sites compromised silencing and revealed cooperation between both proteins. Particularly relevant for the current study are results that support a critical role in PcG silencing for GAGA and PHO sites within the Ubx PRE (Mahmoudi, 2003).
Functional dissection of the Ubx PRE has revealed that a Pc-dependent PRE silencer is contained in the central 567 bp fragment from position 577 to 1143, which includes all PHO and the highest density of GAGA sites. Another study showed that an oligomerized subfragment, corresponding to positions 890-1079 within PRE C, harboring two PHO and five GAGA elements, is able to confer PcG silencing in vivo. Finally, deletion of a 160 bp region corresponding to positions 851-1011 within PRE C impairs maintenance of silencing. The large extent of overlap between the DNA fragments identified in these independent studies strongly suggests that the common region within PRE C represents the critical core of the Ubx PRE. The most noticeable feature of this region is the many alternating GAGA and PHO binding elements. Moreover, it is of interest to note that footprinting analysis revealed the presence of Zeste as well as HB sites within this region, which may also contribute to the in vivo maintenance of repression (Mahmoudi, 2003).
The identification of Zeste as a component of the PRC1 PcG complex, suggests that it may play a direct role in PcG complex recruitment to the Ubx PRE. Further evidence for the involvement of Zeste in the maintenance of Ubx repression as well as activation has been provided by transgene experiments. Finally, the presence of HB sites within the Ubx PRE suggests a potential role for HB, not only during the initiation of Ubx repression, but also during the transition from establishment to maintenance. One attractive possibility is that this transition involves dMi-2 recruitment by HB. It should be noted that in the absence of initiating activation and repression elements, HB-independent PcG repression of the Ubx promoter has been documented (Mahmoudi, 2003).
Although there is substantial evidence for the notion that the proteins discussed above are involved in PcG silencing of homeotic genes, it remains unclear whether they can be sufficient for targeting or whether additional factors are required. One way to determine a minimal set of protein recognition sequences that can mediate PcG silencing will be the generation of synthetic PREs, which should be tested in vivo. The results suggest that, within such a PRE, PHO sites will need to be flanked by GAGA sites in order to facilitate chromatin binding. The proteins GAGA and Zeste may be particularly well adapted for such a purpose. Both GAGA and Zeste form large homo-oligomers that bind cooperatively to the multiple sites present in their natural response elements, such as the Ubx PRE and promoter. This cooperative mode of DNA-binding may allow these proteins to first bind an accessible site within a nucleosomal array and then progressively displace histones during binding to flanking sites. In addition, GAGA and Zeste have both been shown to recruit selective ATP-dependent chromatin remodeling factors. The process of targeting of remodelers to specific DNA elements may enable GAGA and Zeste to create nucleosome-free or remodeled areas, thus facilitating binding of other regulators. It is considerede likely that the remodeling complexes present in the chromatin preparations used in assays, are involved in the observed synergistic binding between PHO and either GAGA or Zeste (Mahmoudi, 2003).
GAGA oligomerization may also promote the communication between the Ubx PRE and promoter. Both elements, which are separated by ~24 kb of intervening DNA, contain a preponderance of binding sites for GAGA. GAGA oligomerization through its POZ domain allows it to form a protein bridge that directs long-range enhancer-promoter association. In fact, GAGA could even mediate enhancer function in trans by simultaneous binding of two separate DNA fragments. Thus, it is tempting to speculate that GAGA may link the Ubx PRE to the Ubx promoter. It should be noted that both the chromatin remodeling and long-range bridging functions of GAGA might accommodate PRE-mediated activation as well as repression (Mahmoudi, 2003).
The interdependence between proteins belonging to antagonistic genetic groups for efficient chromatin binding described it this study will have to be taken into account when interpreting mutational analysis of PRE function. Thus, removal of recognition sequences for the trxG protein GAGA may block its activation function but could also affect binding of the PcG protein PHO. Moreover, recent results suggest additional opportunities for cross-talk during recruitment of non-DNA-binding PcG complexes. Although a clear consensus between different studies is still lacking, there is experimental evidence for PcG complex recruitment by PHO, GAGA and Zeste. Because binding sites for either one of these proteins alone do not confer PRE function, it appears likely that they work in a combinatorial fashion. Depending on their context, the multitude of distinct binding elements that constitute a PRE might be redundant, cooperative or antagonistic to each other. Furthermore, distinct PREs may require different sets of PRE-binding proteins, and additional recruiters may be involved in PcG-silencing. Attractive candidates are GAGA-related factors batman and the PHO-related factor PHO-like (Mahmoudi, 2003).
In conclusion, current evidence suggests that PRE-directed maintenance of gene activation or repression is not achieved by a simple binary switch set by competing trxG and PcG proteins. Although their relative ratios vary considerably and correlate with transcription levels, they coexist at PREs during gene activation as well as repression. Likewise, genetic suppressor studies indicated extensive cross-talk between PcG and trxG proteins. This study has shown that, already at the level of PRE binding, there is strong interdependence between trxG protein GAGA and PcG protein PHO. The results demonstrate a direct biochemical mechanism for the cooperation between PcG and trxG proteins during PRE binding (Mahmoudi, 2003).
Mammalian Polycomb group (PcG) protein YY1 can bind to Polycomb response elements in Drosophila embryos and can recruit other PcG proteins to DNA. PcG recruitment results in deacetylation and methylation of histone H3. In a CtBP mutant background, recruitment of PcG proteins and concomitant histone modifications do not occur. Surprisingly, YY1 DNA binding in vivo is also ablated. CtBP mutation does not result in YY1 degradation or transport from the nucleus, suggesting a mechanism whereby YY1 DNA binding ability is masked. These results reveal a new role for CtBP in controlling YY1 DNA binding and recruitment of PcG proteins to DNA (Srinivasan, 2004).
To determine whether YY1 can recruit PcG proteins to DNA, chromatin immunoprecipitation (ChIP) assays were performed in a transgenic Drosophila embryo system consisting of hsp70-driven GALYY1 and a reporter construct containing the LacZ gene under control of the Ultrabithorax (Ubx) BXD enhancer and the Ubx promoter adjacent to GAL4-binding sites (BGUZ). The BGUZ reporter is expressed ubiquitously during embryogenesis but is selectively repressed in a PcG-dependent manner by GALYY1 and GALPc. Embryos were either left untreated or heat shocked to induce GALYY1 expression. After immunoprecipitation with various antibodies, the region surrounding the GAL4-binding sites in the BGUZ reporter was detected by PCR. Prior to heat shock, no GALYY1 could be observed at the reporter gene. After heat shock, GALYY1 binding to the reporter gene was easily detected. Interestingly, concomitant with GALYY1 binding, there was an increase in binding of the Polycomb (Pc) and Polyhomeiotic (Ph) proteins. Thus, YY1 DNA binding results in PcG recruitment to DNA (Srinivasan, 2004).
Binding of PcG proteins to PRE sequences is known to cause deacetylation of histone H3 and methylation on Lys 9 and Lys 27. Interestingly, induction of GALYY1 binding to the reporter gene resulted in loss of histone H3 acetylation on K9 and K14. Simultaneously, there was a gain of methylation on histone H3 Lys 9 and Lys 27. Therefore, YY1 binding to the BGUZ reporter results in the recruitment of PcG proteins to DNA and subsequent post-translational modifications of histones characteristic of PcG complexes (Srinivasan, 2004).
The presence of PcG proteins and the status of histone H3 modifications at the Ubx promoter region, which is 4 kb downstream of the GALYY1-binding site, were determined. To avoid amplification of the endogenous Ubx promoter, immunoprecipitated samples were amplified with primers spanning the Ubx-LacZ boundary. Interestingly, the presence of Pc and Ph was detected at the promoter after GALYY1 induction. The presence of GALYY1 at this site was also detected. The GAL4 protein alone does not bind to the Ubx promoter region, indicating specificity for YY1 sequences. The induced GAL4 protein was functional, however, because it efficiently bound to the GAL4-binding site in the BGUZ reporter. Binding by GALYY1 could, therefore, be due to either cryptic YY1-binding sites present at the promoter, physical association of GALYY1 with other proteins bound at the promoter, or interactions via looping of DNA between the GAL4-binding sites and the Ubx promoter. Again, induction of GALYY1 resulted in loss of acetylation of H3K9 and H3K14 and simultaneous gain of methylation on H3K9 and H3K27. These results are consistent with studies that have reported spreading of PcG proteins and histone modifications to flanking DNA (Srinivasan, 2004).
PHO and YY1 bind to the same DNA sequence, and PHO-binding sites have been identified in multiple PREs. Therefore, it was reasoned that YY1 would bind to endogenous PREs and perhaps increase recruitment of PcG proteins. For this, the major Ubx PRE (PRED), that contains multiple PHO-binding sites located in the bxd region, was examined. As expected, upon GALYY1 induction, GALYY1 was detected at this endogenous PRE site. In addition, YY1 binding was accompanied by an increase in Pc and Ph signals when compared with no heat shock controls and a loss of H3 K9 and H3 K14 acetylation and gain of H3 K9 and H3 K27 methylation. Thus, YY1 can bind to an endogenous PRE and can augment PcG recruitment (Srinivasan, 2004).
These results clearly indicated that YY1 DNA binding results in recruitment of PcG proteins, histone deacetylases (HDACs), and histone methyltransferases (HMTases) to DNA. To determine whether the Drosophila E(z) protein (which possesses HMTase activity) was involved, whether YY1 transcriptional repression was lost in an E(z) mutant background was examined. The results are consistent with the observation that E(z) specifically methylates histone H3 on Lys 27, which creates a binding site for the chromodomain of Pc. Thus, the repression observed with GALYY1 requires function of the E(z) PcG protein (Srinivasan, 2004).
It has been shown that YY1 interacts with Drosophila CtBP, a well-characterized corepressor molecule. CtBP can also interact with Pc in vivo. These associations led to a proposal that CtBP might play a bridging function between YY1 and PcG proteins. If true, one would expect loss of PcG recruitment to DNA in a CtBP mutant background. Indeed, ChIP experiments in a CtBP03463/+ background showed greatly reduced Pc and Ph recruitment to the BGUZ reporter. In addition, histone H3 remained acetylated and unmethylated. Surprisingly, in a CtBP mutant background, a dramatic loss of GALYY1 DNA binding was observed. However, full-length GAL4 protein was able to bind to DNA equally well in wild-type and CtBP mutant backgrounds, indicating that the effect of CtBP mutation was specific for YY1. This is a very unexpected result because CtBP has never been demonstrated to control DNA binding of another protein. The absence of GALYY1 and PcG proteins bound to the BGUZ reporter in the CtBP mutant background suggested that expression of the LacZ gene should be increased. Indeed, LacZ expression was increased in CtBP mutant as compared with wild-type embryos. Thus, in a CtBP mutant background, GALYY1 does not bind DNA, PcG proteins are not recruited, histones remain acetylated and unmethylated, and transcription is derepressed (Srinivasan, 2004).
To be certain that this effect is not peculiar to the BGUZ reporter, the effect of CtBP mutation on GALYY1 and PcG binding at endogenous PREs was examined. For this, the Ubx PRED, engrailed (en) PRE, and sex combs reduced (scr) PRE were chosen. Strikingly, GALYY1 and Pc binding to all three PREs was greatly reduced in the CtBP mutant background. Reduction in GALYY1 and Pc DNA binding correlated with H3 K9 acetylation at the PRED and En PREs. In contrast, H3 K9 acetylation at the Scr PRE was lost in a CtBP mutant background. These results clearly indicate an essential role for Drosophila CtBP in PcG recruitment to DNA (Srinivasan, 2004).
Collectively, these studies clearly demonstrate PcG recruitment function by the multifunctional transcription factor YY1. This establishes YY1 DNA binding as a key mechanism for targeting PcG proteins to DNA. The loss of YY1 DNA binding and concomitant loss of PcG recruitment to reporters and endogenous PRE sequences in CtBP mutants underscores this mechanism. A model of YY1 and CtBP function is presented. It is proposed that in a CtBP mutant background, YY1 is sequestered by a protein that inhibits its ability to bind to DNA. In a CtBP wild-type background, YY1 is released from this protein, thus enabling it to bind to DNA. DNA binding by YY1 results in recruitment of PcG complexes that cause deacetylation of histones and methylation of histone H3 at Lys 9 and Lys 27. Deacetylation may also be mediated by HDACs directly recruited by interaction with YY1 (Srinivasan, 2004).
The ablation of YY1 DNA binding in a CtBP mutant background was totally unexpected. This represents a new mechanism for controlling YY1 DNA binding and PcG recruitment. The mechanism appears to be exquisitely sensitive to CtBP dose because YY1 DNA binding and PcG recruitment are greatly reduced in heterozygous mutant backgrounds. Heterozygous effects by CtBP on knirps and hairy mutant phenotypes have been observed in other systems, suggesting that CtBP levels are limiting in vivo (Srinivasan, 2004).
The exact role of CtBP in PcG-mediated repression is yet to be elucidated. The results suggest that CtBP is required for the function of a large subset of PREs that require YY1/PHO for PcG recruitment. Like PcG mutants, CtBP mutants in flies show segmentation defects, but homeotic derepression has not been observed. Heterozygous ctbp mutants can reverse pair-rule phenotypes observed in hairy mutants, and homozygotes show bristle and cuticle defects. Furthermore, embryos that are trans-heterozygous for wimp and the ctpb03463 allele die and their cuticle preparations show severe segmentation defects. Similarly, mouse ctbp1 and ctbp2 null mutants show a variety of defects including skeletal abnormalities, but these defects do not precisely match the skeletal posterior transformations seen with mammalian PcG mutants. Based on the multiple PREs affected by CtBP mutation, it is unclear why a more severe CtBP heterozygous mutant phenotype is not observed. Perhaps a low level of PcG binding to DNA remains that is below detection in immunostains of polytene chromosomes, but which is sufficient to mediate biological effects. In support of this possibility, polytene spreads were occasionally observed that stained with Pc antibodies nearly as well as wild-type spreads. This suggests a possible threshold effect for CtBP involvement in PcG recruitment. ChIP studies on many more PRE sequences will be needed to clarify this issue (Srinivasan, 2004).
These results show that modulation of YY1 DNA binding by CtBP is a critical step in the recruitment of PcG proteins to DNA. This mechanism might be differentially used during development to control PcG assembly on PREs. The demonstration of recruitment of PcG proteins by YY1 should assist in the identification of mammalian PREs since the YY1 recognition sequence is well characterized (Srinivasan, 2004).
The Drosophila Sex comb on midleg (Scm) protein is a transcriptional repressor
of the Polycomb group (PcG). Although genetic studies establish Scm as a crucial
PcG member, its molecular role is not known. To investigate how Scm might link
to PcG complexes, the in vivo role of a conserved protein
interaction module, the SPM domain was analyzed. This domain is found in Scm and in another PcG protein, Polyhomeotic (Ph), which is a core component of Polycomb repressive complex 1 (PRC1). Scm-Ph interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize greater than 30 missense mutations in the SPM domain of Scm. Genetic rescue
assays show that Scm repressor function in vivo is disrupted by mutations that
impair SPM domain interactions in vitro. Furthermore, overexpression of an
isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in
flies. Coassembly of Scm with a reconstituted PRC1 core complex shows that Scm
can partner with PRC1. However, gel filtration chromatography showed that the
bulk of Scm is biochemically separable from Ph in embryo nuclear extracts. These
results suggest that Scm, although not a core component of PRC1, interacts and
functions with PRC1 in gene silencing (Peterson, 2004).
Purifications of nuclear complexes and in vitro studies have
identified eight proteins that are core components of two distinct fly PcG
complexes: Esc, E(z), Su(z)12, and NURF-55 in the ESC-E(Z) complex plus Pc, Ph,
Psc, and Sex combs extra/dRING1 in PRC1. One function of the Esc-E(z) complex is histone H3
methylation on K27. Further studies
are needed to address whether the Esc-E(z) complex has additional functions. The
molecular mechanism of PRC1 is not yet known. Studies to date suggest that it
represses transcription through a noncatalytic mechanism that restricts template
access, but it is not yet clear how PRC1 molecularly affects nucleosome array
organization and/or packaging of the chromatin fiber. Since genetic studies in
Drosophila identify at least 15 genes involved in PcG repression, many
additional components need to be fit into the framework of PcG complexes and
functions. In addition to identifying the players, analyses of loss of function
for individual PcG genes distinguishes those repressors with central PcG roles
from those that are more peripheral. In good agreement with the biochemical
studies, loss of function for core subunits of either PcG complex produces
severe homeotic defects. These mutants
show robust Hox misexpression and die as embryos with most segments
transformed into copies of the eighth abdominal segment. By these criteria, Scm
is clearly a central player in the PcG repression system. In
contrast, other repressors such as Asx and Pcl appear more peripheral since
their complete loss from embryos yields significantly weaker homeotic defects (Peterson, 2004).
In this work, a combination of in vivo and in vitro
approaches are presented to address Scm molecular function. Mutational
analysis shows that Scm function absolutely depends upon an intact SPM protein interaction domain. There is a strong
correlation between disruption of protein interactions in vitro
and failure of Scm function in vivo. These results agree with the finding that Scm repressor function in an in vivo tethering assay requires its SPM
domain. The importance of SPM domain interactions is also revealed by PcG
loss-of-function phenotypes produced by overexpression of an isolated SPM domain. It is suggested that this dominant negative reflects
SPM domain interactions critical for PcG repression that are disrupted by this
avidly binding but otherwise nonfunctional competitor. The embryonic lethality
of SPM domain mutants, together with embryonic and
imaginal defects seen with SPM overexpression, indicate that SPM interactions
contribute to PcG repression at both embryonic and postembryonic times. Thus,
these interactions appear required for long-term maintenance of PcG silencing
in vivo (Peterson, 2004).
The biochemical properties of the SPM domain suggest three potential types of
Scm interactions in vivo: (1) binding to PRC1, (2) binding to other fly
SPM domain proteins, or (3) binding to itself.
Although the data do not rule out contributions from any of these, several lines
of evidence favor Scm interaction and function with PRC1. (1) in vivo
evidence derives from studies showing that Scm can repress reporter genes when
tethered by fusion to a DNA-binding domain. Since this repression depends
upon Ph function, Scm cannot repress on its own but rather requires PRC1 to
repress in this context. (2) Substoichiometric quantities of Scm
consistently copurify with tagged PRC1 complexes from both fly and mammalian
extracts. Although
the majority of Scm appears to not be stably bound,
the conserved association of some Scm with purified PRC1 likely reflects
in vivo interactions. (3) No stably associated
partner proteins have been detected that copurify when FLAG-Scm is affinity purified from embryo
extracts. Thus, there is no evidence for a
heteromeric Scm-containing complex that could repress independently of PRC1 (Peterson, 2004).
If Scm does work with PRC1, then what might explain its substoichiometric
association with purified PRC1? One possibility is that Scm assembles only into
a subset of PRC1 complexes, perhaps restricted to certain tissues or times of
development. Such a model has been proposed to explain how Asx contributes to
PcG repression in the embryonic epidermis but not in the central nervous system.
This explanation for Scm, however, is not favored because its requirement in PcG
repression is widespread in both embryonic and imaginal tissues. Another
possibility is that Scm interaction with PRC1 is robust in chromatin but is not
fully preserved during preparation of soluble nuclear extracts used in
purification. In this view, nucleosome arrays might provide a platform that
promotes Scm-PRC1 binding. Indeed, both PRC1 and Scm have affinity in
vitro for nucleosome arrays. Additional in vitro studies
will be needed to address the nature of Scm-PRC1 interactions in the context of
chromatin templates. It is noted that the GAGA factor provides an example of a
protein that is not stably associated with PRC1 in embryo extracts but can
nevertheless help recruit PRC1 to nucleosomal templates in vitro (Peterson, 2004).
At present, the evidence favors a noncatalytic role for PRC1 in PcG
repression. How might Scm, which also lacks recognizable catalytic domains,
contribute to PRC1 mechanism? Recent in vitro studies show that mouse
PRC1 bound to a single nucleosome array can recruit a second chromatin template
that then also becomes repressed.
These bridging interactions between repressed templates
in vitro may reflect the PcG-dependent chromosome-pairing and
chromosome-chromosome interactions frequently observed in vivo.
Thus, one role of PRC1 may be
to promote higher-order chromosome interactions that spread or stabilize
repression. Intriguingly, among the core PRC1 components, the mouse Ph protein
was found most critical for in vitro bridging activity. Since Ph is the key subunit that mediates Scm interaction with PRC1, the possibility is raised
that Scm could facilitate PRC1-mediated long-distance chromatin interactions. In
this view, Scm might work by helping to anchor PRE-promoter and/or PRE-PRE
interactions needed for PcG repression in vivo (Peterson, 2004).
A second type of potential Scm-PRC1 partnership in chromatin has been
proposed on the basis of structural properties of the SPM domain. The SPM domain
of fly Ph, determined by X-ray crystallography, is a five-helix bundle that has
the special property of forming helical self-polymers in vitro. The possibility
of an extended protein polymer that could bind alongside nucleosome arrays has
prompted speculation that SPM proteins might organize higher-order chromatin
arrangements. In such a model, SPM domain-containing proteins or complexes form
a core helical polymer around which the chromatin fiber could be wrapped.
This model, although speculative, is appealing since it
brings structural data to bear upon the long-standing hypothesis that PcG
proteins create extended tracts of repressed chromatin.
Intriguingly, when mixed together, the SPM domains
of Ph and Scm can also form copolymers in vitro.
Thus, PH and Scm could collaborate in
forming the proposed higher-order chromatin structures. In this context, the
dominant-negative properties of overexpressed SPM domain
could reflect disruption of contacts needed to produce PH-Scm chromatin
polymers. To evaluate this model, it will be necessary to test if full-length
PcG proteins or their intact complexes can form polymers in vitro like
those seen for their isolated SPM domains. If so, then further studies would
need to address the existence and roles of such polymers in vivo (Peterson, 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).
The Polycomb and trithorax groups of genes control the maintenance of homeotic gene expression in a variety of organisms. A putative participant in the regulation of this process is the murine RYBP (Ring and YY1 Binding Protein). Sequence comparison between different species has identified the homologous gene in Drosophila, the dRYBP gene. Whether dRYBP participates in the mechanisms of silencing of homeotic genes expression was investigated. dRYBP expression, examined by RNA in situ hybridisation, was found ubiquitously throughout development. Moreover, a polyclonal anti-dRYBP antibody was generated that recognises the dRYBP protein. dRYBP protein is nuclear and expressed maternally and ubiquitously throughout development. To study the transcriptional activity of dRYBP, a fusion protein was generated containing the entire dRYBP protein and the GAL4 DNA binding domain. This fusion protein functions, in vivo, as a transcriptional repressor throughout development. Importantly, this repression is dependent on the function of the Polycomb group genes. Furthermore, using the GAL4/UAS system, dRYBP was over-expressed in the haltere and the wing imaginal discs. In the haltere discs, high levels of dRYBP repress the expression of the homeotic Ultrabithorax gene. This repression is Polycomb dependent. In the wing discs, dRYBP over-expression produces a variety of phenotypes suggesting the overall miss-regulation of the many putative genes affected by high levels of dRYBP. Taking together, these results indicate that dRYBP is able to interact with PcG proteins to repress transcription suggesting that the dRYBP gene might belong to the Polycomb group of genes in Drosophila (Bejarano, 2005).
The mouse homologous gene, RYBP, was identified in a two-hybrid screen for murine Ring1 interacting proteins. RYBP family members include the human YEAF1 homologous gene and the murine and human YAF2 gene coding for structurally related proteins. Although very similar in sequences, they seem to have different functions as transcriptional regulators of the hGABP gene, i.e. YAF2 positively regulates the transcriptional activity of hGABP but YEAF1 negatively regulates this activity (Sawa, 2002; Bejarano, 2005).
dRYBP is expressed maternally and throughout development in all the nuclei of the embryo and the imaginal discs cells. The murine RYBP gene is also expressed ubiquitiously in the mouse embryo (Garcia, 1999). The ubiquitous and nuclear pattern of dRYBP expression coincides with the pattern of expression of the Polycomb group proteins so far described (Bejarano, 2005).
When dRYBP is tethered to DNA sequences, it is able to repress the transcriptional state of minigene reporter constructs. Moreover, GALDB-dRYBP transcriptional repression function requires the products of at least the Pc, Sce and pho genes, suggesting that GALDB-dRYBP represses transcription by interacting with PcG protein complexes. The Pho protein (homologous to mouse YY1) is able to bind DNA in a sequence specific manner and it has been proposed to recruit the PcG complexes to DNA. However, the results show that the transcriptional repression function of GALDB-dRYBP cannot be achieved in the absence of Pho protein. Although silencing in these experimental conditions could formally result solely from the interaction of dRYBP with Pho, the need of Pho to execute the transcriptional repression may also suggest that in the process of maintenance of homeotic gene expression, the Pho protein serve other functions than the recruitment of PcG complexes to DNA (Bejarano, 2005).
Additional evidence for the transcriptional repressor function of dRYBP comes from the experiments of over-expression of dRYBP using the GAL4/UAS system. UbxGAL4/UASdRYBP halteres show partial transformation towards wing which is correlated with the repression of UBX expression in the haltere imaginal discs due to high levels of dRYBP. The partial transformation of the haltere towards wing is not fully understood. It is speculated that the over-expression of dRYBP may also affect genes involved in proliferation that act downstream the Ubx gene. The repressive effect is Polycomb dependent, suggesting that dRYBP transcriptional repression function needs the interaction with Polycomb proteins. Moreover, although no changes have been detected in the levels of engrailed expression, some of the phenotypes observed in enGAL4/UASdRYBP flies are indicative of engrailed repression, revealing again the repressor effect of dRYBP over expression (Bejarano, 2005).
A model has been proposed in which RYBP protein, through its interaction with DNA-binding proteins like YY1, function as a ‘bridge’ to ensure interactions of DNA and non-DNA binding proteins in multimeric protein complexes. It is not yet known if dRYBP serves a similar bridging function in Drosophila. The YY1 protein (homologous to Drosophila Pho) is able to bind DNA in a sequence specific manner and directly interacts with dRYBP. It is speculated that dRYBP, serves a similar bridging function, bridging between DNA binding proteins like Pho and the multimeric PcG complexes. Further work and mutations in the dRYBP gene will be necessary to define whether dRYBP serves this putative bridge function (Bejarano, 2005).
dRYBP over-expression in the wing produces homeotic and non-homeotic phenotypes indicative of miss regulation of a variety of genes. High levels of dRYBP in the wing (i.e. sdGAL4/UASdRYBP flies) produces, among others, transformation towards haltere with the corresponding expression of the Ubx protein in the wing cells, i.e. outside its normal domain of expression. This effect could seem opposite to the repressor effect observed when dRYBP is tethered to DNA (GALDBdRYBP) or when dRYBP is over expressed under the control of the UbxGAL4 line. However, interference with the assembling/recruting of the PcG and trxG complexes either because of sequestration of PcG/trxG proteins, perturbation of the PcG/trxG balance or disruption of the cross regulatory interactions between PcG proteins could perhaps explain the observed expression of UBX protein in the wing disc due to over-expression of dRYBP. Alternatively, over abundance of dRYBP or dRYBP containing complexes might lead to a unique target gene repretoire that lead to the effects observed. Finally, the cross regulatory interactions between the genes patterning the wing, that are perhaps being miss regulated by the high levels of dRYBP could also explain the range of phenotypes observed in the wing due to over expression of dRYBP (Bejarano, 2005).
In conclusion, these results show that dRYBP protein is nuclear, maternal and ubiquotiously expressed throughout development. The results also show that dRYBP functions, in a Polycomb dependent manner, as a transcriptional repressor, suggesting that dRYBP is able to interact with the PcG proteins to repress transcription and therefore might belong to the Polycomb group of genes of Drosophila. Finally, the study of the multiple phenotypes produced by high levels of dRYBP in the wing might be indicative of the involvement of dRYBP on the regulation of many genes as also described for the PcG genes in Drosophila (Bejarano, 2005).
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).
Polycomb group (PcG) epigenetic silencing proteins act through cis-acting DNA sequences, named Polycomb response elements (PREs). Within PREs, Pleiohomeotic (Pho) binding sites and juxtaposed Pc binding elements (PBEs) function as an integrated DNA platform for the synergistic binding of Pho and the multisubunit Polycomb core complex (PCC). This study analyzed the architecture of the Pho/PCC/PRE nucleoprotein complex. DNase I footprinting revealed extensive contacts between Pho/PCC and the PRE. Scanning force microscopy (SFM) in combination with DNA topological assays suggested that Pho/PCC wraps the PRE DNA around its surface in a constrained negative supercoil. These features are difficult to reconcile with the simultaneous presence of nucleosomes at the PRE. Indeed, chromatin immunoprecipitations (ChIPs) and nuclease mapping demonstrated that PREs are nucleosome depleted in vivo. The implications of these findings for models explaining PRE function are discussed (Mohd-Sarip, 2006).
How specialized DNA elements such as PREs can bring a linked gene under epigenetic control remains poorly understood. An important breakthrough