Ultrabithorax

Ubx regulation: Table of contents

Ubx upstream promoter and boundary elements

Polycomb response elements (PREs) can establish a silenced state that affects the expression of genes over considerable distances. The ability of insulator or boundary elements to block the repression of the miniwhite gene by the Ubx PRE has been tested. The gypsy element and the scs element interposed between PRE and the miniwhite gene protect miniwhite against silencing but the scs element is only weakly effective. Blocking the action of gypsy requires su(Hw). When the PRE-miniwhite gene construct is insulated from flanking chromosomal sequences by gypsy elements at both ends, the construct can still establish efficient silencing in some lines but not others. This silencing can be caused by interactions in trans with PREs at other sites. PRE-containing transposons inserted at different sites or even on different chromosomes can interact, resulting in enhanced silencing. These trans interactions are not blocked by the gypsy insulator and reveal the importance of nonhomologous associations between different regions of the genome for both silencing and activation of genes. The similarity between the behavior of PREs and enhancers suggests a model for their long-distance action. Thus blocking elements can prevent communication along a chromatin fiber, and enhance silencing of PREs in trans (Sigrist, 1997).

The suppressor of Hairy-wing [su(Hw)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the su(Hw) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. These studies investigate whether repression resulting from the Polycomb group genes (derived from a gypsy element) can be blocked by the su(Hw) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group response element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. The su(Hw) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome (Mallina, 1998).

The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-7') dominantly transforms parasegment 11 into parasegment 12. This chromosomal region contains both a boundary element and a silencer. Previous studies have suggested that removal of a domain boundary element on the proximal side of Fab-7' is responsible for dominantly transforming gain-of-function phenotype. The Fab-7 boundary element maps to two nuclease hypersensitive sites, HS1 and HS2. This article demonstrates that the Fab-7' deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment (the HS3 site) and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of an artificial BXD, Ubx/lacZ reporter transgene. Both mini-white and Ubx/lacZ silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste' allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans the HS3 nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor. It is suggested that PRE-PRE cooperation, either in trans or in cis, may be an important feature of the silencing process within the BX-C and that boundaries may limit PRE-PRE cooperation (Hagstrom, 1997).

Enhancers are able to activate promoters located several kilobases away, but how this is accomplished is not known. Activation by the wing margin enhancer in the cut gene, located 85 kb from the promoter, requires several genes that participate in the Notch receptor pathway in the wing margin, including scalloped, vestigial, mastermind, Chip, and the Nipped locus. Nipped mutations disrupt one or more of four essential complementation groups: l(2)41Ae, l(2)41Af, Nipped-A, and Nipped-B. Heterozygous Nipped mutations modify Notch mutant phenotypes in the wing margin and other tissues, and magnify the effects that mutations in the cis regulatory region of cut have on cut expression. Nipped-A and l(2)41Af mutations further diminish activation of a wing margin enhancer that has been partly impaired due to a small deletion. In contrast, Nipped-B mutations do not diminish activation by the impaired enhancer, but increase the inhibitory effect of a gypsy transposon insertion between the enhancer and promoter. Nipped-B mutations also magnify the effect of a gypsy insertion in the Ultrabithorax gene. Gypsy binds the Suppressor of Hairy-wing insulator protein [Su(Hw)] that blocks enhancer-promoter communication. Increased insulation by Su(Hw) in Nipped-B mutants suggests that Nipped-B products structurally facilitate enhancer-promoter communication. Compatible with this idea, Nipped-B protein is homologous to a family of chromosomal adherins with broad roles in sister chromatid cohesion, chromosome condensation, and DNA repair (Rollins, 1999).

Database searches reveal homologs of the Nipped-B protein in fungi, worms, and mammals. Only short expressed-sequence tags (ESTs) of Caenorhabditis elegans, mouse, and humans were identified. The human ESTs are from a variety of tissue-specific libraries, suggesting that the human homologs are widely expressed. The combined human ESTs, which do not represent a complete sequence, encode 411 amino acids. Residues 2-232 of the partial human protein overlap Nipped-B residues 1744-1994 with 34% identity and 52% similarity. In order of decreasing homology, the 2157-amino acid Rad9 protein of Coprinus cinereus, the 1583-amino acid Mis4 protein of Schizosaccharomyces pombe, and the 1493-amino acid Scc2 protein of Saccharomyces cerevisiae, are more distantly related. Rad9 residues 669-2071 display 21% identity and 41% similarity to Nipped-B residues 576-1887; Mis4 residues 780-1492 have 19% identity and 41% similarity to Nipped-B residues 1110-1818, and Scc2 residues 697-1291 display 19% identity with 39% similarity to Nipped-B residues 1103-1704. The three fungal homologs show similar levels of homology among themselves, but it is evident that there is a large conserved domain shared by all three proteins. Consistent with the idea that Nipped-B plays an architectural role in enhancer-promoter communication, the fungal homologs of Nipped-B all participate in regulating chromosome structure, with roles in DNA repair, meiotic chromosome condensation, or sister chromatid cohesion. It has been proposed that these three fungal proteins define a new class of chromosomal proteins and have been named adherins to distinguish them from the cohesins that have similar functions (Rollins, 1999 and references).

H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex

The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity (Bowman, 2014).

The Polycomb Group repression system is often described as a cellular memory mechanism, which can impose lifelong silencing of a gene in response to a transitory signal. That view seems valid, but the concept of a PcG regulatory domain is much richer. In the PS6 domain of the BX-C, for example, there are many enhancers to drive Ubx expression in specific cells at specific developmental times, all of which are blocked in parasegments one through five, but active in parasegments 6 through 12. Individual enhancers need not include a segmental address that is specified, for example, by gap and pair-rule DNA-binding factors; their function is segmentally restricted by the domain architecture. Indeed, these enhancers will drive expression in a different parasegment when inserted into a different domain (as in the Cbx transposition). Each domain has a distinctive collection of enhancers; the UBX pattern in PS5 is quite different from that in PS6. Thus, there are two developmental programs for Ubx, one in each of these parasegments, without the need for a duplication of the Ubx gene. Other loci with broad regions of H3K27 methylation may likewise be parsed into multiple domains, once histone marks are examined in specific cell types (Bowman, 2014).

The all-or-nothing H3K27me3 coverage of the BX-C parasegmental domains validates and refines the domain model. In particular, K27me3 is uniformly removed across the PS5 and PS7 domains in PS5 and PS7, even though the activated genes in those parasegments (Ubx and abd-A, respectively) are only transcribed in a subset of cells. It is interesting that both PRC1 and PRC2 components have binding patterns that do not fully reflect function (repression and K27 methylation, respectively), indicating the possibility that function of these complexes is regulated separately from binding. The challenges now are to understand how PcG regulated domains are established, differently in different parasegments, and to describe the molecular mechanisms, including changes in chromosome structure, that block gene activity in H3K27 trimethylated domains (Bowman, 2014).

A variably occupied CTCF binding site in the Ultrabithorax gene in the Drosophila Bithorax Complex

Although the majority of genomic binding sites for the insulator protein CTCF are constitutively occupied, a subset show variably occupancy. Such variable sites provide an opportunity to assess context-specific CTCF functions in gene regulation. This study has identified a variably occupied CTCF site in the Drosophila Ultrabithorax (Ubx) gene. This site is occupied in tissues where Ubx is active (third thoracic leg imaginal disc) but is not bound in tissues where the Ubx gene is repressed (first thoracic leg imaginal disc). Using chromatin conformation capture this site was shown to preferentially interact with the Ubx promoter region in the active state. The site lies close to Ubx enhancer elements and is also close to the locations of several gypsy transposon insertions that disrupt Ubx expression, leading to the bx mutant phenotype. Gypsy insertions carry the Su(Hw)-dependent gypsy insulator and were found to affect both CTCF binding at the variable site and the chromatin topology. This suggests that insertion of the gypsy insulator in this region interferes with CTCF function and supports a model for the normal function of the variable CTCF site as a chromatin loop facilitator, promoting interaction between Ubx enhancers and the Ubx transcription start site (Magbanua, 2014).

Boundary swapping in the Drosophila Bithorax complex

Although the boundary elements of the Drosophila Bithorax complex (BX-C) have properties similar to chromatin insulators, genetic substitution experiments have demonstrated that these elements do more than simply insulate adjacent cis-regulatory domains. Many BX-C boundaries lie between enhancers and their target promoter, and must modulate their activity to allow distal enhancers to communicate with their target promoter. Given this complex function, it is surprising that the numerous BX-C boundaries share little sequence identity. To determine the extent of the similarity between these elements, tests were performed to see whether different BX-C boundary elements can functionally substitute for one another. Using gene conversion, the Fab-7 and Fab-8 boundaries were exchanged within the BX-C. Although the Fab-8 boundary can only partially substitute for the Fab-7 boundary, it was found that the Fab-7 boundary can almost completely replace the Fab-8 boundary. The results suggest that although boundary elements are not completely interchangeable, there is a commonality to the mechanism by which boundaries function. This commonality allows different DNA-binding proteins to create functional boundaries (Iampietro, 2008).

The fact that Fab-7 can substitute for Fab-8 means that everything required to restore Fab-8 function is present in the Fab-7 fragment inserted. However, at the DNA sequence level, the Fab-7 and Fab-8 boundaries share almost no similarity. A detailed analysis of the two sequences using dot-plot and Markov analysis found little in common between the two elements other than GAGA factor-binding sites (six in Fab-7 and two in Fab-8). The GAGA factor binding sites have been shown to be important for Fab-7 enhancer blocking activity in transgenic contexts. However, the role of the GAGA factor in Fab-8 enhancer blocking activity is still unknown. Thus far, the only factor shown to be important for Fab-8 function is the dCTCF factor. It has been shown that deleting the dCTCF-binding sites in Fab-8 impairs its insulator function in transgenic insulator assays. Moreover, dCTCF mutants display phenotypes reminiscent of Fab-8 mutants. Since Fab-7 is one of the few BX-C boundaries to which dCTCF oes not bind, the results show that dCTCF is not absolutely required for Fab-8-like function (Iampietro, 2008).

In the case of the F8>>F7 conversion, it was found that the iab-7 cis-regulatory domain is capable of bypassing a boundary element that it never has to bypass but in the case of the F7>>F8 conversion, it was found that iab-6 is partially blocked by a boundary element that it must normally bypass (Fab-8 is located between iab-6 and the Abd-B promoter). One possible explanation for this discrepancy is that the Fab-8 fragment inserted lacked a specific element required for insulator bypass. Although this is a possibility, it is thought not to be the case. Both the Fab-7 and Fab-8 regions have been extensively scanned for elements allowing insulator bypass. In these attempts, elements called promoter-targeting sequences (PTSs) have been identified that allow enhancers to bypass insulator elements on reporter transgenes. In the current experiments, the smallest characterized boundary deletions were replaced with the smallest characterized insulator fragments. In both cases, molecular data suggest that the fragments that were introduced were separated from any PTS-type activity, but were capable, in transgenic contexts, of being bypassed by known PTS elements. Conversely, the deletions created were chosen to be clean boundary deletions; as much as possible, all known nearby elements, including PTS elements, were left intact. In the F7>>F8 substitution, for example, the entire PTS-6 element that was capable of bypassing the identical Fab-8 insulator fragment is still present (Iampietro, 2008).

Therefore, if no PTS-type elements were deleted, the main difference between the cases tested is context. For example, in the wild-type situation, Fab-8 is located between the iab-7 and iab-8 cis-regulatory domains, whereas in F7>>F8, Fab-8 is placed between the iab-6 and iab-7 cis-regulatory domains. It was recently found that the Fab-7 boundary seems to be regulated along AP axis. If it is assumed that all boundaries behave in a similar manner, then Fab-8 would also be regulated along the AP axis. As this regulation does not seem to come from the boundary element itself, it must come through specific interactions with the nearby cis-regulatory domains. Previous work has pointed to PTS elements as the mediators of this function. However, based on the data and because PTS deletions have little phenotype when deleted, it is believed that there must be something more that inactivates boundary elements. For now, the identity of these elements remains a mystery (Iampietro, 2008).

Regulation at Ubx Polychrome Response Elements

The DNA-binding Polycomb group protein Pleiohomeotic mediates silencing of a Drosophila homeotic gene

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 (PRE_1.6) 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 PRE_1.6 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 PRE_1.6 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 PRE_1.6 (Fritsch, 1999).

Subfragments of the 1.6 kb PRE have been tested for silencing function. PRE silencer is contained in the central 567 bp PRE_D 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 PRE_D 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 PRE_D requires PcG gene function. It was next examined whether Pho protein binds directly to PRE_D. 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 PRE_D 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 PRE_D 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 PRE_D transformant line, a strong additional signal was found at the transposon insertion site. These data suggest that Pho protein binds directly to PRE_D 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 PRE_D 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 PRE_D fragment was inserted into the PBX-IDE reporter gene to obtain PRE_Dphomut. These PRE_Dphomut 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 pho^b 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 PRE_D 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 PRE_Dphomut 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 PRE_Dphomut reporter gene, silencing is probably never established. Conversely, silencing of the PRE_D 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 PRE_D 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).

Structure of a Ubx polycomb response element and in vitro binding of polycomb group complexes containing GAGA factor

Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. The GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs (Horard, 2000).

Dissection of the PRE reveals that it is a compound region containing several sequences that are able (to different extents) to induce variegated expression of the miniwhite gene, respond to PcG mutations, and create new binding sites for PcG proteins on polytene chromosomes. The separate fragments are definitely weaker in activity than the whole. A single copy of a fragment containing different restriction enzyme fragments (BP, AB, and part of HA) silences very effectively, indicating that the different sequences normally cooperate to achieve more complete silencing to a degree that is not attained by multiple tandem copies of one fragment. The different subfragments most likely contribute complementary functions, but it has not been possible to demonstrate that different PcG proteins interact with different subfragments. As with the entire PRE, the response to different PcG mutations depends strongly on the site of insertion of the transposon construct. The genomic context makes therefore a strong contribution not only to the strength of the silencing but also to the relative importance of the different PcG components of the silencing complex. The activity of PRE-containing transposons inserted at different sites suggests that this contribution is due not only to sequences flanking the insertion site but also to the interaction in trans with other genomic PRE sites (Horard, 2000).

Only one of the three subfragments tested in embryos, BP, was able to maintain repression of the Ubx-lacZ reporter gene. This could be due simply to the relative PRE strengths of the different fragments. That is, increasing the number of copies of the other fragments might achieve the same silencing strength. Another possibility is that the complex formed at the BP fragment is qualitatively different from that recruited by the other fragments; for example, it might be able to recruit PcG proteins sufficiently early in embryonic development to have an effect on the Ubx-lacZ gene, while other PRE fragments might be able to institute silencing only at later stages. Different affinities for PcG complexes could also account for the different abilities to create binding sites for PcG proteins on polytene chromosomes. However, the fact that the PF fragment, though able to induce variegation at a high rate and to bind PcG proteins on polytenes, failed to show any detectable PcG complex formation in the immunoprecipitation assays suggests that the nature and composition of the complexes and/or the mode and timing of their recruitment are likely to differ for the different fragments (Horard, 2000).

The in vitro experiments show that GAGAG-containing sequences (GAGAG is the consensus binding site for GAGA protein) are binding sites for PcG complexes and that the GAGA factor is associated with PcG complexes present in the nuclear extracts. Ion exchange chromatography of nuclear extracts confirms that, while PcG proteins elute over a broad range of salt concentrations, the in vitro binding activity constitutes a small minority and copurifies with the GAGA factor. The multiplicity and heterogeneity of PcG complexes present in nuclear extracts would not be detected in affinity-based purification schemes. In contrast, the GAGA factor, along with Polyhomeotic, is found in a multiprotein complex that binds in vitro to PRE regions corresponding to ours. The possibility that some other PcG protein also recognizes the GAGA consensus sequence cannot be excluded, but the association of the GAGA factor with PcG complexes shows that it is most likely involved in at least one mode of PcG binding to PRE DNA. Does this reflect a role for the GAGA factor in PcG silencing in vivo? The GAGA factor was originally identified as a transcription-stimulating factor both in vivo and in vitro and was classified as a trxG protein because it stimulated the activity of homeotic genes while its mutants had phenotypes indicative of homeotic insufficiency. However, some evidence suggests that it can also be associated with repressive functions. The GAGA factor, together with another activator, NTF-1, also binds to an 11-bp element required for the repression of tailless by the torso-dependent pathway. Evidence that it might be involved in PRE function consists of the fact that GAGA mutations decrease the silencing effected by the Fab-7 PRE. In the Ubx gene, the bxd PRE region contains the largest concentration of GAGA binding sites. If each continuous G(AG)n stretch is taken as one binding site, the 1-kb interval containing the core of the PRE contains 13 sites while the next highest concentration (8 sites) is found in a 1-kb region containing the bx PRE (not to be confused with the BX enhancer). Chromatin cross-linking and immunoprecipitation experiments confirm that these regions bind the GAGA factor in vivo. The results suggest that at these sites the GAGA factor is not an antagonist of silencing and is not simply an accessory or a facilitator of PcG complex formation but may, in concert with other factors, contribute to targeting PcG complexes (Horard, 2000).

It was surprising, in view of in vitro results, that the effects of Trl mutations on either miniwhite variegation or the silencing of the Ubx-lacZ reporter are sporadic and strongly dependent on the insertion site. One possible explanation is that, in vivo, the GAGA factor is only one of a set of DNA-binding recruiting proteins and that, while it contributes to, it is not essential for, the assembly of PcG complexes. Chromatographic fractionation of nuclear extracts indicates in fact that only a fraction of the PcG complexes present in embryonic extracts are associated with the GAGA factor. Furthermore, embryos contain an important maternal supply of the GAGA factor, which would mask the effect of a reduced zygotic contribution. Later, other recruiting factors might be involved. Finally, the results cannot exclude the possibility that, although the GAGA factor is (1) a component of PcG complexes, (2) can target their binding in vitro, and (3) is apparently important for the function of the Fab-7 PRE, it is not primarily involved in recruitment at the bxd PRE. Instead, its role might be primarily architectural. The GAGA factor binds to DNA as a multimer that recognizes clustered GAGA consensus sequences, and it has been argued that such binding would be expected to bend DNA in a way incompatible with nucleosome assembly. GAGA binding would then clear the PRE core of nucleosomes and bend it to facilitate interactions among other DNA-binding components (Horard, 2000).

The presence of GAGA binding sites alone appears to be sufficient in vitro to bind a PcG complex since not only the PRE fragments but also the Ubx promoter and the hsp70 promoter bind, though they have no known PcG silencing activity in vivo. In addition, a GAGA-containing oligonucleotide also binds efficiently to PcG complexes. Nevertheless, GAGA protein binding to a DNA sequence is not sufficient to recruit PcG complexes in vivo. Clearly the in vitro binding reaction does not reflect the in vivo activity. The most probable explanation of this discrepancy is that the binding detected in vitro is due to complexes that are preassembled in vivo and are then dissociated from the chromatin during the preparation of nuclear extracts. If the nature and composition of PcG complexes are templated by the PREs at which they are assembled, GAGA-containing PcG complexes would be efficiently targeted to GAGA binding sites in vitro while, in vivo, complex formation would require the de novo recruitment and assembly of PcG complexes, involving other DNA binding components or cofactors. This interpretation is favored because it would also explain the variable compositions of PcG complexes detected at different chromosomal sites. In vivo, the large majority of GAGA binding sites visible on polytene chromosomes are not associated with PcG binding, suggesting that only a small fraction of the GAGA protein is involved in PcG complexes. This interpretation also accounts for the fact that the LexA-GAGA protein cannot recruit PcG complexes to LexA binding sites. It is also noted that the target of PcG complexes in vivo is chromatin, not naked DNA. The presence of nucleosomes might normally increase the selectivity, allowing PcG complexes to assemble only at sites where other recruiting or architectural proteins are also bound (Horard, 2000).

In view of these results, the existence of GAGA sites at the Ubx promoter raises other possibilities. In the presence of a PRE, a GAGA factor bound at the Ubx promoter might participate in the silencing activity by interacting with GAGA-containing PcG complexes recruited at the PRE, mediating or contributing to promoter silencing. Both the hsp70 and hsp26 promoters are efficiently repressed by the presence of a PRE in the same transposon construct. The GAGA factor might contribute to silencing in these cases also. The miniwhite gene, which is also silenced by the PRE, does not contain typical clustered GAGA sites in its promoter region but only a few scattered sites in the transcribed region. The expression of the miniwhite gene is strongly dependent on the site of insertion and on distant enhancers within or outside of the transposon construct. The silencing of these enhancers might be in part responsible for the effect of the PRE on miniwhite expression. Alternatively, other proteins binding to the miniwhite promoter region might interact with PcG complexes (Horard, 2000).

The immunoprecipitation experiments also detected binding that is not competed by GAGA oligonucleotides with PRE fragments that do not contain consensus GAGA binding sites. This implies that other recognition sequences and other DNA-binding proteins are involved in these cases. The recent discovery that Pleiohomeotic (a Drosophila PcG protein homolog of the mammalian YY-1 factor) binds to DNA suggests that it might be one such recruiter of PcG complexes. There are in fact a number of putative Pho binding sites with the minimal consensus GCCAT in the PRE region: one in AB, two in BP (a third site is destroyed by the BglI cleavage), and three in the PF fragment. These bind Pho protein in vitro and are important for PRE activity in vivo. However, none are found in the HH or HA fragments; hence these presumably depend on other recruiting proteins. However, the PF fragment, though it contains three putative Pho sites, is conspicuous for its inability to bind PcG complexes in extracts, suggesting that Pho is either not present in the complex containing Pc and Psc or does not interact directly with it. The fact that the mammalian Pho homolog YY-1 causes sharp bends in the DNA raises the possibility that Pho too might serve a primarily architectural role without necessarily interacting directly with PcG complexes (Horard, 2000).

Although PF does not contain GAGA sites, it is almost as effective in inducing PcG-dependent variegation of the miniwhite gene as the BP fragment and it can generate new PcG binding sites at the site of insertion on polytene chromosomes. Yet PF cannot maintain repression of the Ubx-lacZ reporter gene in embryos. One possible explanation for these results is that PF is the target for yet another PcG recruiting mechanism that either functions poorly under these in vitro binding conditions or depends on proteins that are not present in the embryonic extracts. The fact that the PF fragment can recruit silencing complexes in larval cells but cannot maintain repression in the embryo would be consistent with a requirement for proteins present only at later developmental stages. Another possible explanation is that PF does interact with certain PcG complexes which do not include PC or PSC and hence escaped detection (Horard, 2000).

The picture of the PRE that emerges from these experiments is that of a mosaic of multiple interaction sites which may require different DNA-binding proteins to recruit PcG components. A similar conclusion has been reached, based on deletions that abolish the activity of the bxd PRE and by an in vitro binding approach similar to that reported in this study. GAGA sites are associated with some PREs but not others (e.g., the Mcp PRE). If the GAGA factor acts as a recruiting protein, it is most likely only one of many possible recruiters. Different recruiters might interact specifically with different PcG proteins, accounting for the fact that the binding sites for different PcG proteins on polytene chromosomes do not completely coincide. Nevertheless, the ability of PcG proteins to interact with one another or to enter into a chain of recruitment means that, in most cases, strong binding sites for one PcG protein will be able to recruit at least to some degree the other PcG proteins. The difference between direct and indirect recruitment may be responsible for the fact that a strong chromosomal binding site for one PcG protein is sometimes a weak binding site for another PcG protein (Horard, 2000 and references therein).

pipsqueak encodes a factor essential for sequence-specific targeting of a polycomb group protein complex

pipsqueak is a sequence-specific DNA binding protein that targets a Polycomb group protein complex to Polycomb response elements (PREs). The Polycomb (Pc) group (Pc-G) of repressors is essential for transcriptional silencing of homeotic genes that determine the axial development of metazoan animals. It is generally believed that the multimeric complexes formed by these proteins nucleate certain chromatin structures to silence promoter activity upon binding to PREs. Little is known, however, about the molecular mechanism involved in sequence-specific binding of these complexes. An immunoaffinity-purified Pc protein complex has been shown to contains a DNA binding activity specific to the (GA)n motif in a PRE from the bithoraxoid region of Ultrabithorax. This activity can be attributed primarily to the large protein isoform encoded by pipsqueak (psq) instead of to the well-characterized GAGA factor Trithorax-like. The functional relevance of psq to the silencing mechanism is strongly supported by its synergistic interactions with a subset of Pc-G that cause misexpression of homeotic genes (Huang, 2002).

An ~440-bp DNA fragment from the bithoraxoid region of Ubx can recapitulate both positive and negative effects of trx and Pc, respectively. Immunoaffinity chromatography has been used to purify tagged Pc-G complexes and then their DNA binding activity was assayed. The (GA)n motif in this fragment has been found to be the primary binding site for the Pc-G complexes. Several lines of evidence are presented to show that the DNA binding protein for the Ubx PRE is encoded by pipsqueak (Huang, 2002).

Several lines of evidence are provided to show that a novel DNA binding factor encoded by psq is a constituent of CHRASCH (chromatin-associated silencing complex for homeotics), a previously characterized major Pc-G protein complex. Since CHRASCH also contains a histone modification factor, HDAC1, it is suggested that this complex may represent a fully functional entity that can nucleate certain chromatin structures at and around specific sequences (i.e., PRE) of homeotic genes (Huang, 2002).

The bxd region has been extensively examined for polycomb response elements. Although different fragments ranging from ~400 bp to ~1 kb have been studied, they share a common region represented almost entirely by the B-151 fragment analyzed in this study. Among the three binding motifs of this fragment, it was found that the (GA)_n motif represents the most prominent binding site for CHRASCH. In recent studies, the role of this motif in silencing has been demonstrated in transgenic flies. Thus, it is believed that this motif plays a critical role in anchoring one of the major Pc-G complexes (i.e., CHRASCH). These results, however, are not mutually exclusive to the possibility that other motifs may be required for different functional aspects of PRE (Huang, 2002).

GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element

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).

Hierarchical recruitment of polycomb group silencing complexes

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).

A PRE from the Drosophila Hox gene Ultrabithorax potently silences different heterologous enhancers and promoters that are normally not subject to Polycomb repression

Polycomb response elements (PREs) are cis-regulatory sequences required for Polycomb repression of Hox genes in Drosophila. PREs function as potent silencers in the context of Hox reporter genes and they have been shown to partially repress a linked miniwhite reporter gene. The silencing capacity of PREs has not been systematically tested and, therefore, it has remained unclear whether only specific enhancers and promoters can respond to Polycomb silencing. Using a reporter gene assay in imaginal discs, it has been shown that a PRE from the Drosophila Hox gene Ultrabithorax potently silences different heterologous enhancers and promoters that are normally not subject to Polycomb repression. Silencing of these reporter genes is abolished in PcG mutants and excision of the PRE from the reporter gene during development results in loss of silencing within one cell generation. Together, these results suggest that PREs function as general silencer elements through which PcG proteins mediate transcriptional repression (Sengupta, 2004).

A 1.6 kb fragment encompassing the PRE from the Ubx upstream control region was tested for its capacity to prevent transcriptional activation by enhancers from genes that are normally not under PcG control. For this purpose, three different enhancers were tested in a lacZ reporter gene assay in imaginal discs: dpp^WE, the imaginal disc enhancer from the decapentaplegic (dpp) gene; vg^QE the quadrant enhancer from the vestigial (vg) gene; and vg^BE, the vg D/V boundary enhancer. If linked to a reporter gene, each of these enhancers directs a distinct pattern of expression in the wing imaginal disc and activation by each enhancer is regulated by transcription factors that are controlled by a different signaling pathway. Specifically, the dpp enhancer contains binding sites for the Ci protein and is activated in response to hedgehog signaling, the vg quadrant enhancer contains binding sites for the Mad transcriptional regulator and is activated in response to dpp signaling, and the vg boundary enhancer contains binding sites for the Su(H) transcription factor and is regulated by Notch signaling. The dpp^WE, vg^QE and vg^BE enhancers were individually inserted into a lacZ reporter gene construct that contained the PRE fragment and either a TATA box minimal promoter from the hsp70 gene (here referred to as TATA), or a 4.1 kb fragment of the proximal Ubx promoter (here referred to as Ubx_P), fused to lacZ. In each construct, the PRE fragment was flanked by FRT sites that permit excision of the PRE fragment by flp recombinase. Several independent transgenic lines for each of the six PRE transgenes were generated. From individual transgene insertions, derivative transgenic lines were then generated by flp-mediated excision of the PRE in the germline. Thus expression of individual transgene insertions could be compared in the presence and absence of the PRE by staining wing imaginal discs for ß-galactosidase (ß-gal) activity. In the absence of the PRE, each of the three enhancers tested directs ß-gal expression in a characteristic previously characterized pattern. Each enhancer activated expression in the same pattern from either the TATA box minimal promoter or the Ubx promoter with some minor, promoter-specific differences with respect to the expression levels. By contrast, in most of the parental transformant lines, i.e., those carrying the corresponding reporter gene with the PRE, ß-gal expression is completely suppressed. These observations suggest that the PRE fragment very potently silences each of the six reporter genes. It is noted, however, that, at some transgene insertion sites, efficiency of silencing by the PRE fragment appeared to be impeded by flanking chromosomal sequences; in these cases, it was found that ß-gal expression is activated even in the presence of the PRE (Sengupta, 2004).

To test whether silencing of the reporter genes by the PRE depends on PcG gene function, the PRE-containing transgenes >PRE>dpp^WE-TATA-lacZ and >PRE>vg^QE-Ubx-lacZ were introduced into larvae that carried mutations in the PcG gene Suppressor of zeste 12 [Su(z)12]. Su(z)12 encodes a core component of the Esc-E(z) histone methyltransferase. Silencing of both transgenes is lost in Su(z)12²/Su(z)12³ mutant larvae, and the transgenes express ß-gal expression at levels comparable with the transgene derivatives that lack the PRE fragment. Taken together, these observations suggest that the 1.6 kb PRE fragment from Ubx is a very potent general transcriptional silencer element that represses transcription in a PcG protein-dependent manner. Thus, it appears that this PRE acts indiscriminately to block transcriptional activation by a variety of different activator proteins (Sengupta, 2004),

To test the long-term requirement for the PRE for silencing of these reporter genes, the PRE was excised during larval development and ß-gal expression was then monitored at different time points after excision. Forty-eight hours after induction of flp expression, all six reporter genes showed robust derepression of ß-gal, suggesting that, in each case, removal of the PRE results in the loss of PcG silencing. Among the different enhancer-promoter combinations used in this study, the dpp^W enhancer fused to the TATA box minimal promoter appears to direct the highest levels of lacZ expression; >PRE>dpp^W–TZ transformant lines consistently show the strongest ß-gal staining after excision of the PRE. Therefore >PRE>dpp^W-TZ transformants were analyzed at 4, 8, 12 and 24 hours after induction of flp expression to study the kinetics of this derepression. No ß-gal signal was detected at 4 hours or even at 8 hours after flp induction, but 12 hours after flp induction, all discs showed robust ß-gal expression. Thus, even in the case of the most potent enhancer-promoter combination used (i.e. dpp^W enhancer and TATA box minimal promoter), a delay of 12 hours between flp induction and ß-gal expression was observed. Since the average cell cycle length of imaginal disc cells in third instar larvae is 12 hours, this implies that most disc cells have undergone a full division cycle within this period. Derepression of the reporter gene in this experiment requires several steps: (1) excision of the PRE by the flp recombinase; (2) dissociation of the PRE and PcG proteins attached to it -- possibly by disrupting PcG protein complexes formed between the PRE and factors bound at the promoter, and (3) transcriptional activation by factors binding to the enhancer in the construct. It is possible that one or several steps in this process require a specific process during the cell cycle (e.g., passage through S phase) (Sengupta, 2004),

These experiments here show that three reporter genes, each containing a different enhancer linked to a canonical TATA box promoter, are completely silenced by a PRE placed upstream of the enhancer. The data suggest that PcG proteins that act through this PRE prevent indiscriminately activation by a variety of different transcription factors. The PcG machinery thus does not seem to require any specific enhancer and/or promoter sequences for repression (Sengupta, 2004),

Two points deserve to be discussed in more detail. The first concerns the stability of silencing imposed by a PRE. Previous studies have suggested that transcriptional activation in the early embryo could prevent the establishment of PcG silencing by PREs. More specifically, early transcriptional activation of Hox genes by blastoderm enhancers may play an important role in preventing the establishment of permanent PcG silencing in segment primordia in which Hox genes need to be expressed at later developmental stages. Importantly, none of the three enhancers used in this study is active in the early embryo. Moreover, these enhancers probably do not contain binding sites for specific transcriptional repressors, such as the gap repressors, which are required for establishment of PcG silencing at some PREs in the early embryo. It is therefore imagined that, in these constructs, PcG silencing complexes assemble by default on the 1.6 kb Ubx PRE in the early embryo and that PcG silencing is thus firmly established by the stage when the imaginal discs enhancers would become active. Silencing by the PRE during larval stages therefore appears to be dominant overactivation and cannot be overcome by any of the enhancers used in this study. There is other evidence in support of the idea that PcG silencing during larval development is more stable than in embryos. In particular, a PRE reporter gene that contains a Gal4-inducible promoter is only transiently activated if a pulse of the transcriptional activator Gal4 is supplied during larval development; by contrast, a pulse of Gal4 during embryogenesis switches the PRE into an 'active mode' that supports transcriptional activation throughout development. Furthermore, recent studies in imaginal discs suggest that there is a distinction between transcriptional repression and the inheritance of the silenced state; the silenced state can be propagated for some period even if repression is lost. Specifically, loss of Hox gene silencing after removal of PcG proteins in proliferating cells can be reversed if the depleted PcG protein is resupplied within a few cell generations. Taken together, it thus appears that PcG silencing during postembryonic development is a remarkably stable process. Finally, the results reported in this study also imply that, once PcG silencing is established, Hox genes can `make use of virtually any type of transcriptional activator to maintain their expression; PcG silencing will ensure that activation by these factors only occurs in cells in which the Hox gene should be active. The analysis of Ubx control sequences supports this view; if individually linked to a reporter gene, most late-acting enhancers direct expression both within as well as outside of the normal Ubx expression domain (Sengupta, 2004),

The second point to discuss concerns the repression mechanism used by PcG proteins. Biochemical purification of PRC1 has revealed that several TFIID components co-purify with the PcG proteins that constitute the core of PRC1. Moreover, formaldehyde crosslinking experiments in tissue culture cells showed that TFIID components are associated with promoters, even if these are repressed by PcG proteins. This suggests that PcG protein complexes anchored at the PRE interact with general transcription factors bound at the promoter. One possibility would be that PcG repressors directly target components of the general transcription machinery to prevent transcriptional activation by enhancer-binding factors. Three distinct activators act through the three enhancers used in this study and, according to these results, none of them is able to overcome the block imposed by the PcG machinery. But how do the known activities of PcG protein complexes [i.e., histone methylation by the Esc-E(z) complex and inhibition of chromatin remodeling by PRC1] fit into this scenario? Both these activities may be required for the repression process by altering the structure of chromatin around the transcription start site and thus prevent the formation of productive RNA Pol II complexes. Other scenarios are possible. For example, histone methylation may primarily serve to mark the chromatin for binding of PRC1 through Pc, and PRC1 components such as Psc then perform the actual repression process. Whatever the exact repression mechanism may be, the PRE-excision experiment shows that this repression is lost within one cell generation after removal of the PRE. This implies that changes in the chromatin generated by the action of PcG proteins cannot be propagated by the flanking chromatin (Sengupta, 2004),

YY1 DNA binding and PcG recruitment requires CtBP

Mammalian Polycomb group (PcG) protein YY1 (see Drosophila Pleiohomeotic) 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 (PRE_D), 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 PRE_D, 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 PRE_D 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).

Intergenic transcription through a polycomb group response element counteracts silencing

Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, it was shown that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system (Schmitt, 2005).

A previous study has shown that in wild-type Drosophila embryos, the characterized PREs of the BX-C are transcribed in a spatially and temporally regulated manner which reflects the pattern of activity of their associated genes. The analysis of the transgenic system used in this study indicates that transcription through the Fab-7 element until the beginning of the first-instar larval stage is sufficient for the stable activation of this PRE. Since these data were obtained in a transgenic background, it was next asked whether endogenous PRE sequences are transcribed only during early development or whether transcription persists until later stages (Schmitt, 2005).

In the brain of third-instar larvae, the homeotic genes are expressed in a characteristic pattern along the anterior-posterior axis, following the principle of spatial colinearity. If transcription through PREs is required for the maintenance of epigenetic activation, it would be expected that the bxd, Mcp, and Fab-7 elements would also be transcribed in this tissue. By in situ hybridizations, RNAs spanning the bxd, Mcp, and Fab-7 sequences were detected in a pattern that reflects the expression of the target genes Ubx and AbdB, respectively. PRE transcripts were detected only in the sense direction with respect to the orientation of the coding genes. In contrast to the mRNAs, the noncoding RNAs showed a punctate staining in the brain tissue, suggesting that they are localized to the nuclei. Fluorescent in situ hybridizations in combination with DAPI staining showed the Fab-7 transcripts in the brain of third-instar larvae to appear as single or doublet spots within the cell nuclei. In line with previous studies, in which noncoding transcripts through infra-abdominal (iab) regions of the BX-C were found to be restricted to the nucleus in embryos, the Fab-7, Mcp, and bxd RNAs were detected at discrete loci within the nucleus at this stage. It is expected that these signals represent nascent, noncoding transcripts, suggesting that they may be degraded following their synthesis. To determine the relative amounts of RNA accounting for these signals, the expression levels of total and nascent AbdB mRNA were compared with the level of noncoding Fab-7 RNA in SF4 tissue culture cells by real-time RT-PCR. The results from this analysis showed that the level of Fab-7 RNA is approximately sixfold less abundant than the amount of AbdB target mRNA (Schmitt, 2005).

The finding that PRE transcripts can be detected in the brain of third-instar larvae suggests that in the context of the endogenous BX-C, continued transcription through PREs may be required for the stable maintenance of the epigenetically activated state (Schmitt, 2005).

Apart from the homeotic genes, the PcG/trxG memory system is known to regulate many more targets. It was thus asked whether PcG-regulated gene transcription is accompanied by transcription through the associated PREs at loci other than the BX-C. To test this, RNA probes were designed directed against the genetically characterized engrailed (en) PRE, as well as against three predicted PREs that potentially regulate the genes slouch (slou), spalt major (salm), and tailless (tll). The genetically characterized PRE of the en gene and the predicted PREs of the salm and tll genes are localized in the respective promoter regions, whereas the predicted PRE of the slou gene lies 3 kb upstream of the transcriptional start site. In situ hybridizations showed that the en PRE as well as the predicted PREs of the slou, salm, and tll genes are transcribed in embryos. The transcription through these sequences shows the same spatial regulation as that of the mRNAs of the known (en) and predicted target genes (slou, salm, and tll). For the en, slou, and salm PREs, transcripts of both sense and antisense strands were detected, whereas transcripts of the tll PRE were only detected in the antisense orientation. In addition, the putative PRE transcripts display a nuclear localization similar to that of the bxd, Mcp, and Fab-7 RNAs. Three of the transcribed PRE sequences lie in the promoter regions of their associated genes. Interestingly, the antisense strand of the predicted tll PRE is also transcribed in the optic lobes of the brain in third-instar larvae, where the tll mRNA is also expressed. In contrast, no transcription was detected through the en and salm PREs at this stage, although the mRNAs for both genes are transcribed at high levels (Schmitt, 2005).

These results suggest that the activation of a gene does not result in spurious transcription through its promoter region, but rather that the transcription observed in embryos is involved in triggering the epigenetic activation of the known and predicted PREs tested. The fact that transcripts were detected only through the tll PRE and not through the en and salm PREs later in development suggests that the maintenance of the activated state may be regulated by different mechanisms in different tissues. Alternatively, the en and salm genes might be controlled by more than one PRE, which are differentially deployed in different tissues and would thus show a differential pattern of epigenetic activation and noncoding transcription (Schmitt, 2005).

This work has present evidence that transcription through a PRE prevents silencing and switches the maintenance mode of the element to activity. These results with the transgene system indicate that noncoding transcription is not a consequence of gene activation but is causally related to the prevention of silencing. The process of transcription appears to be central to this mechanism, while the RNAs produced seem to play a minor role, if they do have a function at all (Schmitt, 2005).

Intergenic transcription in the BX-C has a profound phenotypic effect on the expression of the Hox genes. The results with transgenes suggest that the spatially and temporally regulated transcription of noncoding RNAs in the BX-C induces a remodeling of the chromatin, thereby preventing PcG-mediated silencing. The consequence of this is the segment-specific activation of the Hox genes. This is probably especially important in large gene complexes where PREs are located at long distances from the promoters they regulate. It has been shown that the activation of a minimal 219-bp Fab-7 PRE is not accompanied by transcription through the element. However, in this case the minimal PRE was juxtaposed to the promoter, probably benefiting from the open chromatin environment induced by the bound transcription factors. Other results indicate that an 870-bp large Fab-7 PRE, under similar conditions but containing more PcG protein-binding sites, cannot be activated anymore. This suggests that over a certain threshold level of silencing, imposed by the stability of the silencing complexes, chromatin remodeling by transcription is required to remove PcG complexes in order to counteract their silencing activity in a mitotically heritable fashion (Schmitt, 2005).

Hox cluster regulation is only part of the entire PcG/trxG memory system, prompting an analysis of the transcription pattern of characterized en and predicted salm, slou, and tll target genes as well as their respective PRE sequences. RNA in situ hybridizations reveal that these sequences are also transcribed in a pattern that reflects the expression of the cognate target genes. This suggests that the mechanism of epigenetic activation of PREs initially described for the homeotic gene clusters may be required for the regulation of many more PcG target genes than previously thought. Transcription through these PREs can be either uni- (tll PRE) or bidirectional (en, slou, and salm PREs), further suggesting that the induction of epigenetic activation relies on the remodeling of chromatin induced by the transcriptional process, rather than by the noncoding RNAs (Schmitt, 2005).

With the transgenic reporter system, it was shown that the constitutive transcription through the Fab-7 PRE from the actin5c promoter results in the stable epigenetic activation of this element. This raises the question of how this could be achieved mechanistically. As has been proposed for the developmental regulation of globin gene expression and the regulation of VDJ_H-recombination in mice, the opening of the chromatin structure at a transcribed PRE may be induced by the passing of the transcriptional machinery through the regulatory sequence. It has been shown that the elongating RNA polymerase II complex is associated with the SWI/SNF remodeling complex and a histone acetyl transferase activity. Such enzymatic activities linked with the transcription machinery may catalyze the epigenetic modification of chromatin (Schmitt, 2005).

In this respect, it is interesting to note that most of the trxG mutants were initially uncovered as suppressors of the Pc phenotype, and that the combination of PcG with trxG mutations can restore the typical phenotype of the single mutations to wild type. The molecular mechanism behind this antagonism is not clear. Using clonal analysis, it has been shown that the trxG proteins Ash1 and Trx do not function as coactivators of Hox gene expression, but that they are required as anti-repressors to prevent PcG-induced silencing. In contrast, the Brahma complex containing the trxG proteins Brm, Osa, and Mor to acts as a coactivator of transcription, and a subset of trxG genes encode components of the Mediator complex. The finding that transcription through Fab-7 induces the epigenetic activation of this PRE may explain how trxG complexes involved in more general transcriptional processes antagonize the establishment of PcG silencing (Schmitt, 2005).

Interestingly, in budding yeast, intergenic transcription through a promoter has been shown to prevent the binding of a transcriptional activator to its target sequences. It is possible that, in a similar fashion, the transcription through PREs may lead to the displacement of repressive PcG complexes from the chromatin and/or the prevention of PcG recruitment to the PRE in the first place. Enzymatic activities carried by the RNA polymerase II complex may subsequently or in addition modify histones at PREs with positive epigenetic marks like acetyl or methyl moieties. Interestingly, has been shown that the sequential induction of HoxB gene expression in mouse embryonic stem (ES) cells by retinoic acid correlates with the orchestrated looping out of this locus from chromosome territories. This indicates that, in addition to locus-wide changes in the chromatin structure such as histone modifications, the transcriptional activity of genes may be regulated by an additional order of complexity. It is possible that, in addition to inducing 'small-scale' changes in the chromatin structure, transcription through PREs may lead to a subnuclear relocation of the target gene locus from a repressive into a transcriptionally permissive environment (Schmitt, 2005).

Removal or inhibition of binding of PcG silencing complexes to PREs by tissue-specific transcription appears to be an attractive mechanism to counteract the constant pressure of the repressive system acting by default. However, with such a solution, the problem of epigenetic maintenance is simply moved to another level, since the question arises of what prevents the intergenic transcription from being silenced by the PcG. The simple answer -- promoters of noncoding transcripts are not sensitive to PcG/PRE silencing -- is probably not valid. Noncoding transcripts of the BX-C are activated by the same set of early segmentation genes as the Hox protein encoding mRNAs. As such, their subsequent regulation might be subjected to the same regimen of factors as the protein-encoding transcripts. However, the problem of transcriptional memory might be reduced to the problem of how to inhibit PcG silencing in particular cells/tissues, while the rest will be down-regulated by default (Schmitt, 2005).

With the processive transcription as the central issue, the cell cycle might become an important factor for the maintenance of active transcription. In Drosophila, PcG proteins dissociate from the chromosomes at mitosis. Thus, if after mitosis intergenic transcription starts before PcG proteins rebind to the PREs, the chromatin would be turned into an active mode, and would thus be protected from PcG-mediated silencing until the next round of cell division. At this point, it remains an open question whether continuous transcription of noncoding RNAs is required throughout the cell cycle to prevent the PcG complexes from rebinding, or whether the initial setting of positive epigenetic marks by the early transcription process is sufficient to prevent silencing during interphase. This proposed mechanism further reduces the problem of how transcriptional activity is maintained to the problem of how only a positive epigenetic mark is maintained during DNA replication and mitosis. Here, recent advances in studies of histone variants propose some attractive candidate marks. In particular, the histone variant H3.3 associated with the transcription of active genes could be envisaged as a possible positive signal that is locally maintained and propagated at cell division. As has been suggested before, targeted deposition of the H3.3 variant at sites of active transcription may serve to remove repressive epigenetic marks such as methylation. The establishment of stable PcG silencing complexes not only requires a sequence component, but is also accompanied by the methylation of K9 and K27 of histone H3. In contrast, positive marks, which have been shown to be mainly associated with H3.3 compared to H3, would be specifically enriched at a transcribed PRE and transmitted through mitosis. After cell division, these epigenetic marks may then in turn provide a platform for noncoding transcription through the PRE early in the cell cycle, which itself may re-establish the full active chromatin environment, unsuitable for PcG protein binding and silencing. Additionally, the reported result that an activated PRE is still maintained over a certain period after transcription has ceased (by removal of the promoter by the inducible Cre recombinase) suggests that this positive epigenetic signal is quite stable and is only diluted out by multiple cell divisions (Schmitt, 2005).

In summary, it is proposed that transcriptional maintenance during development by the PcG/trxG system is primarily a process of preventing PcG silencing to occur at those target genes that need to be maintained active in a defined cell lineage. The advantage of this mode of action is that a positive epigenetic mark, surviving DNA replication and mitosis, is sufficient to ensure stable and heritable maintenance of gene expression patterns, since the silenced mode is created by default. As such, transcription of intergenic sequences would serve as an anti-silencing mechanism that would continually counteract the initiation of this PcG-mediated silencing. In the future, it will be important to pursue the involvement of the various trxG components in the establishment and maintenance of regulatory transcription mechanisms and to analyze the link to cell cycle control and the identity and propagation of the positive epigenetic marks required to sustain active transcription (Schmitt, 2005).

Synergistic recognition of an epigenetic DNA element by Pleiohomeotic and a Polycomb core complex

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). Next, the effects of PBE mutations on in vivo silencing were tested. Because the site of integration within the genome influences silencing, repression does not occur in all transgenic lines. Therefore, PSS is expressed as the percentage of lines that show repression. Independent lines were establised harboring the mini-white transgene under control of either the minimal 260-bp iab-7 PRE or the PBE mutant PRE (PBE^mt iab-7). 48 homozygous viable lines were raised with the wild-type PRE in front of the mini-white gene. In 46% of these lines, homozygotes (P[w⁺]/P[w⁺]) have much lighter eyes than their heterozygous (P[w⁺]/+) siblings, revealing PSS. In 8% of the lines, the eye color of homozygotes is about the same as that of heterozygotes, reflecting weak PRE-directed silencing. In the remaining 46% of the lines, no PSS was observed and the eyes of homozygotes were darker than that of heterozygotes. In summary, recruitment of a PcG repressing complex is observed in more than half of the generated lines. Strikingly, when the PBEs were mutated, only one line (4.5%) out of a total of 22 analyzed showed strong repression of the mini-white gene in homozygotes, and for five lines (23%), homozygotes had an eye color similar to that of their heterozygous siblings. It is worthwhile noting that in the case of the wild-type iab-7 PRE, the majority of repressed lines showed strong repression (22 of 26). In contrast, the majority of the repressed lines (five of six) harboring the mutant PRE display only weak silencing. Thus, not only is the proportion of repressed lines decreased in the mutant iab-7 PRE lines but the efficiency of repression is also lowered. These results strongly support the notion that the PBE is a critical PRE element, required for the assembly of a functional repressive PcG complex in vivo (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).

Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins

Polycomb group (PcG) and trithorax group (trxG) proteins act as antagonistic regulators to maintain transcriptional OFF and ON states of HOX and other target genes. To study the molecular basis of PcG/trxG control, the chromatin of the HOX gene Ultrabithorax (Ubx) was analyzed in Ubx^OFF and Ubx^ONcells purified from developing Drosophila. PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all constitutively bound to Polycomb response elements (PREs) in the OFF and ON state. In contrast, the trxG protein Ash1 is only bound in the ON state; not at PREs but downstream of the transcription start site. In the OFF state, extensive trimethylation was found at H3-K27, H3-K9, and H4-K20 across the entire Ubx gene; i.e., throughout the upstream control, promoter, and coding region. In the ON state, the upstream control region is also trimethylated at H3-K27, H3-K9, and H4-K20, but all three modifications are absent in the promoter and 5' coding region. These analyses of mutants that lack the PcG histone methyltransferase (HMTase) E(z) or the trxG HMTase Ash1 provide strong evidence that differential histone lysine trimethylation at the promoter and in the coding region confers transcriptional ON and OFF states of Ubx. In particular, these results suggest that PRE-tethered PcG protein complexes act over long distances to generate Pc-repressed chromatin that is trimethylated at H3-K27, H3-K9, and H4-K20, but that the trxG HMTase Ash1 selectively prevents this trimethylation in the promoter and coding region in the ON state (Papp, 2006; Full text of article).

Previous studies have shown that PhoRC contains the DNA-binding PcG protein Pho that targets the complex to PREs, and dSfmbt, a novel PcG protein that selectively binds to histone H3 and H4 tail peptides that are mono- or dimethylated at H3-K9 or H4-K20 (H3-K9me1/2 and H4-K20me1/2, respectively) (Klymenko, 2006). PRC1 contains the PcG proteins Ph, Psc, Sce/Ring, and Pc. PRC1 inhibits nucleosome remodeling and transcription in in-vitro assays and its subunit Pc specifically binds to trimethylated K27 in histone H3 (H3-K27me3). PRC2 contains the PcG proteins E(z), Su(z)12, and Esc as well as Nurf55, and this complex functions as a histone methyltransferase (HMTase) that specifically methylates K27 in histone H3 (H3-K27) in nucleosomes (Papp, 2006).

This study used quantitative X-ChIP analysis to examine the chromatin of the HOX gene Ubx in its ON and OFF state in developing Drosophila larvae. Previous genetic studies had established that all of the PcG and trxG proteins analyzed in this study are critically needed to maintain Ubx OFF and ON states in the very same imaginal disc cells in which their binding to Ubx was analyzed in this study. The following conclusions can be drawn from the analyses reported in this study. (1) The PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all highly localized at PREs, but they are all constitutively bound at comparable levels in the OFF and ON state. (2) The trxG protein Ash1 is bound only in the ON state, where it is specifically localized ~1 kb downstream of the transcription start site. (3) In the OFF state, PRC2 and other unknown HMTases trimethylate H3-K27, H3-K9, and H4-K20 over an extended 100-kb domain that spans the whole Ubx gene. (4) In the ON state, comparable H3-K27, H3-K9, and H4-K20 trimethylation is restricted to the upstream control regions and Ash1 selectively prevents this trimethylation in the promoter and coding region. (5) Repressed Ubx chromatin is extensively tri- but not di- or monomethylated at H3-K27, H3-K9, and H4-K20. (6) Trimethylation of H3-K27, H3-K9, and H4-K20 at imaginal disc enhancers in the upstream control region does not impair the function of these enhancers in the ON state. (7) TBP and Spt5 are bound at the Ubx transcription start site in the ON and OFF state, but Kis is only bound in the ON state. This suggests that in the OFF state, transcription is blocked at a late step of transcriptional initiation, prior to the transition to elongation. A schematic representation of PcG and trxG protein complex binding and histone methylation at the Ubx gene in the OFF and ON state is presented (Papp, 2006).

Unexpectedly, ChIP analysis by qPCR used in this study and in a similar study by the laboratory of Vincent Pirrotta (V. Pirrotta, pers. comm. to Papp, 2006) reveals that the relationship between PcG and trxG proteins and histone methylation is quite different from the currently held views. Specifically, X-ChIP studies have reported that H3-K27 trimethylation is localized at PREs and this led to the model that recruitment of PRC1 to PREs occurs through H3-K27me3 (i.e., via the Pc chromodomain). In contrast, the current study and that by Vincent Pirrotta found H3-K27 trimethylation to be present across the whole inactive Ubx gene, both in wing discs and in S2 cells (V. Pirrotta, pers. comm. to Papp, 2006). No specific enrichment of H3-K27 trimethylation at PREs has been detected; rather, a reduction of H3-K27me3 signals is observed at PREs, consistent with the reduced signals of H3 that are detected at these sites. Consistent with these results, genome-wide analyses of PcG protein binding and H3-K27me3 profiles in S2 cells revealed that, at most PcG-binding sites in the genome, PcG proteins are tightly localized, whereas H3-K27 trimethylation is typically present across an extended domain that often spans the whole coding region. How could the differences between this study and the earlier studies be explained? It should be noted that in contrast to the qPCR analysis used in the current study, previous studies all relied on nonquantitative end-point PCR after 36 or more cycles to assess the X-ChIP results. It is possible that these experimental differences account for the discrepancies (Papp, 2006).

PhoRC, PRC1, and PRC2 are all tightly localized at PREs but they are all constitutively bound at the inactive and active Ubx gene. This suggests that recruitment of PcG complexes to PREs occurs by default. Although all three complexes are bound at comparable levels to the bxd PRE in the inactive and active state and PhoRC is also bound at comparable levels at the bx PRE, it should be pointed out that the levels of PRC1 and PRC2 binding at the bx PRE are about twofold reduced in the active Ubx gene compared with the inactive Ubx gene. Even though there is still high-level binding of PRC1 and PRC2 at the bx PRE, it cannot be excluded that the observed reduction in binding helps to prevent default PcG repression of the active Ubx gene. It is possible that transcription through the bx PRE reduces PRC1 and PRC2 binding at this PRE. Transcription through PREs has been proposed to serve as an 'anti-silencing' mechanism that prevents default silencing of active genes by PREs (Papp, 2006),

The highly localized binding of all three PcG protein complexes at PREs, together with earlier studies on PRE targeting of PcG protein complexes supports the idea that not only PhoRC but also PRC1 and PRC2 are targeted to PRE DNA through interactions with Pho and/or other sequence-specific DNA-binding proteins. In the case of trxG proteins, the binding modes are more diverse. In particular, recruitment of Trx protein to PREs and to the promoter is also constitutive in both states but recruitment of Ash1 to the coding region is clearly observed only at the active Ubx gene. At present, it is not known how Trx or Ash1 are targeted to these sites. It is possible that a transcription-coupled process recruits Ash1 to the position 1 kb downstream of the transcription start site (Papp, 2006).

In contrast to the localized and constitutive binding of PcG protein complexes and the Trx protein, it was found that the patterns of histone trimethylation are very distinct in the active and inactive Ubx gene. The results also suggest that the locally bound PcG and trxG HMTases act across different distances to methylate chromatin. For example, H3-K4 trimethylation is confined to the first kilobase of the Ubx coding region where Ash1 and Trx are bound, whereas H3-K27 trimethylation is present across an extended 100-kb domain of chromatin that spans the whole Ubx gene. This suggests that PRE-tethered PRC2 is able to trimethylate H3-K27 in nucleosomes that are as far as 30 kb away from the bxd or bx PREs. Unexpectedly, it was found that the H3-K9me3 and H4-K20me3 profiles closely match the H3-K27me3 profile. At present it is not known which HMTases are responsible for H3-K9 and H4-K20 trimethylation, but analysis of E(z) mutants indicate that these modifications may be generated in a sequential manner, following H3-K27 trimethylation by PRC2. The molecular mechanisms that permit locally tethered HMTases such as PRE-bound PRC2 to maintain such extended chromatin stretches in a trimethylated state are only poorly understood. However, a recent study showed that the PhoRC subunit dSfmbt selectively binds to mono- and dimethylated H3-K9 and H4-K20 in peptide-binding assays (Klymenko. 2006). One possibility would be that dSfmbt participates in the process that ensures that repressed Ubx chromatin is trimethylated at H3-K27, H3-K9, and H4-K20. For example, dSfmbt, tethered to PREs by Pho, may interact with nucleosomes of lower methylated states (i.e., H3-K9me1/2 or H4-K20me1/2) in the flanking chromatin and thereby bring them into the vicinity of PRE-anchored HMTases that will hypermethylate them to the trimethylated state (Papp, 2006).

These analyses suggest that H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region is critical for Polycomb repression. (1) Although H3-K27, H3-K9, and H4-K20 trimethylation is present at the inactive and active Ubx gene, it is specifically depleted in the promoter and coding region in the active Ubx gene. (2) Misexpression of Ubx in wing discs with impaired E(z) activity correlates well with loss of H3-K27 and H3-K9 trimethylation at the promoter and 5' coding region. It is possible that the persisting H3-K27 and H3-K9 trimethylation in the 3' coding region is responsible for maintenance of repression in those E(z) mutant wing discs cells that do not show misexpression of Ubx. (3) In haltere and third-leg discs of ash1 mutants, the promoter and coding region become extensively trimethylated at H3-K27 and H3-K9, and this correlates with loss of Ubx expression. Previous studies showed that Ubx expression is restored in ash1 mutants cells that also lack E(z) function. Together, these findings therefore provide strong evidence that Ash1 is required to prevent PRC2 and other HMTases from trimethylating the promoter and coding region at H3-K27 and H3-K9. The loss of H3-K4 trimethylation in ash1 mutants is formally consistent with the idea that Ash1 exerts its antirepressor function by trimethylating H3-K4 in nucleosomes in the promoter and 5' coding region, but other explanations are possible (Papp, 2006).

But how might H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region repress transcription? The observation that TBP and Spt5 are also bound to the promoter in the OFF state suggests that these methylation marks do not prevent assembly of the basic transcription apparatus at the promoter. However, the nucleosome remodeling factor Kis is not recruited in the OFF state, and transcription thus appears to be blocked at a late step of transcriptional intiation prior to elongation. It was found that the low-level binding of Pc in the coding region correlates with the presence of H3-K27 trimethylation; i.e., Pc and H3-K27me3 are both present in the OFF state, but are absent in the ON state. One possible scenario would thus be that H3-K27 trimethylation in the promoter and coding region permits direct recruitment of PRC1. According to this view, locally recruited PRC1 would then repress transcription; e.g., by inhibiting nucleosome remodeling in the promoter region. However, several observations are not easily reconciled with such a simple 'recruitment-by-methylation' model. First, peak levels of all PRC1 components are present at PREs and, apart from Pc, very little binding is observed outside of PREs. Second, excision of PRE sequences from a PRE reporter gene during development leads to a rapid loss of silencing, suggesting that transcriptional repression requires the continuous presence of PREs and the proteins that are bound to them. A second, more plausible scenario would therefore be that DNA-binding factors first target PcG protein complexes to PREs, and that these PRE-tethered complexes then interact with trimethylated nucleosomes in the flanking chromatin in order to repress transcription. For example, it is possible that bridging interactions between the Pc chromodomain in PRE-tethered PRC1 and H3-K27me3-marked chromatin in the promoter or coding region permit other PRE-tethered PcG proteins to recognize the chromatin interval across which they should act, e.g., to inhibit nucleosome remodeling in the case of PRC1 or to trimethylate H3-K27 at hypomethylated nucleosomes in the case of PRC2 (Papp, 2006).

The analysis of a HOX gene in developing Drosophila suggests that histone trimethylation at H3-K27, H3-K9, and H4-K20 in the promoter and coding region plays a central role in generating and maintaining of a PcG-repressed state. Contrary to previous reports, the current findings provide no evidence that H3-K27 trimethylation is specifically localized at PREs and could thus recruit PRC1 to PREs; widespread H3-K27 trimethylation is found across the whole transcription unit. The data presented in this study provide evidence that PREs serve as assembly platforms for PcG protein complexes such as PRC2 that act over considerable distances to trimethylate H3-K27 across long stretches of chromatin. The presence of this trimethylation mark in the chromatin that flanks PREs may in turn serve as a signal to define the chromatin interval that is targeted by other PRE-tethered PcG protein complexes such as PRC1. The results reported here also provide a molecular explanation for the previously reported antirepressor function of trxG HMTases; selective binding of Ash1 to the active HOX gene blocks PcG repression by preventing PRC2 from trimethylating the promoter and coding region. It is possible that the extended domain of combined H3-K27, H3-K9, and H4-K20 trimethylation creates not only the necessary stability for transcriptional repression, but that it also provides the molecular marks that permits PcG repression to be heritably maintained through cell division (Papp, 2006).

Architecture of a polycomb nucleoprotein complex; Chromatin immunoprecipitation and nuclease mapping demonstrate that PREs are nucleosome depleted

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 was the identification of Pho as a sequence-specific PcG protein. Subsequent research firmly established that Pho forms a critical component of the 'PRE code.' Another building block of PREs, the PBE is located directly downstream of Pho binding sites. Pho and PCC interact only weakly in solution, but docking onto Pho/PBE modules drives the assemblage of a stable Pho/PCC/PRE silenceosome. The mechanistical properties of silenceosome and enhanceosome formation are strikingly similar. Both involve synergistic interactions between a stereo-specific arrangement of binding sites and a reciprocal network of protein-protein interactions. This study investigated the architecture of a PcG silenceosome (Mohd-Sarip, 2006).

The results revealed that Pho/PCC contacts the bxd PRE over 400 bp and wraps the PRE DNA around its surface in a constrained negative supercoil. It has been found that Pho/PCC binding to the PRE can overcome chromatinization. Moreover, the resulting DNase I digestion pattern of the Pho/PCC/PRE complex suggested the absence of nucleosomes. It is estimated that a Pho/PCC oligomer can wrap more DNA around its surface than a nucleosome. The extensive contacts between Pho/PCC and PRE DNA together with the left-handed wrapping are likely to affect histone-DNA interactions (Mohd-Sarip, 2006).

The 400 bp bxd PRE core is nucleosome poor in vivo, as revealed by nuclease mapping and quantitative ChIP assays. Likewise, the PREs from the Abd-B cis-regulatory domains were found to be nuclease hypersensitive in chromatin digests. Other researchers have also suggested that the core PRE region is largely devoid of nucleosomes. While this work was in progress, ChIP analysis by others independently established that PREs are depleted for histones. The iab-7 PRE sequences required for the pairing-sensitive silencing of mini-white in transgene assays closely coincide with the nuclease-hypersensitive region bound by Pho/PCC. Finally, there appears to be a good correlation between PRE activity and the extent of nuclease hypersensitivity (Mohd-Sarip, 2006).

These findings dovetail nicely with the results of recent genome-scale determination of nucleosome positioning in yeast. These studies suggested that RNA polymerase II promoters comprise a nucleosome-free region flanked by positioned nucleosomes, bearing a stereotyped pattern of histone modifications. It is proposed that, like promoters and enhancers, PREs are in a nucleosome-depleted conformation in vivo (Mohd-Sarip, 2006).

It is suggested that PcG-directed gene silencing is a multistep process, initiated by silenceosome formation on the PRE. The next step requires the establishment of a silenced state onto PRE-linked genes. PCC-histone interactions, modulated by covalent histone modifications, are likely to be the main driving force of sequence-independent spreading over a target gene. Thus, it is imagined that histone modifications would generally follow, rather than precede, Polycomb nucleocomplex formation on PREs. Collectively, the available evidence enforces the notion that a cooperative network of contextual protein-DNA and protein-protein interactions nucleates silenceosome formation. This work presents a view of the architecture of a Pho/PCC/PRE nucleoprotein complex and provides a framework for models explaining PRE function (Mohd-Sarip, 2006).

The Mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila

Chromosome organization inside the nucleus is not random but rather is determined by a variety of factors, including interactions between chromosomes and nuclear components such as the nuclear envelope or nuclear matrix. Such interactions may be critical for proper nuclear organization, chromosome partitioning during cell division, and gene regulation. An important, but poorly documented subset, includes interactions between specific chromosomal regions. Interactions of this type are thought to be involved in long-range promoter regulation by distant enhancers or locus control regions and may underlie phenomena such as transvection. This study used an in vivo microscopy assay based on Lac Repressor/operator recognition to show that Mcp, a polycomb response element from the Drosophila bithorax complex, is able to mediate physical interaction between remote chromosomal regions. These interactions are tissue specific, can take place between multiple Mcp elements, and seem to be stable once established. It is speculated that this ability to interact may be part of the mechanism through which Mcp mediates its regulatory function in the bithorax complex (Vazquez, 2006).

The isolation of mutants that suppress Mcp-dependent silencing of mini-white could potentially uncover chromosomal proteins that play a role in chromosome-chromosome interaction. One such mutation, grappa (gpp), has been described in detail previously. gpp encodes the Drosophila homologue of the yeast Dot1p, a Histone H3 methyltransferase that modulates chromatin structure and gene silencing in yeast. In Drosophila, the dominant grappa allele gpp^1A is homozygous viable. When tested on various double recombinant P[Mcp, mini-white] chromosomes, long-distance Mcp-mediated mini-white silencing is often (but not always) suppressed in heterozygous gpp^1A flies. If occurring, suppression is always enhanced in a homozygous gpp^1A background. Therefore, the colocalization of inserts OM4 and OM7 were examined in gpp^1A/gpp^1A flies. Mcp elements were paired in >90% of the nuclei, a frequency similar to that observed in flies wild-type for grappa. Although limited, these results suggest that gpp^1A does not prevent the establishment or maintenance of chromosome-chromosome interactions (Vazquez, 2006).

This suggests that pairing may be an initial necessary step in the regulatory process mediated by Mcp and that grappa acts subsequently to induce chromatin changes required for silencing. In the absence of additional data, however, other possibilities cannot be excluded. For example, the timing of pairing could be critical to allow developmentally regulated factors to associate to, and repress transcription around the Mcp element. In such a model, gpp^1A could be delaying the onset of pairing, resulting in reduced levels of silencing. Additional studies will be necessary to establish the series of events that lead to pairing-dependent silencing of Mcp-associated genes (Vazquez, 2006).

Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex

In Drosophila, the function of the Polycomb group genes (PcGs) and their target sequences (Polycomb response elements [PREs]) is to convey mitotic heritability of transcription programmes -- in particular, gene silencing. As part of the mechanisms involved, PREs are thought to mediate this transcriptional memory function by building up higher-order structures in the nucleus. To address this question, in vivo the three-dimensional structure was studied of the homeotic locus bithorax complex (BX-C) by combining chromosome conformation capture (3C) with fluorescent in situ hybridization (FISH) and FISH immunostaining (FISH-I) analysis. It was found that, in the repressed state, all major elements that have been shown to bind PcG proteins, including PREs and core promoters, interact at a distance, giving rise to a topologically complex structure. This structure is important for epigenetic silencing of the BX-C, since it was found that major changes in higher-order structures must occur to stably maintain alternative transcription states, whereas histone modification and reduced levels of PcG proteins determine an epigenetic switch that is only partially heritable (Lanzuolo, 2007).

Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene

Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, chromatin immunoprecipitation studies were performed using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. It was found that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. These results are discussed in light of current models for recruitment of PcG complexes to chromatin targets (Wang, 2010).

How might SCM fit in molecularly with the other PcG components Although in vitro associations of SCM with PRC1 subunits have been described, the ChIP analyses here indicate that SCM can associate with the Ubx PRE despite the loss of PRC1. Similarly, although SCM can bind to the PHO-RC subunit SFMBT in a pairwise assay, SCM localization at the PRE does not appear to be dependent on PHO. Taken together, these ChIP results are consistent with biochemical studies that reveal SCM separability from PHO-RC, PRC1, and PRC2 in fly embryo extracts (Wang, 2010).

An intriguing finding from the matrix of molecular epistasis tests is that SCM exhibits recruitment properties very similar to those of PHO. Specifically, both SCM and PHO can localize to the Ubx PRE independent of all other PcG components tested, and loss of either SCM or PHO diminishes PRC2 and PRC1 association with the PRE. This similarity suggests that SCM may function, like PHO-RC, at an early step in PcG recruitment. In this context, it is worth emphasizing the striking overall similarities between SCM and the PHO-RC subunit SFMBT. Perhaps SCM partners with a yet-to-be identified PcG DNA-binding protein, akin to the functional partnership of SFMBT with PHO. Indeed, since PHO-binding sites are insufficient for PRE function in vivo and many other DNA-binding proteins have been implicated in Drosophila PcG silencing, there is abundant evidence that PRE recognition involves more than just PHO-RC. The common view is that many PREs contain a composite of PHO sites plus additional types of factor-binding motifs. At present, little is known about the nature of SCM-containing complexes beyond the detection of an approximately 500-kDa moiety in fly embryo extracts. It will be informative to characterize stably associated SCM partner proteins and evaluate their potential roles in binding to PRE DNA (Wang, 2010).

Although the ChIP assays presented in this study emphasize SCM separability from other PcG components, SCM must still integrate with its PcG cohorts to achieve gene silencing. This interdependence is highlighted by in vivo assays where robust silencing of a miniwhite reporter by a tethered form of SCM is disrupted if the PRC1 subunit PH is compromised by mutation. Despite advances in understanding biochemical activities of individual PcG complexes, it is not yet clear how their multiple functions are integrated to achieve gene silencing. Further studies will be needed to determine how SCM functions in concert with other PcG components at target chromatin (Wang, 2010).

Ultimately, a precise understanding of SCM function requires deciphering the mechanistic contributions of each of its three identified domains. SCM contains a C-terminal SPM domain, two mbt repeats, and two Cys₂-Cys₂ zinc fingers. Strikingly, each of these domains is also present in SFMBT, suggesting that the overall biochemical roles of these two PcG components may be very similar. Indeed, a recent study provides evidence of functional synergy between SCM and SFMBT (Grimm, 2009). In addition, the PH PcG protein possesses two of these three homology domains. This presents the curious situation of three different PcG proteins related by shared domains yet with none appearing to reside in a stable common complex in nuclear extracts (Wang, 2010).

There are currently in vitro and in vivo data on roles of the SPM domain and mbt repeats but little knowledge yet about the zinc fingers. The SPM domain is a subtype within the broader category of SAM domains that mediate protein interactions. The SCM version of this domain is capable of robust self-binding and cross-binding to the PH version in vitro. The importance of SPM domain interactions in vivo is emphasized by PcG phenotypes observed after overexpressing a dominant-negative isolated SPM domain in developing flies (Peterson, 2004). However, it remains unclear precisely what SPM interactions contribute to the PcG silencing mechanism. The simple idea that they constitutively glue PcG complex subunits together is at odds with the biochemical separabilities in embryo extracts. Perhaps SPM interactions function primarily directly at chromatin targets, where they could sponsor contacts among different PcG complexes rather than among subunits within the same complex. Such chromatin-specific interactions could contribute to intralocus loops, which have been hypothesized to exist at PcG silenced loci (Wang, 2010).

The functional significance of the SCM mbt repeats is reflected by partial loss-of-function alleles that alter the first repeat and by Hox gene silencing defects observed after disruption of the second repeat. Structural determinations and in vitro binding studies have revealed that mbt repeats are modules for binding to methylated lysines. Since trimethylated H3-K27 is a prominent feature of PcG-silenced chromatin, the mbt repeats could, at first glance, play a role akin to that of the PC chromodomain. However, there are important differences between the substrate-binding properties of these mbt repeats and the PC chromodomain. First, the mbt repeats prefer mono-and dimethylated lysines, whereas the chromodomain prefers the trimethylated form. An intriguing hypothesis is that this mono/di preference could reflect a 'grappling hook' function whereby hypomethylated nucleosomes are recognized and brought into proximity for trimethylation by PRC2. Another distinction is that the binding mode of mbt repeats is not much influenced by peptide sequence context, whereas chromodomain binding features extensive contact with residues flanking the methylated lysine. Consistent with this, the SCM mbt repeats lack binding preference for any particular histone tail lysines. Thus, mbt repeats provide a pocket for methyl-lysine binding, but it is not yet clear if the relevant substrate for SCM is a particular methylated histone residue or even a nonhistone protein. Certainly, the in vitro binding preferences could be modified by additional associated factors in vivo (Wang, 2010).

A sequence alignment of the Cys₂-Cys₂ fingers present in SCM, SFMBT and PH shows that this zinc finger is a distinct subtype that adheres to the consensus sequence CXXCG-X_n-K/R-X-F/Y-CSXXC. These fingers do not appear to function by binding DNA, since sequence-specific binding is not observed in vitro for any of them. Thus, their molecular role is unknown, but their common inclusion in these related fly PcG proteins suggests some key contribution to PcG chromatin function. Curiously, both the SCM and SFMBT human homologs appear to have lost their Cys₂-Cys₂ fingers, whereas all three human PH homologs have retained them. Thus, if these zinc fingers are critical in PcG silencing, then they apparently can be supplied from different combinations of PcG proteins in flies and in mammals. It will be important to test the genetic requirement for the SCM zinc fingers in Drosophila and to further define the mechanistic contributions of all three SCM functional domains to PcG chromatin silencing (Wang, 2010).

Stalled Hox promoters as chromosomal boundaries

Many developmental control genes contain stalled RNA Polymerase II (Pol II) in the early Drosophila embryo, including four of the eight Hox genes. Evidence is presented that the stalled Hox promoters possess an intrinsic insulator activity. The enhancer-blocking activities of these promoters are dependent on general transcription factors that inhibit Pol II elongation, including components of the DSIF (Spt4, and Spt5) and NELF complexes. The activities of conventional insulators are also impaired in embryos containing reduced levels of DSIF and NELF. Thus, promoter-proximal stalling factors might help promote insulator-promoter interactions. It is proposed that stalled promoters help organize gene complexes within chromosomal loop domains (Chopra, 2009b).

Hox genes are responsible for the anterior-posterior patterning of most metazoan embryos. They are typically organized in gene complexes containing a series of cis-regulatory DNAs, including enhancers, silencers, and insulator DNAs . In Drosophila, the eight Hox genes are contained within two gene complexes: the Antennapedia complex (ANT-C), which controls the patterning of anterior regions, and the Bithorax complex (BX-C), which controls posterior regions. The proper spatiotemporal transcription of Hox genes is achieved by the coordinated action of linked cis-regulatory DNAs that are organized in a colinear fashion across the ANT-C and BX-C complexes (Chopra, 2009b).

Chromosomal boundary elements, or insulators, are essential for the orderly regulation of Hox gene expression. They are thought to ensure proper cis-regulatory 'trafficking,' whereby the correct enhancers interact with the appropriate target promoters. Insulators might also help control the levels of transcription by attenuating enhancer-promoter interactions. Insulators are sometimes associated with promoter targeting sequences (PTS), which can facilitate enhancer-promoter interactions by modulating the activities of neighboring insulators (Chopra, 2009b).

Recently, long-range cis-regulatory interactions have been mapped in Drosophila Hox complexes using the DamID technique, chromosomal conformation capture (3C) assays, and transgenic approaches. These studies suggest that the Fab7 and Fab8 insulators are associated with the Abd-B promoter under repressed conditions, even though they map >30-50 kb downstream from the promoter. These long-range interactions depend on the CTCF boundary-binding protein, thereby raising the possibility that insulators interact with one another and organize Abd-B cis-regulatory DNAs within chromosomal loop domains. Similarly, the prototypic insulators flanking the heat-shock puff locus, scs and scs', have also been shown to interact with one another. Additional insulator-insulator loops have also been documented. These loops are thought to facilitate the interactions of remote enhancers and silencers with appropriate target promoters. This study presents evidence that Hox promoters with stalled RNA Polymerase II (Pol II) possess an intrinsic insulator activity, which might help foster the formation of insulator-promoter chromosomal loop domains (Chopra, 2009b).

Four of the eight Hox genes contained in the ANT-C and BX-C contain stalled Pol II. Interestingly, all four stalled genes map at the boundaries of the two Hox complexes. In contrast, internal Hox genes (pb, Dfd, and Scr within the ANT-C, and abd-A within the BX-C) lack stalled Pol II. This arrangement of stalled Hox genes raises the possibility that stalling contributes to the chromosomal organization of Hox complexes. All four stalled Hox genes (lab, Antp, Ubx, and Abd-B) were tested for enhancer-blocking activity in transgenic embryos, along with the promoter regions of two nonstalled genes (Scr and abd-A). Test promoters were placed 5' of lacZ and inserted between a divergent white reporter gene and 3' iab-5 enhancer (IAB5) (Chopra, 2009b).

IAB5 regulates Abd-B expression in posterior regions of the early embryo, corresponding to the primordia for parasegments 10-14. IAB5 is a robust enhancer, and can activate lacZ and white even when positioned far from the reporter genes. This assay was used to reveal an intrinsic enhancer-blocking activity of the eve promoter region. eve/lacZ fusion genes block the ability of IAB5 to activate a distal CAT reporter gene. However, mutagenized eve promoter sequences lacking a critical proximal GAGA element failed to block IAB5-white interactions. Similarly, the Abd-B proximal promoter (Abd-Bm) and Ubx promoter regions block activation of distal white expression, whereas the abd-A promoter does not interfere with the activation of white expression in the presumptive abdomen by the IAB5 enhancer (Chopra, 2009b).

These results suggest that the stalled Abd-B proximal promoter and Ubx promoters possess an enhancer-blocking activity, whereas abd-A does not. A similar trend was observed for Hox promoter sequences from the ANT-C. The Antp and lab promoters block IAB5-white interactions, whereas the Scr promoter (which lacks stalled Pol II) does not interfere with the activation of white expression in the presumptive abdomen. Stalled genes from the tinman complex (Tin-C), which encode NK homeobox proteins responsible for patterning mesodermal lineages, were also examined. All of the stalled promoters from the Tin-C contain insulator activities. In contrast, nonstalled promoters from lbl and C15 lack such activities when tested in similar transgenic assays. Even the Hsp70 promoter, the classic example of Pol II pausing, displayed insulator activity when tested in similar enhancer-blocking transgenic assays (Chopra, 2009b).

The preceding experiments suggest that stalled Hox gene promoters contain enhancer-blocking activities. However, an alternative possibility is that stalled promoters are 'stronger' than the white promoter, and are able to sequester the shared IAB5 enhancer. To distinguish between competition and insulator activities, the IAB5 enhancer was placed between the divergently transcribed white and lacZ reporter genes. When the white promoter sequence was placed 5' of the lacZ reporter gene, the shared IAB5 enhancer worked equally well to activate both white and lacZ expression. Similar results were obtained when the leftward lacZ reporter gene was placed under the control of either the stalled Abd-B or Ubx promoters. In all of these cases, both white and lacZ are expressed equally well in the presumptive abdomen. These results suggest that stalled promoters do not block enhancer-promoter interactions by a competition mechanism. Rather, they work like insulators and block such interactions only when positioned between the distal enhancer and target promoter (Chopra, 2009b).

To determine whether stalled Pol II is important for the enhancer-blocking activities of Ubx and Abd-B, mutant embryos were examined with reduced levels of critical Pol II elongation factors. Ubx and Abd-B were selected for further studies since optimal expression of both genes depends on the Pol II elongation factors Cdk9 (pTEFb) and Elo-A (Chopra, 2009a). It was reasoned that destabilization of stalled Pol II might reduce the enhancer-blocking activities of the Ubx and Abd-B promoter regions. However, reductions in Cdk9 and Elo-A are expected to stabilize, not destabilize, Pol II stalling since both are positive factors that promote elongation (Saunders, 2006). Indeed, reductions in Cdk9 or Elo-A activity do not alter the enhancer-blocking activities of the Ubx and Abd-B promoters (Chopra, 2009b).

To investigate the link between Pol II stalling and enhancer blocking, two negative elongation factors were examined: NELF (Lee, 2008) and DSIF (Wada, 1998; Yamaguchi, 1998; Kaplan, 2000). The NELF-E protein binds to the short nascent transcripts protruding from the active site of Pol II after transcription initiation and promoter clearance, and thereby inhibits Pol II elongation (Wu, 2005; Lee, 2008). Both NELF and DSIF are thought to help stabilize Pol II at the pause site, typically 20-50 base pairs (bp) downstream from the transcription start site (Saunders, 2006; Gilchrist, 2008; Lee et al. 2008). Since Pol II elongation factors are encoded by essential genes, it is not possible to examine the lacZ/white reporter genes in homozygous mutant embryos. Instead, the transgenes were expressed in embryos derived from heterozygous females, and thereby contain half the normal levels of NELF and DSIF (Spt) subunits. Reductions in Nelf-E, Nelf-A, Spt4, and Spt5 cause clear disruptions in the enhancer-blocking activities of both the Ubx and Abd-B promoters, as seen by the strong activation of the distal white reporter gene. In contrast, white expression is blocked when the same transgenes are expressed in a wild-type background. The simplest interpretation of these results is that reduced levels of the NELF and DSIF inhibitory complexes destabilize stalled Pol II at the pause site. Reduced pausing results in diminished enhancer-blocking activities. There is a similar loss in the enhancer-blocking activities of the eve promoter and Fab7 insulator when the transgenes are expressed in embryos containing reduced levels of the GAGA factor, Trl. It is conceivable that the GAGA factor also contributes to the enhancer-blocking activity of the Ubx promoter since Trl/+ embryos display augmented expression of white (Chopra, 2009b).

In principle, the augmented expression of the white reporter gene might not result from the impaired function of the stalled insulators, but might arise from enhanced activity of the white promoter. To investigate this issue, Pol II chromatin immunoprecipitation (ChIP) assays were performed, coupled with quantitative PCR (qPCR) assays. In DSIF and NELF mutant embryos, there is no increase in Pol II levels at either the white promoter or intronic regions as compared with wild-type embryos. These results suggest that augmented expression of white is due to diminished insulator activities of stalled promoters in embryos containing reduced levels of negative Pol II elongation factors (Chopra, 2009b).

It has been suggested that insulators might work, at least in part, via promoter mimicry. To explore this issue, the impact of reductions in NELF and DSIF on the activities of two known insulators, Fab7 and Fab8, from the BX-C, were examined. Previously published transgenic lines were used that contain Fab7 or Fab8 inserted between the IAB5 and 2XPE (twist) enhancers attached to a leftward lacZ reporter gene and rightward white reporter. In wild-type embryos, the reporter genes are activated only by the proximal enhancer. Thus, white is activated solely in the mesoderm by the 2XPE enhancer, while lacZ is activated in the presumptive abdomen by IAB5. The distal enhancers are blocked by the Fab7 or Fab8 insulators. Consequently, IAB5 fails to activate white and the 2XPE enhancer fails to activate lacZ (Chopra, 2009b).

Very different results are observed when the transgenes are crossed into mutant embryos containing reduced levels of NELF or DSIF (Spt) subunits. There is a loss in the enhancer-blocking activities of the Fab7 and Fab8 insulators and, as a result, white and lacZ display composite patterns of expression in the mesoderm and abdomen since they are now activated by both enhancers. These results suggest that negative Pol II elongation factors are required for the enhancer-blocking activities of the Fab7 and Fab8 insulators (Chopra, 2009b).

It is proposed that insulators interact with stalled promoters to form higher-order chromatin loop domains, similar to those created by insulator-insulator interactions. Perhaps proteins that bind insulators interact with components of the Pol II complex at stalled genes. Indeed, the recent documentation that the BEAF insulator protein binds to many of the same sites as NELF is consistent with a physical link between stalled Pol II and insulators (Jiang, 2009). The resulting chromatin loops can prevent the inappropriate activation of stalled genes by enhancers associated with neighboring loci. As discussed earlier, stalled Hox genes are located at the boundaries of the ANT-C and BX-C. This arrangement might help ensure that cis-regulatory sequences located outside the complexes do not fortuitously interact with genes contained inside the complex and vice versa. The demonstration that stalled Hox promoters possess an intrinsic insulator activity adds to the intricacy of the chromosomal landscapes that control Hox gene expression in both arthropods and vertebrates (Chopra, 2009b).

Stalled Hox promoters may help promote higher-order chromatin organization within the Hox loci (see illustration). These results suggest that the stalled promoters contain intrinsic insulator activity that requires NELF and DSIF proteins, and this may help define higher-order loops within gene complexes such as the Hox complex. The stalled Pol II along with the NELF and DSIF complex may interact with putative insulator sequences, as seen for the Abd-B promoter and the Fab7. These experiments also suggest that that putative insulator sequences also require NELF and DSIF proteins, and this could be due to sharing of these proteins via the formation of higher-order loops. Such loop domains may help in proper regulation of genes and prevent any aberrant activation from neighboring enhancers, thus favoring proper gene regulations at the higher-order level (Chopra, 2009b).

Ubx regulation: Table of contents

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