Promoter Structure

ph-p has a 5' proximal promoter that contains at least six sites to which Engrailed binds. The consensus Engrailed-binding site is TCAATTAAAT. There is a second promoter region known as the D fragment located between ph-p and ph-d. This contains two regions (D1 and D2) separated by over 500 bp. D1 and D2 each have several Engrailed-binding sites. The consensus Engrailed binding site in D1 is TCAATTAAAT. D2 contains several higher affinity TAAT stretches that bind Engrailed (Serrano 1995).

The polyhomeotic (ph) gene is a member of the Polycomb group of genes (Pc-G), that are required for the maintenance of the spatial expression pattern of homeotic genes. In contrast to homeotic genes, ph is ubiquitously expressed and it is quantitatively regulated. ph is negatively regulated by the Pc-G genes, except Psc, and positively regulated by the antagonist trithorax group of genes (trx-G), suggesting that Pc-G and trx-G response elements (PREs and TREs) exist at the ph locus. In this study, PREs and TREs at the ph locus that function in transgenic constructs have been functionally characterized. A strong PRE and TRE has been identified in the ph proximal unit as well as a weak one in the ph distal unit. The PRE/TRE of both ph units appear atypical compared with the well-defined homeotic maintenance elements because the minimal ph proximal response element activity requires at least 2 kb of sequence and does not work at long range. Chromatin immunoprecipitation experiments on cultured cells and embryos have been used to show that Pc-G proteins are located in restricted regions, close to the ph promoters, that overlap functionally defined PRE/TREs. The data suggest that ph PRE/TREs are cis-acting DNA elements that modulate rather than silence Pc-G- and trx-G-mediated regulation, enlarging the role of these two groups of genes in transcriptional regulation (Bloyer, 2003).

A fragment, termed P{418}, that is located upstream of php and includes 50 bp downstream of the php transcription start, acts as a PRE in pairing-sensitive repression (PSR) assays, and exhibits variegated repression of white. Because Pc, Psc, and Ph bind to the ph locus in polytene chromosomes, polytene chromosome immunostaining was performed on the P{418}T40 transgenic line. In P{418}T40 line, the transgene maps at cytological location 9D in the X chromosome. No endogenous Ph, Pc, or Psc protein binding is observed at this location in wild type w1118 X chromosomes. However, a strong additional binding site is observed in the P{418}T40 transgenic line at the insertion point of the transposon for Ph, Pc, and Psc. Therefore, this PRE is sufficient to recruit these proteins, meeting one of five formal criteria for PREs (Bloyer, 2003).

The ph locus contains two PRE/TREs that recruit Pc-G proteins in vivo as shown by polytene staining of transgenes and ChIP analysis of the endogenous locus; transgenes containing the PRE/TREs exhibit pairing-sensitive repression (PSR), repression of the white reporter in eye discs, and their activity is sensitive to mutations in Pc-G and trx-G genes. Thus, by these criteria, which have been used for the majority of PREs studies, ph PRE/TREs are authentic. However, it was of concern that PSR and white repression assays of transgene activity might not reflect regulation of the endogenous ph locus. For the reasons below, it is argued that the assays used in this study do correlate with endogenous regulation of ph. First, ph mutations affect steady state endogenous ph mRNA levels as has been shown by using in situ hybridization in embryos as an assay, demonstrating that ph is regulated quantitatively in vivo. Secondly, in phlac+3 embryos, in which a white gene is inserted in a P transposon near the php transcriptional start, the white gene exhibits identical regulation to that of php itself, arguing that expression of white faithfully reflects regulation of ph. Third, the expression of php was examined in wild-type and PcK/+ females using quantitative RT-PcR. A 2.12 ± 0.05 (n = 4) increase in php expression was observed in the PcK/+ mutants, showing that the endogenous ph locus and the transgenes used in this study are sensitive to PcG mutations. An important criterion of PRE activity is the ability to maintain spatially regulated embryonic silencing of homeotic genes. However, the ability of the ph PRE/TREs to maintain silencing of homeotic loci was not tested in these assays. Based on comparison of ph PREs to other PREs tested in the same assays, there are some structural and functional differences between the ph and homeotic PREs revealed by thius analysis (Bloyer, 2003).

Detailed analysis of the php PRE/TRE shows that this element is modular, and contains at least three regions of differential sensitivity to Pc-G and trx-G mutations. Recently, it has been proposed that homeotic 'maintenance elements'(MEs) are composed of several small DNA modules that are bound by several subsets of Pc-G and TRX-G proteins. In other PREs and MEs, the PSR is separable from silencing modules. Pc-G-dependent silencing and PSR are always linked in the ph PRE: both silencing and PSR are lost when the minimal 2-kb P{C4-812} PRE/TRE is dissected into several subfragments. It was not possible to separate PSR from silencing of white, but this may be because these studies lacked sufficient resolution (Bloyer, 2003).

Unusually, it was found that the smallest fragment retaining silencing and PSR activity is 2-kb. This is much larger than minimal fragments showing activity reported from previously described MEs or PREs. Reduced activity might be detected as a lower percentage of transgenic lines showing silencing or PSR, reduced strength of silencing or PSR, or both. The bxd PRE, the iab-7 PRE, and the engrailed PRE all exhibit detectable activity as 191-, 260-, and 181-bp fragments respectively. Because at least three 1-kb fragments were examined in the region showing PRE/TRE activity, it is unlikely that each fragment breaks in a key sequence that prevents activity. If so, there would have to be three key sequences contained within the 418 fragment, one for each unique breakpoint in the fragments tested (822, 824, and 827). The results strongly suggest that the structure of the php PRE/TRE is different from previously characterized PREs or MEs (Bloyer, 2003).

There is not a perfect correspondence between sites of Pc-G binding as detected by ChIP, and functional activity of transgenes. Peak-binding for Pc, Ph, Psc, and GAF mapped to a 0.9-kb fragment which showed no silencing activity and no sensitivity to Pc-G and trx-G mutations when tested in isolation in the P{C4-811} transgenic lines. In both cells and embryos, Pc-G and GAGA factor (GAF) binding overlaps the conserved regulatory sequences found in both the php and phd PREs. However, neither of these conserved sequences were sufficient for PRE activity in these assays. The P{C4-827} and P{C4-824} fragments overlap, and both contain the upstream 350-bp conserved region, but neither demonstrates PRE activity. Similarly, P{C4-811} that contains the downstream conserved region also lacks PRE activity. It is not known why these differences exist; but it is suggested that proteins not assayed in the ChIP experiments, and/or unconserved sequences, must contribute to PRE function. In addition, there were differences between binding of Pc-G proteins in cultured cells and in embryos, which may reflect the different physiology of developing embryos versus cultured cells or experimental variation of the technique (Bloyer, 2003).

The isolated ph PRE/TREs do not completely reproduce the ph wild type regulation even if most of the Pc-G and trx-G response elements are conserved in the minimal P{C4-812} PRE/TRE. An opposite eye color phenotype was observed in a Sce and Pc mutant background when the endogenous ph regulation was compared with transgenic lines containing isolated ph PRE/TREs. It may be simply that the effects of Pc-G mutations on ph regulation are indirect. It may be that different Pc-G complexes function differently in the endogenous and exogenous chromosomal locations. Using an in vivo-functional assay it has been shown that ph and Psc may have a different silencing function than Pc and Sce. Consistent with this, in this study, ph and Psc consistently show strong genetic effects on ph regulation. The results are in accordance with the idea that ph/Psc in one case and Pc/Sce in the other may play different roles in Pc-G silencing complexes, and that the function of these complexes may depend on chromosomal context. An alternative, but not necessarily mutually exclusive explanation for the opposing response of php PRE/TRE transgenes and the endogenous ph locus to Pc-G mutations might be that the isolated PRE/TREs lack distant cis-DNA regulatory elements that can act on ph wild type regulation within the endogenous chromosomal context. In this model, activity of the php PRE/TRE would be modulated by the distant cis-regulatory element, so that transgenes, lacking this sequence would behave differently. These hypotheses cannot be distinguished with the current data (Bloyer, 2003).

The MEs of homeotic loci in Drosophila work at distances of tens of kilobases. The php PRE/TRE does not silence when separated from the promoter by a 4-kb ph intron, as in the P{C4-815} and P{C4-819} lines. The 418 fragment contains the endogenous ph promoter; the 3' end of the fragment is 50 bp downstream of the transcription start site as inferred from analysis of the php cDNA, and the 418 fragment promotes expression of lacZ. One possibility is that the ph promoter prevents interaction of the 418 fragment with the distal white promoter, when the 418 fragment is separated from the white promoter by 4 kb (Bloyer, 2003).

Another possibility is that php sequences downstream of the PRE may contain enhancer elements counteracting the PRE or a chromatin insulator element that may prevent spreading of silencing to distal regions. Alternatively, the downstream sequences could contain a promoter and a truncated-transcription unit that could be spliced. These possibilities were eliminated by showing that, when the bacterial lacZ gene that permits homeotic PRE function was inserted between the 418 fragment and the white gene, silencing by the ph PRE/TRE was abolished. While sequences in the php intron may interfere with 418 PRE activity, the lacZ sequence has already been demonstrated to be free of sequences that interfere with homeotic PRE-mediated silencing. Therefore, the simplest explanation is that silencing induced by the php PRE/TRE is determined by a short-range repression mechanism and that silencing cannot spread over long distances. This result is consistent with the ChIP binding studies showing that Pc-G proteins do not spread past the php and phd PRE/TREs. The ability of this PRE to silence a reporter gene only when it is close to the PRE but not when it is located 4 kb away may depend on the inability of Pc-G to spread across large distances from the ph PRE (Bloyer, 2003).

The fact that ph is maternally and ubiquitously expressed yet regulated by Pc-G genes, raises the question of how the php PRE/TREs function. Previous studies suggest that Pc-G and TRX-G act at the ph locus by modulating the ph transcription level, rather than by silencing. ph is likely not the only gene that is modulated by the Pc-G and TRX-G. One example of this type of regulation concerns the gene toutatis (tou), a newly defined trx-G gene. The tou transcript is strongly and ubiquitously expressed throughout development, the tou locus is bound by Ph and Psc proteins and is quantitatively misexpressed in ph mutants. ph and tou may be the first members of a novel class of Pc-G and TRX-G target genes that may be modulated instead of repressed in an all or none fashion (Bloyer, 2003).

One common feature of genes regulated quantitatively by Pc-G and TRX-G may be that their PRE/TREs are located close to promoters. These PRE/TREs may have a short-range action depending on a direct contribution of Pc-G and TRX-G complexes to the efficiency of the transcription machinery assembly on the promoter. In contrast, PRE/TREs located far away from promoters and enhancers may depend on distant interactions that stabilize long-range architecture (Bloyer, 2003).

The regulation of ph by its PRE/TREs is apparently different from regulation of homeotic loci by PREs, because ph expression is ubiquitous, whereas homeotic loci are silenced, at least in some portions of the embryo. Nevertheless, ph and homeotic PREs may have some features in common. The bxd PRE does silence Ubx expression in anterior parasegments, but it also acts within parasegments to modulate Ubx expression. In wild type embryos, there is an anterior gradient of Ubx expression within parasegments from anterior high to posterior low. There are also variations in Ubx expression levels between parasegments; expression of Ubx is highest in parasegment 6, but lower in more posterior parasegments. Both the variation of Ubx expression within and between parasegments is abolished in embryos mutant for esc, Pc, or ph, arguing that Pc-G genes, and by implication the bxd PRE, also has a role in modulation of homeotic gene expression. One could argue that the effects of Pc-G mutations on Ubx expression are indirect. However, evidence is available from studies of bxd transgene expression in embryos that small fragments of the bxd PRE modulate levels of expression as well as spatial restriction of expression of reporter genes. Therefore, it is argued that, depending on the cellular context, homeotic PREs can silence in anterior parasegments, or modulate gene expression in posterior parasegments. Whether the ph PRE/TRE can silence in transgenes containing homeotic loci, or whether homeotic PREs can modulate gene expression, have not been directly tested. ph and Psc/Su(z)2 loci (2D and 49F, respectively) are strongly bound, on polytene chromosomes, by Psc but only weakly by an antibody anti-trimethylated lysine 9 of histone H3 (H3me3K9). Interestingly, Psc and H3meK9 antibodies colocalize at most Pc-G binding sites, including the BX-C and ANT-C loci. This result suggests that Pc-G-silenced loci are marked by H3me3K9, whereas Pc-G nonsilenced target are not. It will be interesting to determine whether the ph PRE differs from that of homeotic PREs because it does not silence, and whether the differences can be correlated to histone methylation. Taking these results together, it is proposed that modulation of gene expression is part of the normal repertoire of PREs. Is it possible that ph PREs are simply weak PREs compared with homeotic PREs, because they reduce rather than silence expression? Where it is possible to make direct comparisons between ph PREs and homeotic PREs in the same assays (PSR and white repression), ph PREs act similarly to homeotic PREs in terms of number of lines exhibiting variegating expression, the strength of the variegation, and the magnitude of responses to Pc-G or trx-G mutations, arguing that ph PREs are not weaker than homeotic PREs, at least in these assays (Bloyer, 2003).

One could imagine that the mechanism of modulation by PREs is similar to silencing, but based on the number, type and position of binding sites present, coupled with local differences in concentration of PRE binding factors, complete repression cannot be established. Understanding how Pc-G proteins act at different PREs is a key challenge for future research (Bloyer, 2003).

Transcriptional Regulation

Early polyhomeotic expression is activated directly by bicoid and engrailed and inhibited by oskar. Bicoid is effective over all but the most posterior 10% of the embryo. The negative autoregulation of ph starts at the blastoderm stage. As the number of functional copies of ph increases in the same genome, a concomitant reduction of the transcription of each copy is observed. This regulation is ensured positively by the trithorax group and negatively by the Polycomb group gene products acting simultaneously in the same cells. It is likely that an equilibrium between these two states of chromatin activity ensures an accurate level of ph transcription (Fauvarque, 1995).

The Drosophila Engrailed homeoprotein has been shown to directly activate a Polycomb-group gene, polyhomeotic, during embryogenesis. A study of the molecular mechanism involved in this activation detected two different types of Engrailed-binding fragments within the polyhomeotic locus. The P1 and D1 fragments contain several 'TTAATTGCAT' motifs, whereas the D2 fragment contains a long 'TAAT' stretch to which multiple copies of Engrailed bind cooperatively. Another homeodomain-containing protein, Extradenticle, establishes protein-protein interactions with Engrailed on the D2 fragment. Both types of Engrailed-binding sites (P1 or D1 and D2), as well as Extradenticle, are necessary to obtain activation by Engrailed. In vivo, normal polyhomeotic expression depends on extradenticle expression. Moreover, in the absence of Extradenticle, overexpression of Engrailed protein represses polyhomeotic expression (Serrano, 1998).

On the basis of these results, a model is proposed to explain ph regulation by En. In the absence of Exd, En protein is probably bound to P1 and D1, while D2 might be bound either by En or by other homeodomain-containing proteins. Under such conditions, no activation of ph is observed. This situation might represent what happens in the embryo prior to full germband extension. When Exd is present, activation of ph occurs. Although Exd is expressed prior to the time at which En activates ph, Exd might not be active at those earlier stages: Exd activity has been shown to be regulated by nuclear import and was described as exclusively cytoplasmic in the embryo before germband extension. Because the En-binding sites are so far apart within the ph locus and because D2 is required for activation, acting as an enhancer on both ph transcription units, it is further suggested that DNA bending might be involved in activation. In particular, En binding to P1 and D1 could be involved in bringing the proximal and distal ph promoters close to D2 in the presence of Exd, allowing ph activation from both transcription units. En binding to P1 and D1 prior to germband extension might thus prepare the chromatin for rapid activation. At least two lines of evidence support the idea that D2 interacts with P1 as well as D1, to mediate activation of both the proximal and the distal transcription units: (1) the P1-D2 and D1-D2 fusions behave similarly in CAT assays and (2) the in vivo activation was observed with the phlac enhancer trap, which corresponds to an insertion in the proximal transcription unit of ph (Serrano, 1998).

Targets of Activity

Mutations in several Polycomb group genes cause maternal-effect or zygotic segmentation defects, suggesting that Pc group genes may regulate the segmentation genes of Drosophila. Individuals doubly heterozygous for mutations in polyhomeotic and six other Pc group genes show gap, pair rule, and segment polarity segmentation defects. Posterior sex combs and polyhomeotic interact with Krüppel and enhance embryonic phenotypes of hunchback and knirps, and polyhomeotic enhances even-skipped (McKeon, 1994).

ph-p and ph-d regulate homeotic genes like Scr and Ubx as well as segmentation genes ftz and eve (Dura 1988). Polyhomeotic maintains transcription of engrailed (Serrano 1995).

When two to six copies of a white promoter-Alcohol dehydrogenase (Adh) reporter fusion gene are introduced into the genome, the transgene's expression is progressively reduced, both in larvae and adults, rather than the expected gene dosage effect. In addition, multiple transgenes reduce endogenous Adh transcripts, a result that is strongly analogous to "cosuppression" phenomena described in many plant species but which has not been previously observed in animals. Silencing of the Adh gene is not influenced by zeste-dependent transvection but strongly affected by the Polycomb and Polycomblike mutations. Polycomb and polyhomeotic proteins bind to the chromatin at the sites of the repressed w-Adh transgenes revealing that the cosuppression process initiates accumulation of Pc-G proteins in the absence of a canonical Polycomb response element (Pal-Bhadra, 1997).

Regulation of proboscipedia in Drosophila by homeotic selector genes

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Rusch, 2000).

During the late phase, two PcG genes, Psc and ph, have been identified that are involved in maintaining repression of pb outside its normal domain of expression. This result supersedes a previous report that the PcG genes do not regulate pb. No trxG genes have been identified that are required for the maintenance of pb expression. To function, the PcG genes are thought to assemble on DNA in large multimeric complexes. Unlike the genes of the BX-C, which are regulated, to a greater and lesser extent, by all the PcG genes that have been tested, pb is not regulated by the majority of known PcG genes. Assuming that the PcG genes function similarly at the pb locus, the implication is that not all multimeric complexes can be equal. However, it is not clear how these differences are established. One possibility is that complexes composed of different combinations of PcG genes are formed at different times during development, thereby regulating different loci. Interestingly, a vertebrate homolog of Psc has been shown to bind a specific DNA sequence. This exact sequence is also found in the regulatory elements of the pb reporter construct, indicating that Psc may bind directly, though this remains to be shown (Rusch, 2000).

polyhomeotic controls engrailed expression and the hedgehog signaling pathway in imaginal discs

Polycomb group (PcG) genes maintain cell identities during development in insects and mammals and their products are required in many developmental pathways. These include limb morphogenesis in Drosophila, since PcG genes interact with identity and pattern specifying genes in imaginal discs and clones of polyhomeotic (ph) null cells induce abnormal limb patterning. Such clones are associated with ectopic expression of engrailed, hedgehog, patched, cubitus interruptus and decapentaplegic, in a compartment specific manner. The results also reveal negative engrailed regulation by ph in both disc compartments: ph silences engrailed in anterior cells and maintains the level of engrailed expression in posterior ones. It is suggested that PcG targets are not exclusively regulated by an on/off mechanism, but that the PcG also exerts negative transcriptional control on active genes (Randsholt, 2000).

polyhomeotic is expressed in all imaginal disc territories. ph is required for limb patterning. Induction of ph null clones in larvae, by irradiating ph heterozygotes, leads to appendage pattern defects resembling those caused by ectopic expression of the hedgehog pathway. Adults that are heterozygous for ph null (pho) mutations and that have been irradiated as larvae, show only small ph null clones, no larger than 16 cells, that are not homeotically transformed. Irradiation of such animals performed during late second or early third larval instars (L2 or L3) causes the appearance of small vesicles of tissue trapped inside the body cavity and easily visualized in the wing. Animals irradiated earlier during larval development can show pattern abnormalities of all their appendages: this is particularly striking in the anterior wing compartment. Wing phenotypes range from slight margin or vein deformations to blisters and symmetrical mirror duplications of anterior compartment elements (Randsholt, 2000).

One copy of P[phd+] rescues lethality and homeotic transformations of pho. Furthermore, in the presence of a P[phd+] transgene, pho/1 irradiated flies never exhibit limb pattern defects, indicating that these are indeed induced by the ph null clones. To understand the origin of these abnormalities, the expression patterns of a series of lacZ reporters were examined in wing discs where ph null clones had been induced. The anterior-specific pattern disruptions in irradiated pho/1 animals mimic exactly those caused by ectopic expression of either engrailed, hedgehog or decapentaplegic, all genes that control A/P identity specification and limb morphogenesis. These developmental processes depend on interactions between posterior cells (which express en, the en-related gene invected and hh), and anterior cells, which respond to the Hedgehog signal by activating dpp through Ci in a stripe of cells at the A/P compartment border (Randsholt, 2000).

Starting with en-lacZ, ectopic en-lacZ expression is rapidly detected (three to four cell divisions after irradiation) in the shape of a spotted pattern in anterior disc compartments. Larger spherical anterior en-lacZ expressing domains are seen in late L3 discs that had been irradiated during L1. The shape of these ectopic en-lacZ domains suggests that they correspond to cells that are minimizing their contact surface with the rest of the disc. When ph null cells are induced in a hh-lacZ background, ectopic hh-lacZ expression is detected with the same time-lag and in a similar pattern in the anterior compartment. A ptc-lacZ reporter, whose peak expression is normally restricted to anterior cells along the compartment boundary, reveals ectopic ptc-lacZ expression in irradiated pho/1 discs in both compartments. A similar experiment performed in a ci-lacZ background detected ectopic ci-lacZ expression in the posterior compartment, despite the presence of Engrailed in these cells. Activation of dpp along the A/P boundary is the normal result of hh signaling, so dpp expression was monitored with a dpp-lacZ reporter. Ectopic dpp-lacZ expression is consistently seen in the anterior wing compartment of irradiated pho/1;dpp-lacZ/1 discs. dpp-lacZ expression was never detected in the posterior compartment. This anterior-specific induction of dpp-lacZ makes dpp a likely candidate for causing the anterior appendage defects of irradiated pho/1 animals. From these data, it has been concluded that loss of ph product leads to misregulation of hh pathway genes in both compartments. Additional experiments have shown that maintenance of A/P cellular identities in the developing appendages requires not only ph, but also several PcG products (Randsholt, 2000).

It was important to determine whether anterior activation of hh is a consequence of anterior engrailed activation or reflects an independent effect of loss of ph on the hh gene. The latter situation would agree with the fact that hh is upregulated in a trans-heterozygous mutant context for both ph and en. The data collectively suggest that anterior deregulation of hh in a ph mutant background can be independent of engrailed. It is suggested that Ci expression is increased when the ph level decreases (Randsholt, 2000).

Double-immunostaining of irradiated discs from different genetic backgrounds allows a precise determination of which genes are deregulated by the absence of functional Ph. pho cells were followed by Myc expression after irradiation of pho/Myc larvae. Staining of discs from pho/Myc;en-lacZ/1 larvae show that the anterior ph null cells form bubble-like shapes that are in a different plane from the rest of the disc. This shows that the pho cells are sorting-out from the disc surface and explains the origin of the vesicles of tissue trapped between the wing surfaces or in the body cavities of irradiated pho/1 adults. Furthermore, all cells that do not express Myc in the anterior compartment, and thus are ph null, express beta- Galactosidase, hence ectopic Engrailed. This shows that ph negatively regulates engrailed in the anterior compartment and that the induction of En is cell autonomous in all anterior compartment ph null clones. Ectopic engrailed expression allowed for the identification of the ph null cells in the anterior compartment (Randsholt, 2000).

Similar results were obtained when clones were induced in a hh-lacZ or ptc-lacZ background, showing that both hh and ptc are autonomously induced in anterior compartment ph null cells. In discs from pho/1 larvae stained with anti-En and anti-Ci antibodies, ci expression is present in some but absent in other anterior compartment ph clones. The presence of En in pho cells might secondarily lead to repression of ci when sufficient En has accumulated. Finally, in discs from pho/1;dpp-lacZ/1 larvae, all the ph null clones in the anterior compartment are associated with non-autonomous dpp-lacZ expression in the surrounding cells. Furthermore, similar to what has been observed for ci, ectopic dpp-lacZ expression is detected in some of the anterior ph null clones. These results suggest that the effect of ph on dpp expression is indirect, and that the relative levels of En and Hh within the clones are likely responsible for the expression or non-expression of dpp. Indeed, En is a strong repressor of dpp, whereas Hh stabilizes the activator form of Ci, which promotes dpp expression (Randsholt, 2000).

To conclude, loss of ph in the anterior compartment leads to sorting-out of the ph null cells and to misregulation of en and hh. This changes the identity of the cells toward cells that are not quite posterior either since they strongly express ptc, and sometimes even ci and dpp (Randsholt, 2000).

Discs from irradiated pho/phlac+2 larvae stained with antibodies against Ci and beta-Galactosidase exhibit cell autonomous expression of Ci in all posterior ph null cells, recognized by the fact that they do not express beta-Galactosidase. ph null cells also sort-out in the posterior compartment. Similarly, posterior ph null clones are found to be associated with ectopic ptc expression. Irradiated discs from pho/phlac+2 larvae labeled with Ptc and beta-Galactosidase antibodies exhibit Ptc in all the ph null cells. Ectopic expression of either ci or ptc allows the posterior ph null clones to be identifed by the presence of these markers. Discs from pho/1 larvae stained with antibodies against En and Ci suggest that loss of ph (detected in this case by ectopic Ci expression) is likely to have an effect on engrailed expression in posterior clones. Indeed, the posterior ci-expressing cells apparently express en at a higher level than the surrounding wild-type cells. The clonal cells sort-out, and they are not on the same focal plan as surrounding cells (Randsholt, 2000).

Enhanced En expression is also revealed in clones induced in pho/1 larvae and labeled with antibodies directed against Ptc and En. It is noteworthy that discs from irradiated pho/1;dpp-lacZ/1 larvae labeled with anti-beta-Galactosidase antibody never show dpp-lacZ expression in the posterior compartment. Indeed, pattern defaults of irradiated pho/1 flies occur anteriorly, which is in agreement with the presence of ectopic dpp expression associated only with anterior pho clones. Together these analyses reveal that posterior pho clones also sort-out. Loss of ph in posterior cells induces misregulation of ci and ptc, which are anterior specific genes, suggesting that ph is involved in maintenance of posterior cell identity. Several sets of data indicate that engrailed expression is affected by loss of ph in both compartments, suggesting that ph participates in the repression of engrailed in the anterior compartment, but also in the maintenance of a certain level of engrailed expression in posterior cells (Randsholt, 2000).

Negative regulation of engrailed in the anterior compartment is complex. Indeed, engrailed does not depend on transcriptional repression by the PcG alone; the groucho gene product, for one, also participates in silencing of en in anterior cells. The data presented here indicate that posterior ph null cells that have lost all Ph product express engrailed more strongly than their wild-type neighbors, suggesting that posterior compartment regulation of engrailed also involves more than a simple on/off mechanism. ph could intervene in this regulation either through direct regulation of en or the loss of ph could deregulate other genes that in turn control the expression level of en. Alternatively, ph could, together with other PcG products, maintain a rate of en transcription in posterior cells, possibly by regulating chromatin structure or accessibility. The repressive mechanism controlling en expression in the posterior compartment might, as in mammalian cell systems, change the probability that a given promoter is transcribed. The fact that Ph and Psc can bind transcribed loci in cell cultures suggests that control of gene activity by PcG products could extend to the regulation of active genes. The data from ph null clones provides further evidence that such a regulation does indeed take place during Drosophila development, and suggests that it plays a crucial role in the regulation of selector genes whose wildtype function requires, like engrailed, a strict control of their expression level (Randsholt, 2000).

General transcription factors bind promoters repressed by Polycomb group proteins

To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) was used to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. PcG proteins interact in vitro with GTFs. It is suggested that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription (Breiling, 2001).

For X-ChIP analysis of promoter regions, the following PcG target genes were chosen: Abdominal-B (Abd-B, B-promoter), iab-4, abdominal-A (abd-A, AI-promoter) and Ultrabithorax (Ubx), all located in the Bithorax complex (BX-C), engrailed (en) and empty spiracles (ems). Also chosen were RpII140 (the subunit of RNA polymerase II with relative molecular mass 140,000 [Mr 140K]) and brown (bw): these last two do not reside in PC binding sites on polytene chromosomes and thus are most probably not PcG regulated. Expression of these genes in Drosophila SL-2 culture cells was assessed by polymerase chain reaction with reverse transcription (RT-PCR) and it was found that Abd-B and RpII140 are transcribed whereas iab-4, abd-A, Ubx, en, ems and bw are inactive (Breiling, 2001).

Acetylation of Histone H3 and Histone H4 is considered to be a mark for ongoing transcription. Thus, the promoters of the genes were screened for the presence of amino-terminally acetylated H4 and H3 by X-ChIP. Two antisera were used, one that recognizes H4 acetylated at lysine 12 and one or more other lysines, and one that recognizes H3 acetylated at lysines 9 and/or 18. H4 was found generally acetylated across the promoter regions analysed, in some cases with reduced levels in upstream and downstream regions. H3 is strongly acetylated in the active Abd-B and RpII140 promoters, whereas the inactive loci (iab-4, abd-A, Ubx, en, ems and bw) showed a decrease (5-10 times less than the H3 signal in the active Abd-B and RpII140 promoters) or absence of acetylation both at the core promoters as well as downstream of the initiator. Thus, H3 is acetylated in the active but underacetylated in the inactive promoters, whereas H4 acetylation shows no such changes. Acetylation of histones H3 and H4 seems to be regulated independently across the BX-C, consistent with results in other systems (Breiling, 2001).

The same promoter regions were analyzed by X-ChIP using antibodies against the PcG proteins Polycomb (PC) and Polyhomeotic (PH), dHDAC1, TBP, TFIIB and TFIIF (RAP 30 subunit, associated with RNA polymerase II). All six proteins were found in the core promoter regions (200 base pairs [bp] around the initiator) of the Abd-B, iab-4, abd-A, Ubx, en and ems transcription units. PC was found in most regions both upstream and downstream of the transcription start site (Breiling, 2001).

The presence of PC and PH at the active Abd-B-B promoter is striking, although precedented. Co-localization of PcG proteins and trithorax-group (trxG) proteins, which have been identified as suppressors of the PcG, has been reported for most PcG-bound regulatory regions of the BX-C, including promoters. PcG and certain trxG proteins might simultaneously be needed for changing and maintaining opposite transcriptional states. Thus, coincidental association of repressors and activators with active genes might act to guarantee regulated levels of transcription (Breiling, 2001).

RpII140 was expected to be active and not PcG controlled, and indeed GTFs and also dHDAC1, but not PcG proteins, were found bound to the core promoter. As a putative non-PcG-repressed target, the bw gene was chosen. Again, TBP, TFIIB and dHDAC1 but not TFIIF were found. PC, but not PH, binds to the bw promoter. However, no PC was found downstream of the bw promoter, unlike the other PcG-controlled genes. This and the absence of PH suggest that brown is not a canonical PcG-controlled gene, and PC here might have an as-yet-unknown function, not requiring other PcG proteins (Breiling, 2001).

The acetylation state of the promoters investigated could not be correlated with the presence of dHDAC1. dHDAC1 might be present at active promoters together with histone acetyl transferases (HATs) to maintain an appropriate steady-state level of histone acetylation. Five Drosophila HDACs have been identified so far: dHDAC1 (also known as RPD3), dHDAC3, dHDAC4, dHDAC6 and dSIR2 -- and any of these might contribute to the acetylation patterns observed. dHDAC1 might also regulate transcriptional activity by deacetylating transcription factors. GTFs, in particular TFIIE and TFIIF, are substrates for histone acetyl transferases, and transcriptional regulation involves acetylation of developmentally controlled transcription factors. The presence of dHDAC1 at promoters could also be due to the association with Topoisomerase II, which is bound to PcG binding sites (Breiling, 2001).

The close mapping to the same DNA regions suggests a physical interaction of PcG proteins with TBP and other GTFs. Using antibodies against TBP it was shown that the PcG proteins PC, PH and PSC are co-precipitate in a DNA-independent manner with TBP from embryonic nuclear extracts and from nuclear extracts from SL-2 cells. The long proximal isoform of PH (PH 170p) has been found to interact with other PcG proteins and this protein co-purifies with PC, PSC and TBP. Only PC, not PH or PSC, is co-precipitated with TFIIB, whereas no co-purification was observed of PcG proteins with TFIIF (Breiling, 2001).

PcG proteins at repressed promoters may prevent activation of RNA Polymerase II, otherwise committed to transcribe. This hypothesis was tested by dsRNA interference (RNAi), a targeted destruction of messenger RNA, to see whether the inhibition of PcG protein synthesis would lead to de-repression of inactive genes. After prolonged treatment of SL-2 cells with Pc and ph dsRNAs, PC protein is no longer detectable in cellular extracts by Western blotting, and the amount of PH is significantly lower than in non-treated cells. With the same kinetics, PcG-regulated promoters, which are inactive in non-treated cells (iab-4, abd-A, Ubx, en and ems), become de-repressed upon treatment with Pc or ph dsRNAs. In contrast, bw does not show any change of expression state, like Abd-B and RpII140, underlining the specificity of PcG control. Remarkably, an incubation time of 8 days is necessary to observe a significant de-repression of PcG target genes. It appears that PcG proteins are rather stable and cells have to divide several times (eight times assuming a duplication time of roughly 24 h) with inhibited PcG protein synthesis, before an effect on transcription is seen (Breiling, 2001).

The major conclusion from this work is that promoters constitute a key target of PcG function. Evidence is provided that, unexpectedly, GTFs are retained at PcG-repressed promoters and that PcG proteins may function through direct physical interactions with GTFs. This mechanism of transcriptional regulation may provide both transcriptional competence and the flexibility necessary for the rapid re-arrangement of patterns of gene expression in response to developmental signals. Thus, the presence of GTFs and some trxG proteins at PcG-repressed promoters would allow a relatively fast re-activation of these genes, as differentiation processes require. In this context, PcG proteins would need to be continuously present at target gene promoters to constitutively inhibit transcription, a prediction supported by the finding that PcG-repressed genes are re-expressed in cells depleted of PcG proteins by dsRNA interference (Breiling, 2001).

A cellular memory module conveys epigenetic inheritance of hedgehog expression during Drosophila wing imaginal disc development

In Drosophila, the Trithorax-group (trxG) and Polycomb-group (PcG) proteins interact with chromosomal elements, termed Cellular Memory Modules (CMMs). By modifying chromatin, this ensures a stable heritable maintenance of the transcriptional state of developmental regulators, like the homeotic genes, that is defined embryonically. It was asked whether such CMMs could also control expression of genes involved in patterning imaginal discs during larval development. The results demonstrate that expression of the hedgehog gene, once activated, is maintained by a CMM. In addition, the experiments indicate that the switching of such CMMs to an active state during larval stages, in contrast to embryonic stages, may require specific trans-activators. Thes results suggest that the patterning of cells in particular developmental fields in the imaginal discs does not only rely on external cues from morphogens, but also depends on the previous history of the cells, as the control by CMMs ensures a preformatted gene expression pattern (Maurange, 2002).

Immunoprecipitation using cross-linked chromatin (XChIP) allows the mapping of in vivo DNA target sites of chromatin proteins. Because one Polycomb (PC, a member of the PcG) binding site on polytene chromosomes coincides with the cytological position of hh at 94E, this method was applied to ask whether there are PC and GAGA factor (GAF/Trl, a member of the trxG) binding sites in the hh genomic region. These two factors had previously been found to be hallmarks of CMMs, and the GAF has been shown to be associated with some PcG complexes and necessary for the silencing function of PREs. Initially the immunoprecipitated material was hybridized to a genomic stretch of 45 kb encompassing the hh gene. This led to the identification of PC/GAF-binding sites in regions close to the transcription unit. To further fine-map the location of the PC/GAF-binding sites, the region around the hh gene was subdivided into 1-kb-sized PCR fragments (from 4 kb upstream of the hh transcription start site according to the transcript CG4637 from Flybase, to 13.4 kb downstream to the end of the gene). Slot-blot hybridizations of immunoprecipitated material revealed two main sites where PC and GAF are strongly enriched. The first site (A) is located in a region between 0.07 and 1.06 kb upstream of the transcription start site, whereas the second binding site (B) is found in a region spanning the second exon of the hh gene and spreading about 0.4 kb on both sides of the exon. On both sites a substantial overlap was observed between PC- and GAF-binding sites. The presence of this particular arrangement of PC- and GAF-binding sites in the hh genomic region suggests that these PcG and trxG proteins directly control hh expression (Maurange, 2002).

To investigate this at the functional level, the accessibility of the hh promoter region to a trans-activating factor was assessed. It is known that a PRE placed in the vicinity of an Upstream Activating Sequence (UAS) is able to counteract GAL4 binding, preventing expression of the reporter gene (Zink, 1995; Fitzgerald, 2001). Advantage was taken of the availability of an EP line possessing a UAS site close to the endogenous hh transcription start site to test whether the hh-PREs could inhibit the activation of transcription induced by GAL4. The EP3521 line (termed here EP-hh) possesses an EP transposon containing several UAS sites, and is inserted in the hh promoter region (-0.36 kb). The endogenous hh gene is not transcribed in salivary glands. By using an hs-GAL4 line, which is known to be leaky at 25°C, weak expression of GAL4 in larval salivary glands is observed. When hs-GAL4 is crossed to a line containing UAS-hh integrated randomly in the genome, in situ stainings reveal that at 25°C, by the action of GAL4, the hh mRNA is present in high amounts in all the salivary gland cells. However, when hs-GAL4 is crossed to the EP-hh line, in which the UAS sites are juxtaposed to the presumptive PRE, hh transcription was observed in only a very few cells situated mainly at the base of the glands. It was reasoned, because in most cells transcription is inhibited, that the PcG proteins binding the PREs in the vicinity of the hh promoter block the accessibility of GAL4 to the UAS sites. Accordingly, reducing the amount of some of the PcG proteins in the cells by repeating the experiment with flies heterozygous for the Pc3 allele or with males hemizygous for the ph409 allele induces partial derepression of transcription of the endogenous hh gene in a substantial number of gland cells. These results indicate that the repression observed in most of the salivary gland cells in the EP line is caused by the action of the PcG proteins through their binding to the identified PREs. As such, these experiments demonstrate that the transcription of hh is directly repressed by the PcG proteins (Maurange, 2002).

When UAS-en is misexpressed at the D-V boundary in a wild-type genetic background using vg-GAL4, it induces hh expression in most of the cells of the wing pouch except in a stripe along the A-P boundary where hh seems to be repressed. Whereas UAS-en is strongly misexpressed at the D-V boundary, the endogenous en gene is weakly misactivated in some cells of the anterior wing pouch (Maurange, 2002).

Repeating the same experiment in a genetic background hemizygous mutant for an hypomorphic allele of polyhomeotic (ph409) leads to a broader domain of expression of hh. Remarkably, the region along the A-P boundary seems to be less refractory to activation of hh transcription, given that the territory of the repressed domain is reduced. Endogenous en is itself overexpressed in the anterior compartment. This is consistent with the findings demonstrating that en expression can be derepressed in a PcG gene mutant background. In this case in the anterior wing pouch cells, the activation of en transcription by Hh is probably more efficient than in a wild-type background because en cannot be correctly silenced by PH (Maurange, 2002).

The same experiment repeated in a genetic background now doubly heterozygote for the trxG genes trithorax (trxE2) and brahma (brm2) consistently shows that hh expression is activated at the D-V boundary, but can hardly be maintained through cell divisions in the anterior compartment, because with in situ staining, the Hh signal progressively fades away from the D-V boundary. As expected, in such a case, en expression in the anterior compartment is restricted to the D-V boundary, because Hh might not be present in a sufficient amount to activate transcription of the endogenous en gene in the subsequent wing pouch cells (Maurange, 2002).

Furthermore, it is known that PcG-mediated silencing is enhanced at higher temperature, and this hyperrepressed state can be inherited through cell divisions. Based on these observations, it was reasoned that raising embryos at 28°C instead of 18°C would make the Pc-mediated silencing more difficult to derepress, and influence the activation of hh transcription by En. vg-GAL4; UAS-en embryos were allowed to develop at 28°C until the beginning of second instar larvae, when the D-V boundary is established in wing discs and UAS-en is expressed there. As expected, stainings on third instar imaginal discs reveal ectopic clones of wing pouch cells expressing hh. However, the frequency of cells expressing hh is lower than in discs of larvae grown at 18°C, indicating that the Pc-mediated silencing was harder to erase at 28°C. Nevertheless, in contrast with trxG mutant flies, once the transcription has initially been activated in this case, it is maintained in the subsequent daughter cells as suggested by the presence of clones spreading from the D-V midline to the limits of the wing pouch (Maurange, 2002).

These experiments demonstrate that once initiated by En, the maintenance of the transcriptional state of hh to the daughter cells can be attributed to the action of the PcG and trxG proteins. It is concluded that the CMM activity of the hh upstream region described in the transgenic assay is also efficient when considered in its natural chromatin environment and is responsible for the inheritance of the initial transcriptional state of hh from the initiation to the completion of the wing pouch development (Maurange, 2002).

Polycomb group-dependent Cyclin A repression in Drosophila

Polycomb group (PcG) and trithorax group (trxG) proteins are well known for their role in the maintenance of silent and active expression states of homeotic genes. However, PcG proteins may also be required for the control of cellular proliferation in vertebrates. In Drosophila, PcG factors act by associating with specific DNA regions termed PcG response elements (PREs). This study investigated whether Drosophila cell cycle genes are directly regulated by PcG proteins through PREs. A PRE was isolated that regulates Cyclin A expression. This sequence is bound by the Polycomb (PC) and Polyhomeotic (PH) proteins of the PcG, and also by GAGA factor (GAF), a trxG protein that is usually found associated with PREs. This sequence causes PcG- and trxG-dependent variegation of the mini-white reporter gene in transgenic flies. The combination of FISH with PC immunostaining in embryonic cells shows that the endogenous CycA gene colocalizes with PC at foci of high PC concentration named PcG bodies. Finally, loss of function of the Pc gene and overexpression of Pc and ph trigger up-regulation and down-regulation, respectively, of CycA expression in embryos. These results demonstrate that CycA is directly regulated by PcG proteins, linking them to cell cycle control in vivo (Martinez, 2006).

Given the well-described nature of homeotic gene silencing by PcG proteins (i.e., stable maintenance of repression throughout development), PcG genes would not appear at first glance to be obvious candidates for factors controlling the dynamic expression of cell cycle genes. Indeed, actively proliferating cells must reexpress their rate-limiting division components with each cell cycle. It was observed, however, that RNAi-mediated depletion of PC in cycling S2 cells modifies their cell cycle profile, although it does not affect the overall rate of cell proliferation. The Drosophila CycA gene was identified as a direct target in vivo for PC, PH, and GAF in cycling S2 and embryonic cells. In ChIP experiments, the PcG-binding element was precisely mapped in the CycA gene to a region spanning from the promoter to the first intron. This CycA region shares some but not all properties with homeotic PREs. First, the sequence is sufficient to silence the mini-white reporter gene in vivo, producing a characteristic eye variegation phenotype. Second, as expected for a PRE, mini-white silencing is genetically dependent on the activities of the PcG and trxG genes. Third, it was demonstrated that the endogenous CycA gene is repressed in a Pc-dependent manner during embryonic development: In homozygous Pc mutants CycA expression is derepressed in late (stages 11/12/13) embryos. Finally, stable repression of CycA in normal embryos can be visualized as a colocalization between the CycA locus and PcG bodies that gradually increases, reaching a maximum at the time when cells totally stop dividing and begin to differentiate. Together, these results are consistent with PcG proteins playing a functional role in the stable repression of the CycA gene in vitro and in vivo (Martinez, 2006).

In addition, Pc and ph overexpression in rapidly proliferating cells during early embryonic development causes a systematic decrease in the expression of the CycA gene. This suggests that PcG proteins may play a dual molecular role in the regulation of CycA, acting as stable silencing factors in mitotically quiescent cells and as modulators of promoter output in proliferating cells (Martinez, 2006).

In these experiments, PcG members bound the CycA PRE in actively dividing S2 cells. This binding is most likely functionally relevant, since depletion of PC in S2 cells reproducibly modified the cell cycle division profile, correlating with increased CycA levels. An accumulation of cells in the G2/M phase of the cell cycle was found in PC-depleted cells in comparison to control cells. This accumulation is reminiscent of the phenotype observed in the Drosophila dally mutant, in which the cell division pattern is altered in the nervous system and G2/M progression is disrupted in specific sets of dividing cells in the larval brain and eye disc. In this mutant, lamina precursor cells retain high levels of CycA for a prolonged period of time. Although these experiments do not allow a precise definition of exactly which step within the G2/M transition is abnormal, it is proposed that elevated levels of CycA, or an abnormally long persistence of CycA, might cause a delay in exit from mitosis. Accumulation of CycA has been previously shown to accelerate the G1/S transition. Consistent with this finding, in these experiments the population of S2 cells in G1 and S phases was largely decreased after PC depletion (Martinez, 2006).

The implication of PcG members in cell cycle control during active proliferation is surprising. Interestingly, Beuchle (2001) removed individual PcG proteins from clones of proliferating cells in imaginal discs and showed that Psc-Su(z)2 and ph0 mutant clones are large and round, reminiscent of clones of mutations that cause disc tumors. While the exact nature of the defect was unknown, it could be rescued by resupplying Psc and Su(z)2 several hours after the induction of the clone. This suggests that the effects produced by altering PcG-mediated regulation of cell proliferation/growth might be reversible (Martinez, 2006).

In the current experiments, Pc and ph overexpression in cycling embryonic cells were found to silence endogenous CycA expression. This result demonstrates that the effect of PcG proteins on the endogenous CycA PRE is dose-dependent in cycling cells, and suggests that CycA maintains an intrinsic capacity to be silenced despite being normally transcribed. In normal proliferating cells, induced transcription through the CycA locus, which would necessarily transverse the PRE, might be sufficient to counteract the PRE silencing activity of the CycA PRE. Indeed, it has recently been shown that intergenic transcription through a PRE counteracts silencing (Martinez, 2006).

The results suggest that the CycA PRE might present dual functional properties depending on whether cells are cycling or are arrested in the cell cycle. The CycA PRE might behave as a transcriptional attenuator element in cycling cells and as a stable silencer in a subset of mitotically quiescent cells. Recent data suggest the existence of functionally distinct PcG protein complexes that differ in composition as a function of developmental stage and cellular proliferation status. It would thus be of great interest to biochemically characterize the composition of PcG complexes present at different phases of the cell cycle or during different developmental stages in Drosophila (Martinez, 2006).

Although PcG proteins can repress CycA in mitotically arrested embryonic cells, this does not account for all aspects of stable CycA repression. For example, terminally differentiated cells of the salivary glands from third instar larvae do not express CycA, but neither the endogenous gene nor the isolated PRE are able to attract PcG proteins in this tissue. This situation is similar to the hh gene, which is a known target of PcG proteins. Another chromatin-silencing activity must therefore be responsible for this silencing. One possible candidate is the recently described dREAM complex (Korenjak, 2004), which contains the Drosophila E2F and RBF (pRb homolog) factors and binds to silent E2F-binding-site-containing genes during development, including in salivary glands. Whether or not this is the case, silencing of the CycA gene seems to be regulated in a complex manner that might change during different phases of the cell cycle and might depend on the developmental stage and the tissue under analysis (Martinez, 2006).

In addition to CycA being regulated by PcG members, the converse might also be possible; i.e., PcG-binding and/or silencing activity might be regulated in a cell cycle-dependent manner. In a preliminary genetic analysis involving trans-heterozygous allelic combinations, it was found that the homeotic phenotypes of extra sex combs in the T2 and T3 thoracic legs in males and the pigmentation of the A4 tergite (Mcp phenotype) associated with mutations in the Pc and ph genes are enhanced when combined with a CycA mutation. This may suggest the existence of a feedback regulatory loop between PcG genes and CycA (Martinez, 2006).

From studies in vertebrates, it is clear that PcG proteins repress p16ink4a and p19arf, although a strict demonstration of direct repression is still missing. It is not known whether plutonium, the putative Drosophila homolog of p16ink4a, is silenced by PcG proteins. However, a 'ChIP-on-chip' analysis was carried out of the binding profiles of PC, PH, and GAF proteins in a region covering 10% of the Drosophila melanogaster genome. This analysis led to the identification, among others, of several potential PcG target genes that play a role in the control of proliferation and growth. These include the escargot (esg), elbowB (elB), and no ocelli (noc) genes, in addition to a p53-like factor encoded by bifid. Interestingly, esg and elB, as well as the known PcG target gene hh, have been coidentified as potential tumor suppressors in a protein overexpression screen. Finally, recent evidence suggests that hh regulates both proliferation and differentiation in the developing Drosophila retina (Martinez, 2006).

Together with the role of PcG proteins in the regulation of CycA, this evidence suggests that PcG proteins may be globally involved in the coupling of cell proliferation with growth or differentiation during development in Drosophila and perhaps also in vertebrates. This intriguing possibility warrants future investigation (Martinez, 2006).

PRE-mediated bypass of Two Su(Hw) insulators targets PcG proteins to a downstream promoter

Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. In contrast, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter (Comet, 2006).

The present work suggests two complementary mechanisms for promoter silencing by PcG proteins. (1) The data show directly that PcG proteins recruited at a PRE can spread over several kilobases along the flanking chromatin. Therefore, promoters located within short distances from PREs might be silenced by PcG spreading and interference with the transcription machinery. However, PcG spreading induced by the Ubx PRE did not extend beyond few kilobases in these experiments, and ChIP on chip also showed limited extension of PcG protein binding from known PREs. This limited spreading might depend on genomic sequences or proteins bound to them that might attenuate chromatin association of PcG proteins. Thus, spreading alone might not be sufficient for silencing promoters located several tens of kilobases away, as in the case of the Ubx gene, suggesting that additional mechanisms allow PcG proteins to gain access at distant promoters. It was found that pairing of two Su(Hw) insulators can induce promoter association of PcG complexes without PcG-mediated coating of the insulated domain. (2) This suggests an additional mechanism of PRE-dependent promoter silencing, whereby PREs located at large distances from their promoters might contact them via looping of intervening domains. This looping might be favored by natural regulatory elements present at these loci, which might play a role similar to the pair of Su(Hw) insulators used in this study (Comet, 2006).

The endogenous distribution of PcG proteins might reflect spreading from a PRE into the flanking genomic region as well as their ability to bypass insulators. At the two endogenous target loci en and ph, where PREs are located in the promoter region, the distribution of PC and PH suggests spreading from the PREs. The distribution of PC and PH was characterized at Ubx, a locus where the PRE is over 20 kb upstream from the Ubx promoter. In addition to Ubx, this region contains the bxd locus, driving the production of noncoding transcripts. PC and PH binding shows a peak at the bxd transcription start site downstream to the PRE, in addition to the previously described peaks corresponding to the PRE and the Ubx promoter. Furthermore, binding of PH and PC drops between the bxd peak and the Ubx promoter and rises again at the promoter. This distribution is consistent with spreading from the PRE for short-distance chromatin silencing, and direct targeting of PRE bound PcG complexes to the downstream promoter to drive silencing over larger distances (Comet, 2006).

The sharp transitions in PcG protein binding detected at insulators are surprising, especially considering that the PRC1 complex is larger than 1 MDa, a size equivalent to several nucleosomes. The block in PcG spreading might depend on a physical barrier imposed by protein complexes tightly bound to the insulator. The bypass of the insulated domain might be explained by topological features imposed by insulators on three-dimensional chromatin folding. The Su(Hw) and Mod(mdg4) proteins that regulate the Su(Hw) insulator are organized into discrete “insulator bodies” in the cell nucleus. PcG proteins are also organized into “PcG bodies” that might be the sites of PRE-mediated silencing. A single Su(Hw) insulator located near a PRE might thus exclude the downstream domain from the PcG body associated to the PRE. A second insulator paired with the first one in the insulator body might bring the downstream promoter at the PRE-associated PcG body, while excluding from it the intervening chromatin domain. This type of regulation of three-dimensional chromatin folding by insulator elements might modulate gene expression at a number of loci in Drosophila and other species (Comet, 2006).

Drosophila Polycomb complexes restrict neuroblast competence to generate motoneurons

Similar to mammalian neural progenitors, Drosophila neuroblasts progressively lose competence to make early-born neurons. In neuroblast 7-1 (NB7-1), Kruppel (Kr) specifies the third-born U3 motoneuron and Kr misexpression induces ectopic U3 cells. However, competence to generate U3 cells is limited to early divisions, when the Eve+ U motoneurons are produced, and competence is lost when NB7-1 transitions to making interneurons. This study found that Polycomb repressor complexes (PRCs) are necessary and sufficient to restrict competence in NB7-1. PRC loss of function extends the ability of Kr to induce U3 fates and PRC gain of function causes precocious loss of competence to make motoneurons. PRCs also restrict competence to make HB9+ Islet+ motoneurons in another neuroblast that undergoes a motoneuron-to-interneuron transition, NB3-1. In contrast to the regulation of motoneuron competence, PRC activity does not affect the production of Eve+ interneurons by NB3-3, HB9+ Islet+ interneurons by NB7-3, or Dbx+ interneurons by multiple neuroblasts. These findings support a model in which PRCs establish motoneuron-specific competence windows in neuroblasts that transition from motoneuron to interneuron production (Touma, 2012).

This study used multiple genetic approaches to investigate the timing and specificity of competence restriction by PRCs in Drosophila neuroblasts. The data show that PRCs establish motoneuron competence windows in two distinct neuroblast lineages, regulating the production of both Eve+ and HB9+ Islet+ motoneurons. This provides a mechanistic explanation for the loss of competence that has been previously described in NB7-1 and NB3-1. The experiments manipulating the timing of Pdm and Cas expression show that this mechanism is not limited to fate specification by Kr but is involved in establishing a broad motoneuron competence window. Consistent with this model, there appears to be little restriction of competence in a lineage that produces exclusively interneurons (NB3-3) and, correspondingly, PRC activity does not affect the ability of Kr to alter interneuron fates in this lineage. In addition, whereas Ph gain-of-function is sufficient to inhibit production of HB9+ Islet+ motoneurons by NB3-1, the production of HB9+ Islet+ interneurons by NB7-3 and of Dbx+ interneurons by multiple neuroblasts are unaffected (Touma, 2012).

The initial screen revealed a requirement for a subset of PRC1 and PRC2 genes in the regulation of competence. Lack of a statistically significant phenotype for other genes might be due to dosage: all embryos are heterozygous for the mutant allele and there is maternal contribution of Polycomb group and Trithorax group transcripts. Subsequent studies primarily used the Su(z)123 (null allele) and phd401, ph-p602 (ph-d401 is hypomorphic, ph-p602 is null) mutants. Su(z)12 is a component of PRC2 and Ph is a component of PRC1, allowing assessment the roles of each PRC complex. Su(z)12 loss-of- function extended competence to the end of the NB7-1 lineage. Su(z)12 is an essential co-factor of the E(z) H3K27 methyltransferase and levels of Su(z)12 activity correlate with the extent of H3K27 methylation at target genes. This suggests that the degree of competence restriction is determined by the levels of H3K27 methylation at genes required for motoneuron production. Progressive restriction of competence was still observed in the ph-d401, ph-d602 mutants, which was likely to be due to residual Ph activity. However, competence in these mutants is not completely lost until nearly twice the number of neuroblast divisions have occurred than are normally associated with loss of competence (nine divisions in ph mutants versus five in wild type). It is hypothesized that PRC-induced chromatin modifications accumulate over multiple neuroblast divisions and must reach some threshold for inhibiting motoneuron fates, similar to the accumulation of H3K27 trimethylation at the Neurog1 locus during competence restriction in mammalian cortical progenitors. Without testing additional Polycomb group and Trithorax group genes as homozygous mutants and generating maternal nulls (which in some cases might not survive to the relevant stages of neurogenesis), precisely which Polycomb group proteins are necessary for the restriction of competence cannot be precisely identifed. The core components of PRC1 and PRC2 are likely to be ubiquitously and constitutively expressed throughout neurogenesis, and this has been confirmed for Pc and Ph. However, cell type-specific PRC complexes and developmentally regulated changes in PRC composition have been described previously, suggesting that PRC1 or PRC2 co-factors might regulate the timing of competence restriction. It will be interesting to test the role of co-factors that are known or predicted to recruit PRC2 to specific genes, such as the PhoRC complex, Pipsqueak and Grainy head (Touma, 2012).

The sequential generation of motoneurons followed by interneurons has been observed during nervous system development of many insects. Clonal analysis of Drosophila neuroblasts suggests that motoneurons are always produced first, as demonstrated for NB7-1 and NB3-1, although precise birth order data are lacking for most other lineages. In the mammalian spinal cord, motoneurons and interneurons are produced from spatially segregated populations of progenitors that develop along the dorsal-ventral axis of the neural tube. Drosophila lacks this spatial segregation of motoneuroncommitted or interneuron-committed progenitors. Instead, temporal changes allow single progenitors to produce mixed lineages. PRCs appear to work in parallel to the temporal identity transcription factors by establishing competence windows in which temporal identity factors can specify motoneuron fates. Competence windows might represent a 'quality control' mechanism in which PRCs reinforce the timing of fate specification, similar to the role proposed for miRNAs during Drosophila development. Competence windows might also allow temporal identity factors to be 'redeployed' at later divisions. The majority of neuroblasts express Kr and Cas a second time and this study has confirmed that NB7-1 re-expresses Kr when interneurons are being produced. The function of Kr during later neuroblast divisions remains to be determined. If PRC activity alone were responsible for blocking a Kr-specified motoneuron late in the NB7-1 lineage, at least one ectopic U3 might be expected in ph-d, ph-p hemizygous or Su(z)12 homozygous mutant embryos. However, no altered U motoneuron fates were observed in such mutants. There are at least two potential explanations for this result. First, residual PRC activity in these mutants might allow sufficient changes in chromatin states to block endogenous Kr from specifying a motoneuron. This possibility is supported by data showing a dosage-sensitive relationship between Kr and PRC levels in specifying U3 fates, and the eventual loss of competence in ph mutants subjected to heat shock-induced pulses of Kr. Alternatively, there might be an additional transcription factor (or factors) that specifies interneuron fates in the NB7-1 lineage. This interneuron fate determinant could have a dominant effect, such that even when PRC activity is reduced, interneuron fates (or an Eve- 'hybrid' fate) prevail. Conversion to an Eve+ motoneuron might therefore only occur in a combined PRC loss-of-function and Kr gain-of-function background (Touma, 2012).

In both NB7-1 and NB3-1, later-born motoneuron fates are preferentially inhibited in Ph gain-of-function experiments, supporting a link between the number of neuroblast divisions and the restriction of motoneuron competence. The timing of competence restriction might also be regulated by the temporal identity factors themselves. Previous studies of competence in NB7-1 and NB3-1 have shown that constitutive expression of Hb can maintain neuroblasts in a fully competent state. In addition, precocious Pdm expression can inhibit Kr expression and block U3 fates in NB7-1 and RP3 fates in NB3-1. How Hb or Pdm might interact with Polycomb or Trithorax complexes during the regulation of competence remains to be determined (Touma, 2012).

In an attempt to identify PRC target genes that affect competence, NB7-1 fates were analyzed in embryos with wor-GAL4 driving expression of Kr in combination with the following candidates: the anterior-posterior patterning Hox genes Ultrabithorax, abdominal A, Antennapedia and Abdominal B, the nervous system-expressed Hox gene BarH1, the neuroblast fate determinant gooseberry, and the cell cycle regulator Cyclin A. No extension of competence was detected when these PRC targets are coordinately overexpressed with Kr. It would be technically very challenging and beyond the scope of this work to identify direct PRC targets in NB7-1 or NB3-1. However, clues are provided by previous studies that identified PRC targets in Drosophila embryos). One interesting set of PRC targets is a group of genes involved in motoneuron formation or function: eve, islet, HB9, Nkx6 (HGTX - FlyBase), zfh1 and Lim3. All motoneurons that innervate dorsal muscles express Eve, most motoneurons that innervate ventral muscles express some combination of Lim3, Islet, HB9 and Nkx6, and all somatic motoneurons express Zfh1. None of these genes is sufficient to confer motoneuron fates on their own, and some (eve, HB9, islet) are also expressed in subsets of interneurons. It is possible that PRCs silence the transcription of multiple genes that establish motoneuron fate 'combinatorial codes.' Relevant PRC target genes might be coordinately regulated by the temporal identity transcription factors (as suggested by the ability of high levels of Kr to partially overcome competence restriction) or transcription of these targets might depend on indirect interactions (Touma, 2012).

In mammalian embryonic stem cells, PRCs maintain pluripotency by inhibiting transcription of developmental pathway genes. These genes contain 'bivalent' histone modifications, with PRC-associated H3K27 methylation and Trithorax-associated H3K4 methylation keeping developmental regulators silenced but poised for activation. During differentiation of embryonic stem cells into neural progenitors, neural development genes lose PRC-associated modifications but retain H3K4 methylation, resulting in increased transcription. Although PRC silencing maintains pluripotency in embryonic stem cells, PRCs are likely to have an additional role in restricting fate potential once a progenitor becomes lineage committed. This was recently demonstrated for mouse embryonic endoderm progenitors, which undergo a fate choice for liver or pancreas development. The regulatory elements of liver and pancreas genes have distinct chromatin patterns prior to commitment to either lineage, and EZH2 [an ortholog of Drosophila E(z)] promotes liver development by restricting the expression of pancreatic genes. Similar chromatin 'prepatterns' might exist for motoneuron and interneuron genes in newly formed Drosophila neuroblasts, with subsequent PRC activity selectively silencing motoneuron genes in NB7-1 and NB3-1. PRC activity has also been shown to regulate the timing of terminal differentiation in mouse epidermal progenitors and the transition from neurogenesis to astrogenesis in mouse cortical progenitors. The identification of a related mechanism in Drosophila neuroblasts suggests that temporal restriction of fate potential is a common function of PRCs. Drosophila embryonic neuroblasts will provide a useful system for addressing several outstanding questions regarding PRC regulation of fate potential, including how PRCs are recruited to target genes, the composition of the relevant silencing complexes, and how PRC activity is temporally regulated (Touma, 2012).

Protein Interactions

The Sex comb on midleg (Scm) and Polyhomeotic (Ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and Ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, it has been shown that the SPM domain mediates direct interaction between Scm and Ph. Binding studies with isolated SPM domains from Scm and Ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-Ph and Scm-Scm domain interactions are much stronger than the Ph-Ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analyses of site-directed point mutations have identified residues that are important for SPM domain function. These binding properties, their predicted alpha-helical secondary structures, and the conservation of hydrophobic residues prompts comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. Scm and Ph proteins co-localize at polytene chromosome sites in vivo (Peterson, 1997).

The SAM (sterile alpha motif) domain is a 65- to 70-amino acid sequence found in many diverse proteins whose functions range from signal transduction to transcriptional repression. The SAM domain of the Drosophila Polycomb group protein, Polyhomeotic (Ph), is capable of binding to itself in vitro. A number of near relatives of the Ph SAM domain from fruit fly, mouse, and yeast have been tested and all are capable of self-binding. Heterologous interactions are seen among a subset of SAM domains, including Ph, Sexcombs on middle leg (Scm), and RAE28. Several conserved amino acid residues were mutated in the Ph SAM domain, and the effects on self-binding and heterologous association were demonstrated. L33, L41, and 162 are shown to be important determinants of the binding interface, while W1 and G50 are most likely essential for the structure of the domain (Kyba, 1998a).

The proximal Polyhomeotic gene product has 193 amino-terminal amino acids that are absent from distal Ph; in addition, it makes use of internal initiation to give an alternate product that is shorter by 244 amino acids. A notable feature of this unique proximal domain is the presence of a PxxPxxPxxP motif (amino acids 156 to 165) with proline spacing the same as that of the polyproline type II helix recognized by the SH3 domain. Ph also has many glutamine repeats and a serine/threonine-rich region. Near the carboxyl terminus are two blocks of sequence (amino acids 1297 to 1388 and 1511 to 1576) that are shared with the mammalian Ph homologs. The first sequence, named H1, consists of 28 highly conserved amino acids followed by an unusual C4 zinc finger with intercysteine spacing Cx2C...Cx3C. The second sequence has been variously referred to as H2 or SEP (for the mouse homolog); as SPM (for the PcG protein Sex combs on middle leg as well as for the human Ph homologs HPH1 and HPH2), and as SAM (for a variety of yeast signal transduction proteins). This domain can mediate homotypic and heterotypic self-association between Ph and Scm proteins in vitro. In view of this result, the domain is referred to as a self-association motif (SAM), but the specific subset of SAMs with greatest similarity to Ph and Scm is called SPM. The only internal region of sequence dissimilarity between the proximal and distal Ph are the 52 amino acids immediately preceding the SPM domain (Kyba, 1998b).

Polyhomeotic and Posterior sexcombs are shown to coprecipitate with Polycomb from nuclear extracts. The domains required for the association of Psc with Ph and Pc were analyzed by using the yeast two-hybrid system and an in vitro protein-binding assay. Psc and Ph interact through regions of sequence conservation with mammalian homologs, i.e., the H1 domain of Ph (amino acids 1297 to 1418) and the helix-turn-helix-containing region of Psc (amino acids 336 to 473). Psc contacts Pc primarily at this region of Psc, and secondarily at the ring finger (amino acids 250 to 335). The Pc chromobox is not required for this interaction. There is also a discussion of the implication of these results for the nature of the complexes formed by Polycomb group proteins (Kyba, 1998b).

The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. A complex, termed PRC1 (Polycomb repressive complex 1), has been purified that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocks the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF does not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 is active on nucleosomal arrays formed with tailless histones (Shao, 1999).

It is apparent from the composition of PRC1 that there must be other PcG complexes in addition to PRC1. PRC1 purified via either tagged PH or PSC contains Pc, Psc, Ph-p, Ph-d, and Scm, as well as several other proteins. PRC1 does not contain Pcl and E(z). Previous studies using immunoprecipitation, in vitro binding, and/or yeast two-hybrid analysis have shown that Pc, Psc, and Ph interact with each other, and that Scm interacts with Ph. E(z) and Esc have been shown to interact with each other by similar approaches, and E(z) separates from PRC1 during chromatography. Similarly, mammalian homologs of PcG can also be separated into roughly two complexes, one containing homologs to Pc, Psc, and Ph, and the other containing homologs to E(z) and Esc. Another argument that E(z) and Esc form a separate complex with a distinct function is based on the observation that homologs to these genes are found in the C. elegans genome, whereas homologs to Pc, Ph, or Psc are not. The activities of PRC1 suggest that it may be directly involved in creating the repressed state, and that it may require other complexes for targeting. Through screens for homeotic derepression, 14 PcG genes have been well characterized genetically. It is possible that a subset of these genes are required for direct repression, while other PcG proteins function in targeting, regulation of repression activity, or maintenance of the repressed state through mitosis. How PcG proteins are recruited to their targets is still unknown, but several proteins have been suggested as candidates for this function, such as Esc and E(z), and sequence-specific DNA-binding proteins Pho, Trithorax-like, Hunchback (Hb), and the Hb interacting protein dMi-2. PRC1 does not contain E(z) and Trithorax-like. Using antibodies against a region of human YY1 that is conserved in Pho, it was found that Pho is unlikely to be in PRC1; an antibody made specifically against Pho is needed to verify this result. Due to the lack of antibodies, whether PRC1 contains Esc, Hb, or dMi-2 was not tested (Shao, 1999 and references).

Two members of the Pc-G, Polycomb and Polyhomeotic, are constituents of a soluble multimeric protein complex. Size fractionation indicates that a large portion of the two proteins are found in a distinct complex of molecular weight 2-5 x 10(6) Da. During embryogenesis the two proteins show the same spatial distribution. Polycomb and Polyhomeotic have exactly the same binding patterns on polytene chromosomes of larval salivary glands (Franke, 1992).

A Polycomb protein with deleted internal histidine repeats cannot bind to four particular target loci, but otherwise does not change the remaining overall binding pattern. In contrast to the dotted subnuclear localization of the wild-type protein, the nuclear distribution of mutant proteins becomes homogeneous. Surprisingly, in Pc mutants the Polyhomeotic protein is also redistributed in the nucleus (Franke, 1995).

Polyhomeotic binds to 100 sites distributed through the genome, all of which are shared by Polycomb protein. Another Pc-G gene, Posterior sex combs, also binds to these sites. It is suggested that Polyhomeotic forms a multimeric complex with Polycomb to repress homeotic genes (Cheng 1994).

Antibody staining reveals that the Polycomblike protein is found on larval salivary gland polytene chromosomes at approximately 100 specific loci, the same loci to which the Polycomb and Polyhomeotic proteins bind. These data add further support for a model in which Polycomb group proteins form multimeric protein complexes at specific chromosomal loci to repress transcription at those loci (Lonie, 1994).

In Drosophila, the Polycomb group genes are required for the long-term maintenance of the repressed state of many developmentally crucial regulatory genes. Their gene products are thought to function in a common multimeric complex that associates with Polycomb group response elements (PREs) in target genes and regulates higher-order chromatin structure. The chromodomain of Polycomb is necessary for protein-protein interactions within a Polycomb-Polyhomeotic complex. Posterior sexcombs protein coimmunoprecipitates Polycomb and Polyhomeotic, indicating that all three are members of a common multimeric protein complex. Immunoprecipitation experiments using in vivo cross-linked chromatin indicate that these three Polycomb group proteins are associated with identical regulatory elements of the selector gene engrailed in tissue culture cells. Polycomb, Polyhomeotic, and Posterior sexcombs are, however, differentially distributed on regulatory sequences of the engrailed-related gene invected. High-resolution mapping shows that Pc binding is maximal in a 1.0-kb element, 400 bp upstream of the inv start of transcription. Pc binding sites in en are found in a fragment that contains repetitive elements. The Pc binding sites and the repetitive elements are separable. In fact, Pc associates with two distinct elements, one covering the first intron and the other 1 kb upstream from the start of transcription. Both these regions have been implicated in regulation of en expression during embryogenesis. The binding site upstream of en overlaps with a number of pairing-sensitive elements which have been suggested to mediate PcG repression. Ph and Psc are present at both Pc binding sites in the en upstream region and first intron. The common Pc-Ph-Psc complex does not appear to funcion at inv: no Psc is associated with inv and Ph is associated with a much more restricted element than Pc (Strutt, 1997).

The B promoter of Abdominal-B is devoid of all three PcG proteins. Ph and Psc are not associated with the peak Pc binding element A (overlapping the gamma promoter). However, other fragments in the vicinity of gamma and C promoters are associated with Ph and/or Psc, and it may be that this regulatory region is unusually complex and contains several PREs that regulate the different Abd-B promoters. Both Ph and Psc are enriched for a restriction fragment in the 3' region of Abd-B, which is relatively poorly enriched by Pc. This element is strongly associated with GAGA factor. In the empty spiracles gene Psc is associated with an upstream fragment, covering a previously identified ems enhancer element. Pc and Ph are not found at this transcribed locus. These results suggest that there may be multiple different Polycomb group protein complexes which function at different target sites. Polyhomeotic and Posterior sexcombs are also associated with expressed genes. Polyhomeotic and Posterior sex combs may participate in a more general transcriptional mechanism that causes modulated gene repression, whereas the inclusion of Polycomb protein in the complex at PREs leads to stable silencing (Strutt, 1997).

Polycomb Group complexes assemble at polycomb response elements (PREs) in vivo and silence genes in the surrounding chromatin. To study the recruitment of silencing complexes, various Polycomb Group (PcG) proteins have been targeted by fusing them to the LexA DNA binding domain. When LexA-Pc, -Psc, -Ph or -Su(z)2 are targeted to a reporter gene, they recruit functional PcG-silencing complexes that recapitulate the silencing behavior of a PRE: silencing is sensitive to the state of activity of the target chromatin. When the target is transcriptionally active, silencing is not established but when the target is not active at syncytial blastoderm, it becomes silenced. The repressed state persists through embryonic development but cannot be maintained in larval imaginal discs even when the LexA-PcG fusion is constitutively expressed, suggesting a discontinuity in the mechanism of repression. These proteins also interact with other PC-containing complexes in embryonic nuclear extracts. In contrast LexA-Pho is neither able to silence nor to interact with Pc-containing complexes. Analysis of pho mutant embryos and of PRE constructs whose Pho-binding sites are mutated suggests that, while Pho is important for silencing in imaginal discs, it is not necessary for embryonic PcG silencing (Poux, 2001).

These results show that several PcG proteins, targeted by fusion to a DNA-binding domain, can recruit a repressive PcG complex. Several new conclusions can be drawn from these experiments. (1) The recruitment of the silencing complex cannot occur before blastoderm. The alpha1-tubulin-LexA-Pc construct reveals that even when the protein is deposited in the egg during oogenesis, as well as being zygotically expressed, it does not block the initiation of transcription from the Ubx promoter. These results suggest that the LexA-PcG protein cannot establish repression at this early stage, just as the endogenous PcG proteins known to be present in the normal pre-blastoderm embryo do not prevent the initiation of Ubx transcription. Thus, PcG silencing directed by a PRE or by LexA-Pc appears only to set in after blastoderm. One possible explanation is that the assembly of a functional PcG complex at the PRE or at the LexA-binding sites is a multistep process that requires time and is not accomplished until after blastoderm. Another interesting possibility is that the state of the chromatin in nuclei whose very rapid nuclear divisions are just beginning to slow down, cannot yet support the establishment of PcG silencing, for example, because the nucleosomes still bear the deposition-associated histone acetylation pattern. A similar argument might explain why centric heterochromatin is not detectable until blastoderm (Poux, 2001).

(2) The repression established by the LexA-PcG protein is not unconditional but it is sensitive to the state of activity of the target. Like a genuine PRE complex, the LexA-PcG protein establishes silencing only in cells in which the reporter gene is inactive, thus discriminating between active and inactive chromatin targets. The later-acting Ubx H1 enhancer can still function but only in the progeny of cells that were active at early times. The fact that, like the endogenous PRE, the action of the LexA-PcG protein distinguishes between active and silent chromatin, indicates that the discrimination occurs after the binding of the first PcG protein. The sensitive step could be the assembly of a sufficient nucleus of PcG proteins or still later, the involvement of other factors that effect the silencing. It has been shown that Pc-containing complex purified from embryonic nuclear extracts can prevent chromatin remodelling in vitro if it is bound to chromatin before the addition of purified SWI/SNF complex but not if it is added simultaneously or afterwards. If the activation of the Ubx-lacZ reporter gene by the enhancers involves the recruitment of the Drosophila SWI/SNF complex, this observation could help to understand how the LexA binding sites function as a genuine synthetic PRE possessing at least one aspect of the cellular memory displayed by endogenous PREs (Poux, 2001).

(3) Nevertheless, the LexA-binding sites do not constitute a fully functional PRE. Silencing by LexA-PcG proteins is much less effective during larval development. It is possible that the activity of the Ubx H1 enhancer in imaginal discs is more difficult to repress, e.g. because of very high activator concentration. More likely, once the H1 enhancer is active, it is much more difficult to repress by LexA-Pc induced at later times. It cannot be explained, however, why neither daily heat shocks, nor constitutive expression of LexA-Pc can maintain a continuity between embryonic silencing and larval silencing. Repeated heat shocks do eventually reduce the level of expression of the reporter in imaginal discs but the memory of the early domains of repression is lost. The apparent discontinuity in PcG silencing between the embryo and the larva, suggests that there might be a real mechanistic difference between the two states. There are also differences in the maintenance properties of embryos and larvae. One possible explanation is that additional proteins, recruited at a true PRE but not by the LexA fusion proteins, might be necessary for continuous repression at postembryonic developmental stages (Poux, 2001).

LexA proteins unable to repress PcG proteins Psc, Su(z)2 and Ph are as effective as Pc in recruiting a silencing complex, implying that any one of the 'core' PcG proteins can reconstitute a silencing complex. In contrast, LexA-Gaga and -Pho do not silence the reporter gene. That LexA-Gaga does not silence is hardly surprising. Many promoters, including the hsp70 promoter, the alpha1-tubulin promoter or the Ubx promoter itself, contain Gaga-binding sites but are not thereby targets for PcG silencing in vivo. The possibility that the LexA-Gaga fusion protein is defective in some respect cannot be excluded, although it is able to bind to the normal endogenous sites on polytene chromosomes and participate in PcG complexes. Most likely, however, Gaga/Trithorax-like factor by itself cannot recruit PcG complexes. It is supposed that, like many other nuclear factors, Gaga can have either a stimulating or a repressing activity, depending on the binding of other proteins. In the chromatin context of the PRE, however, Gaga interacts with PcG proteins to form a stable complex (Poux, 2001).

A goal of modern biology is to identify the physical interactions that define 'functional modules' of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate 'on' or 'off' state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes, but these complexes have not been described owing to inherent difficulties in purification. A major PcG complex, PRC1, has been purified to 20%-50% homogeneity from Drosophila embryos. Thirty proteins have been identified in these preparations, then the preparation has been further fractionated and Western analyses have been used to validate unanticipated connections. The known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery (Saurin, 2001).

The inheritance of established expression patterns of certain genes during multiple cell divisions is essential for the correct development of an animal. In Drosophila, the expression patterns of the homeotic genes that govern body segment identity are established early in embryogenesis by the products of the gap and pair-rule genes, but are maintained throughout the rest of development by proteins of the PcG and trithorax group (trxG). The trxG maintains the transcriptionally active state of the homeotic genes, whereas the PcG prevents ectopic expression by maintaining a repressive state. The PcG genes encode components of multiple complexes. One of these complexes, Polycomb repressive complex 1 (PRC1), contains the Polycomb (PC), Polyhomeotic (PH) and Posterior sex combs (PSC) proteins (Saurin, 2001).

To better understand the mechanisms of this cellular memory system, an epitope-tag strategy was used to purify PRC1 over 3,000-fold from Drosophila embryos. This complex has been extensively washed in 1 M salt and has a high specific activity in functional analyses; however, contaminating proteins remain associated. Extensive efforts to fractionate this complex to homogeneity in reasonable quantity were blocked by unacceptably low yields on a wide variety of subsequent purification steps. The advent of genome-wide sequence analysis provided an alternative route to identify the components of PRC1. Using mass spectrometry and the recently completed Drosophila genome, almost all of the proteins were identified in the highly fractionated material derived from the M2-affinity column. The sensitivity of Western analysis was used to validate of the association of proteins during subsequent chromatography steps (Saurin, 2001).

The presence of the previously identified PcG proteins PH, PC, and PSC was confirmed by mass spectrometry. In addition, a Drosophila homolog of the mammalian RING1 protein (dRING1) was identified; dRING1 has been found to colocalize with PC on polytene chromosome preparations and its mammalian counterparts have previously been shown to associate with mammalian PcG proteins. Thus, the PcG complement of PRC1 is made up from PH, PSC, PC, dRING1 and sub-stoichiometric amounts of Sex Combs on Midleg (SCM) (Saurin, 2001).

Of the remaining proteins identified by mass spectrometry, the presence of several dTAFII proteins and Zeste is particularly striking. Zeste is a sequence-specific DNA-binding factor, with binding sites in the promoter and regulatory regions of some homeotic genes. The dTAFII proteins were initially identified in the general transcription factor TFIID, a central component for transcriptional initiation, but are also found in histone acetyltransferase complexes. The dTAFII proteins identified by mass spectrometry and Zeste all appear approximately stoichiometric with the PcG complement in the M2 fraction, and maintain a quantitative association with PcG proteins on gel filtration and heparin agarose (Saurin, 2001).

PRC1 fractionates as an extremely large complex, and contains several other proteins in addition to the PcG, dTAFII and Zeste proteins described above. The sequence information provides speculative information on the identity of these proteins, but further work is needed to validate each of these associations. The constitutively expressed Heat shock cognate 3 and 4 (HSC3 and HSC4) proteins were found. The requirement for HSCs in PcG action during development has been demonstrated genetically in flies, where a mutant allele of HSC4 enhances the homeotic phenotype of PC-heterozygous flies. Proteins were found that have been linked to histone deacetylase complexes, including HDAC (RPD3), dMi-2, dSin3A, p55 and SMRTER, a functional homolog of the human SMRT/N-CoR corepressors. While dMi-2 has been linked genetically to PcG repression, and HDAC and p55 have been found present in the Esc/E(z) PcG complex, further studies are clearly needed to examine their association with PRC1. These proteins are present in low stoichiometry: cofractionation of these proteins with PcG on subsequent steps could not be accurately assessed owing to lack of signal, and PRC1 has low deacetylase activity when acetylated core histones and histone peptides are used as substrate (Saurin, 2001).

The most surprising connection revealed in this study is that between PcG proteins and several dTAFIIs. TAFII proteins have previously been found in TFIID and in histone acetyltransferase complexes, and in both contexts have been linked to transcriptional activation. This study suggests that they may also function in PRC1-mediated PcG repression. PcG complexes are targeted to specific genes by sequences called Polycomb response elements (PREs), but are also known to associate at promoters. The presence of dTAFII proteins in PRC1 provides a direct physical connection between PcG proteins and components of the general transcription machinery that bind at promoters. In addition, several of these dTAFIIs have similarities with core histone proteins (dTAFIIs 62, 42 and 30beta) and have been biochemically and structurally demonstrated to associate with each other in a histone octamer-like substructure. Although quite speculative, the structural similarities between the dTAFII42/62 heterotetramer with the histone H3/H4 heterotetramer might indicate a direct role in interacting with nucleosomes and/or DNA to help maintain a stable association of PcG proteins across the numerous rapid cell divisions of the embryo (Saurin, 2001).

The presence of Zeste in PRC1 may serve to assist in the targeting of PcG proteins to repressed loci. Indeed, Zeste can be found localized with PcG proteins at some PcG-repressed loci and recent data demonstrate that Zeste is directly involved in the maintenance of the repressed state of some of these loci. Zeste binds to both PRE and promoter sequences, and thus may serve to bridge the connection of the PcG proteins to these elements. Zeste has also been shown to interact directly with the BRM complex of the trxG. Zeste thus appears be involved in both PcG function and trxG function, consistent with previous genetic studies implying a role in activation and repression (Saurin, 2001).

Polycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd) PRE contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, DNA binding activities have been identified that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)(3) repeats for MHS-70 binding in vitro. Mutation of d(GA)(3) repeats within MHS-70 in the context of the 5.1-kb bxd PRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)(3) repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)(3) repeats, the Trl/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations in Trl and psq enhance homeotic phenotypes of ph, indicating that Trl/GAGA factor and Psq are enhancers of Polycomb that have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of the bxd PRE by d(GA)(n) repeat binding activities mediates PcG-dependent silencing (Hodgson, 2001).

The results of the sequence-specific analysis suggest that d(GA)n-specific binding factors are present in a complex defined by electrophoretic mobility, termed complex 2. Therefore, antibodies directed against two nuclear factors that bind d(GA)n sequences, Trl/GAF and Psq, were tested in binding reactions with a bxd fragment termed MHS-70. In the presence of increasing amounts of antibody to Trl/GAF, the mobility of complex 2 was significantly retarded, migrating close to the sample well. Antibodies to Psq caused a modest but detectable retardation of complex 2. Neither of these antibodies alters the mobility of a second complex, complex 1. These results show that complex 2 contains detectable levels of Trl/GAF and Psq. The significantly reduced mobility of complex 2 in the presence of anti-TRL/GAF antibody presumably results from the ability to induce multimeric aggregates of DNA-TRL/GAF complexes. To show that the complexes formed by MHS-70 and the competing oligomers are equivalent, the formation of complex 2 with synthetic oligomers was tested with antibodies to Trl/GAF, Ph, and Psq. Antibodies to Ph, Trl/GAF, and Psq supershift complex 2 in synthetic oligomer binding reactions (Hodgson, 2001).

Polyhomeotic proximal (Php), Trl/GAF, and Psq have multiple isoforms. To determine which of these isoforms are potential components of complex 2, Western analysis of the Separose AS, BR0.6 and Q0.15 fractions was undertaken. Q0.15 is enriched for isoforms of Trl/GAF P67 plus Trl/GAF P54, Php105 plus Php64, and Psq P70. Taken together with the antibody supershift analysis, these results show that the distinct isoforms of Trl/GAF, Php and Psq coelute with complex 2 and suggest that these isoforms constitute potential subunits of complex 2 binding activities. It has been shown that the full-length isoform of PhP, Php-170, coimmunoprecipitates with the PcG proteins Pc, Psc, Su(z)2, and Scm. Western analyses of the three fractions described above show that there are no detectable levels of Pc, Su(z)2, Psc, or Sex Combs on Midleg (Scm) in Q0.15, indicating that these PcG proteins do not coelute with complex 1 and 2 binding activities. These results suggest that the complex 2 activity is a novel PcG activity containing distinct isoforms of PHP, TRL/GAF, and Psq (Hodgson, 2001).

Genetic interactions between PcG genes are monitored by the enhancement of PcG mutations, providing a sensitive genetic assay for genes required in PcG-mediated silencing. Therefore, the ability of Trl and Psq mutations to enhance the extra sex combs phenotype of ph was tested. Trl enhances the extra sex combs phenotype of Pc. Similarly, Trl62 enhances the extra sex combs phenotype of ph2 and ph409. The effects of psqlola-Delta18 and psq2403 on enhancement of ph2 and ph409 were tested. There is strong enhancement of the expressivity of the extra sex combs phenotype. These results are consistent with a role for Trl/GAF and Psq in PcG-mediated silencing of homeotic loci and indicate that Trl/GAF and Psq are enhancers of PC that have sequence-specific DNA binding activity (Hodgson, 2001).

Native and recombinant polycomb group complexes establish a selective block to template accessibility to repress transcription in vitro

Polycomb group (PcG) proteins are responsible for stable repression of homeotic gene expression during Drosophila melanogaster development. They are thought to stabilize chromatin structure to prevent transcription, though how they do this is unknown. An in vitro system has been established in which the PcG complex PRC1 and a recombinant PRC1 core complex (PCC; a stable complex of the core PcG subunits of the PRC1 complex—Polycomb, Polyhomeotic, Posterior Sex Combs, and dRING1) are able to repress transcription by both RNA polymerase II and by T7 RNA polymerase. Assembly of the template into nucleosomes enhances repression by PRC1 and PCC. The subunit Psc is able to inhibit transcription on its own. PRC1- and PCC-repressed templates remain accessible to Gal4-VP16 binding, and incubation of the template with HeLa nuclear extract before the addition of PCC eliminates PCC repression. These results suggest that PcG proteins do not merely prohibit all transcription machinery from binding the template but instead likely inhibit specific steps in the transcription reaction (King, 2002).

These results are consistent with the hypothesis that an interaction with the template is primarily responsible for PcG repression, rather than an interaction with the general transcription machinery. PRC1 represses transcription of both RNA Pol II and T7 RNA polymerase, and repression of both of these polymerases is quantitatively similar, suggesting that repression requires neither a specific interaction with either polymerase nor one with eukaryotic transcription factors. In addition, the enhancement of repression observed for Pol II transcription on a chromatin template in comparison to naked DNA is also quite similar to the enhancement observed with T7 transcription. Since T7 transcription does not involve activators or any of the general transcription factors required for Pol II transcription, it seems unlikely that PRC1 represses transcription predominantly by interacting with or sequestering these factors. The simplest way to explain how PRC1 and PCC behave in this system is that they interact with the template to block steps required for transcription (King, 2002).

Though this study indicates that PcG complexes are capable of repressing transcription in vitro through an interaction between the PcG complexes and the template, it is still unknown whether interactions with the general machinery also play a role in PcG repression. Indeed, the finding that PRC1 contains a number of TAF proteins, which are also a part of the TFIID complex, raises the possibility of interactions between PRC1 and general factors at the promoter. However, PCC, which contains only PcG proteins, is sufficient for repression in this system, implying that the TAF subunits of PRC1 are not essential for the repression observed here. The TAF subunits may be involved in targeting PcG activity, since TAF proteins are capable of interacting with promoter sequences and with transcription factors (King, 2002).

The Psc subunit of PCC has substantial activity as a single protein, suggesting that Psc may be central to the mechanism of transcriptional repression by PcG complexes. This is in accord with previous results that show that Psc on its own is sufficient to inhibit chromatin remodeling by Swi/Snf in vitro and raises the possibility that a template interaction mediated mainly by Psc is responsible for both of these in vitro activities. The Ph subunit also has a small amount of activity on its own, which is consistent with in vivo studies that have also indicated that Psc and Ph may be central to the mechanism of repression. When Psc and its homolog Su(z)2, or Ph, are removed from imaginal disc clones by recombination, homeotic gene expression is disrupted more quickly and to a greater extent than it is when other PcG genes including Pc are removed. The other subunits of PCC that do not appear to inhibit transcription might function primarily in other aspects of repression that are not limiting in this assay, such as the targeting of complexes to specific genes (King, 2002).

Polyhomeotic is part of a complex that includes Lola like and Trithorax like

Epigenetic inheritance to maintain the expression state of the genome is essential during development. In Drosophila, the cis regulatory elements, called the Polycomb Response Elements (PREs) function to mark the epigenetic cellular memory of the corresponding genomic region with the help of PcG and trxG proteins. While the PcG genes code for the repressor proteins, the trxG genes encode activator proteins. The observations that some proteins may function both as PcG and trxG members and that both these groups of proteins act upon common cis elements, indicate at least a partial functional overlap among these proteins. Trl-GAGA was initially identified as a trxG member but later was shown to be essential for PcG function on several PREs. In order to understand how Trl-GAGA functions in PcG context, the interactors of this protein were sought. lola like, aka batman, was identified as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene. These observations suggest a possible mechanism for how Trl-GAGA plays a role in maintaining the repressed state of target genes involving lolal, which may function as a mediator to recruit PcG complexes (Mishra, 2003).

lolal was originally identified in the Drosophila gene disruption project. Later on this mutation was also found to enhance the homeotic phenotype of polyhomeotic, ph, and renamed batman (Faucheux, 2003). lolal encodes a protein of 127 amino acids that contains a BTB domain of about 90 amino acids, leaving only few residues at both ends of the protein for any other functional motif/domain. Another PcG protein, Esc, consists almost entirely of six WD40 repeat motifs. Unlike multi-domain proteins, the ones made of a single domain alone may function as adaptor modules to bring together two different molecules/complexes. Trl-GAGA is known to activate transcription of several genes. In this context, lolal may function to inhibit this activation role of Trl-GAGA, in a way similar to MyoD-Id interaction. Further studies will be required to differentiate between these mechanisms (Mishra, 2003).

One of the key steps in the PcG/trxG mediated maintenance is the recruitment of the multi-protein complex of correct composition onto the PRE. Recent studies have shown that more than one or perhaps several recruiting processes take place in concert. It is likely that different recruitment possibilities provide the necessary variation that is needed for the establishment and maintenance of varying transcriptional states at hundreds of different loci. These studies identify a new member in this process. Trl-GAGA bound to specific sites on the PREs recruits LOLAL, which in turn, through direct or indirect means, incorporates Polyhomeotic into the complex. This raises a question whether the other recruiting agents like pho, zeste, and others, function in cooperation or competition with each other. Also, it is not clear if the complex is assembled de novo on the PREs, a pre-assembled complex is recruited or partly assembled sub-complexes are recruited. Since large complexes of PcG proteins can be isolated, it is concluded that such structures, once assembled are stable. It is not clear though if these complexes are stable during cell division or they assemble each time a cell divides (Mishra, 2003).

Genetic interaction studies show that lolal interacts with a variety of PcG and trxG mutations,. This underscores the important role of this protein in the regulation of developmental genes. Interestingly, lolal interactions with ph mutation leads to transformation of 2nd (and some times 3rd) leg to 1st leg, an apparent anteriorization type of homeotic transformation in thoracic but in the abdominal region same combination leads to posteriorization type of homeotic transformation, pigmentation of A4 (A4-->A5) reduction in the size of A6 (A6-->A7). However, appearance of sex comb in 2nd and 3rd legs is also known to be due to derepression of Scr in posterior segments thereby explaining this phenotype as due to loss of the repression function of the PcG proteins. Furthermore, trxG and PcG mutations upon interaction with lolal can give a similar phenotype. In lolal context, Asx and trg both show partial A6-->A5 transformation in the abdominal region. Pc is involved in pairing dependent silencing complex recruited by iab-7PRE. ph is also involved in the PS function of iab-7PRE. While it was known that lolal enhances the homeotic phenotype of ph, it is demonstrated that both ph and lolal are involved in establishing the repressive complex at the iab-7PRE. This indicates that lolal and ph function in coordination to set up a repressive complex. Taken together, these results suggest that lolal may be acting along with Trl-GAGA or with other partners in different complexes in a locus or stage specific manner. Depending on the context it could be an activator or repressor function. Since not only 'ON' or 'OFF' but also several 'levels of expression states' for a given hox gene or indeed other regulated loci are maintained, it is likely that a unique combination of trxG and PcG proteins may be needed for each varying level of expression state of a given locus (Mishra, 2003).

Affinity pull-down experiments show that GAGA, Lolal, Ph and Pc proteins coexist in a complex. This is also in agreement with the genetic interaction studies, where interaction was found of lolal with ph and Pc. Genetic and biochemical studies also suggest that, like Trl, lolal could not specify the kind of complex to be assembled. It is likely, therefore, that specificity of the GAGA partner, PcG or trxG member, does not come from lolal. It is even possible that lolal could also be a multifunctional adapter of Trl-GAGA in assembling multi protein complexes. The specificity may come from yet another factor or even from the transcriptional activity around the locus. Recent observations that transcription process itself may contribute to the cellular memory may support this view. This might bring together the ability of GAGA factor to support transcription and recruitment of multi-protein complexes and nucleosome remodeling activity in one mechanistic context (Mishra, 2003).

Trl-GAGA has been suggested to be involved in creating a nucleosome free region. The first step in establishing a PcG complex may be this Trl-GAGA mediated nucleosome remodeling of the chromatin on the PRE region to create a more accessible DNase I hypersensitive site. The recruitment of a protein complex to the accessible region may take place through GAGA factor or by other factors that can anchor the complex onto DNA. As the next step Lolal could mediate recruitment of initial complex, for example, Esc-E(z) protein complex, which modifies nearby histone tails to covalently mark the region for the recruitment of another complex, like PRC1. Consistent histone modifications and remodeling may be needed to maintain the chromatin conformation. These studies would place Lolal as the factor binding to the DNA bound GAGA even when the rest of the complex is not recruited and therefore serves to help in subsequent recruitment steps. In this context, the exact function of proteins like Lolal becomes very important. Further studies will be required to clarify these issues (Mishra, 2003).

Polyhomeotic stably associates with molecular chaperones Hsc4 and Droj2 in Drosophila Kc1 cells

Polycomb group (PcG) proteins silence target loci in Drosophila. Although the mechanism of PcG-mediated silencing remains unknown, there is considerable evidence that PcG proteins act via multiple complexes. Polyhomeotic Proximal, PHP, the major isoform of the proximal product of the polyhomeotic locus, was epitope-tagged at both termini (F-PHP-HA) and a stable Kc1 cell line was generated in order to isolate F-PHP-HA-associated proteins. Using either column chromatography followed by immunoaffinity precipitation or a double immunoaffinity precipitation procedure, multiple proteins were observed that stably associate with F-PHP-HA. Sequencing the five major bands identified PHP-170 and PHP-140 isoforms, Polycomb, Heat shock cognate 4 (Hsc4), and a novel Drosophila J class chaperone that has been termed Droj2. Mutations in both chaperone genes enhance homeotic transformations in PcG genes, suggesting that they have a role in silencing. Minor components of F-PHP-HA-associated proteins include TBP, TAFII42, TAFII85, and p55. However, unlike in PRC1, neither Psc, TAFII62, Modulo, dMI-2, nor Rpd3/HDAC1 associate with F-PHP-HA. The role of chaperones and F-PHP-HA-associated proteins in PcG-mediated silencing and the evidence for different complexes containing Polyhomeotic in vivo are discussed (Wang, 2003).

Hsc4 is a member of the actin superfamily of ATPases, termed Actin-related proteins (Arps). Arps are subunits of ATP-dependent remodeling enzymes and nuclear histone acetyltransfereases, so Arps might function as conformational switches that regulate chromatin remodeling complexes. An important conclusion from these results is that the chaperones Hsc4 and Droj2 associate with PHP in vivo. These results are consistent with previous biochemical results showing that Hsc4 and Hsc3 are found in PRC1 and genetic results showing that hsc70.4 mutations enhance the homeotic phenotypes of PcG mutations. What might be the function of chaperones in PcG-mediated repression? One possibility is that Hsc4 is needed for proper folding or stability of PcG proteins. If so, mutations in hsc70.4 might reduce the available PcG protein, leading to enhancement of PcG phenotypes. If this model is correct, it is surprising that Hsc4 association with F-PHP-HA is stable, rather than being released once proper folding has been achieved. An alternative hypothesis is that Hsc4 protein might be required for stepwise assembly of complexes. In this model, one could explain retention of Hsc4 by F-PHP-HA as a requirement for the Hsc4 protein to nucleate assembly of a complex, or to act as a scaffold, similar to the requirement of Arp1p assembly of dynactin, or signaling complexes (Wang, 2003 and references therein).

It would be interesting to determine the identity of proteins that associate with F-PHP-HA in wild-type cells, and in cells mutant for hsc4. If Hsc4 is required for activity but not assembly, the proteins associating with PHP ought to be the same in each case. But if Hsc4 is required for assembly, then different proteins should associate with PHP in wild-type and mutant cells. It is possible to assemble a core repressive complex containing Ph, Pc, Psc, and dRing1 that is active in blocking SWI/SNF-mediated chromatin remodeling but that does not contain Hsc4. This suggests that Hsc4 is required for PHP interaction with proteins other than Pc, Psc, or dRing1, or that Hsc4 is required for functions other than antagonizing SWI/SNF remodeling (Wang, 2003).

Droj2 is a novel J-domain chaperone. Eukaryotic DnaJ chaperones recruit Hsp70 by binding to its ATPase domain, and accelerate the ATP-hydrolysis step of the chaperone cycle. However, J-domain proteins also participate in cell cycle control mediated by DNA tumor viruses, regulation of protein kinases, or control of GAP-binding proteins. The genetic evidence suggests that Droj2 has a role in PcG-mediated silencing. One appealing possibility is that Droj2 and Hsc4 have a specific role in PcG-mediated silencing. The product of the phd transcription unit (PHD) does not associate with F-PHP-HA, and deletions uncovering Droj2 do not enhance the homeotic phenotypes of mutations in ph401, a phd-specific deletion. These observations suggest that Droj2 has a direct role in PHP-containing complexes and does not have an indirect effect on PHP-mediated silencing (Wang, 2003).

Structural organization of a sex-comb-on-midleg/polyhomeotic copolymer

The polycomb group proteins are required for the stable maintenance of gene repression patterns established during development. They function as part of large multiprotein complexes created via a multitude of protein-protein interaction domains. This study examines the interaction between the SAM (sterile alpha motif) domains of the polycomb group proteins polyhomeotic (Ph) and Sex-comb-on-midleg (Scm). Ph-SAM polymerizes as a helical structure. Scm-SAM also polymerizes, and a crystal structure reveals an architecture similar to the Ph-SAM polymer. These results suggest that Ph-SAM and Scm-SAM form a copolymer. Binding affinity measurements between Scm-SAM and Ph-SAM subunits in different orientations indicate a preference for the formation of a single junction copolymer. To provide a model of the copolymer, the structure of the Ph-SAM/Scm-SAM junction was determined. Similar binding modes are observed in both homo- and hetero-complex formation with minimal change in helix axis direction at the polymer joint. The copolymer model suggests that polymeric Scm complexes could extend beyond the local domains of polymeric Ph complexes on chromatin, possibly playing a role in long range repression (Kim, 2005).

The results clearly demonstrate that Scm-SAM can form a polymer in vitro, and there is considerable evidence that the same polymer is an important aspect of the biological function of Scm. First, the high affinity of the intersubunit interaction is a strong indication that polymerization is a normal function of Scm-SAM in vivo. Second, it is hard to see how polymerization could be an in vitro artifact, because similar polymer architectures have now been seen for SAM domains from three divergent transcriptional repressors (TEL, Ph, and Scm). Moreover, polymer blocking mutations in TEL render the protein unable to repress transcription, suggesting that polymerization is required for repressive function in TEL. Third overexpression of an isolated Scm-SAM generates an Scm defect in vivo. It is easy to envision that an overabundance of the isolated SAM domain could infiltrate endogenous Scm polymers. Finally, a set Scm-SAM mutants have been identified; these mutant domains fail to self-associate. When these mutants were introduced into the full-length Scm protein, they failed to complement Scm mutants in Drosophila (Kim, 2005).

The SAM domain mutants that fail to self-associate and cannot rescue Scm mutant flies are readily rationalized by this polymer structure. Five mutants were found to be defective both in vitro and in vivo: I45T, G47D, M59Delta, M62R, and K71E. The sites of the mutations are localized to the interface seen in the Scm polymer structure. Ile-45 and Gly-47 are found in the mid-loop (ML) binding surface. Both residues are highly buried in the monomer structure (Ile-45 is 91% buried, and Gly-47 is 100% buried). The I45T and G47D mutations are therefore likely to distort the structure of this critical binding surface. Met-59 and Met-62 are both located on helix 4, which contributes to both the ML and end-helix (EH) binding surfaces. Thus, helix 4 is a particularly important region for polymer formation. Moreover, Met-59 is an important hydrophobic residue in the interface. Deletion of Met-59 would therefore remove an important contribution to the interface and would necessarily distort the local structure. Met-62 is 98% buried in the monomer, making it difficult to accommodate the M62R substitution without some sort of structural distortion. Finally, Lys-71 is located on the EH interface and makes a salt bridge across the polymer interface to Asp-46. Thus a K71E mutation would eliminate this interaction and introduce unfavorable electrostatic repulsion. Overall, the results argue that the observed polymer structure is biologically relevant (Kim, 2005).

SAM domain polymerization must be regulated in some fashion to facilitate complex assembly and disassembly. Yan, a member of the Ets family of transcription factors, contains a SAM domain that polymerizes in the same fashion as Ph- and Scm-SAM as well as the closely related ortholog of Yan, TEL-SAM. Yan-SAM can be depolymerized via its interaction with the SAM domain of its regulator, Mae. Mae-SAM binds to a polymerization interface of Yan-SAM with 1000-fold greater binding energy than Yan-SAM has with itself thereby effectively competing away Yan-SAM self-association and ultimately leading to the down-regulation of Yan activity. Regulation of either Ph- or Scm-SAM polymer by each other in the same fashion as Yan/Mae appears unlikely because Ph/Scm-SAM lack the large disparity in binding affinities. It is possible that polymerization is regulated by some still unidentified Mae-like protein that can cap Scm and Ph polymers, or that SAM polymerization is regulated internally by another domain with Scm and Ph (Kim, 2005).

Work on TEL raises the intriguing possibility that polymerization is regulated by covalent modification with small ubiquitin-like modifier (SUMO). TEL is sumoylated at a lysine residue at the edge of the polymeric binding interface. Examination of the site of modification in the context of the TEL polymer structure strongly suggests that polymer formation and sumoylation are mutually incompatible. Thus, it would be expected that SUMO would disrupt TEL-SAM polymers. In this light, it is interesting to note that the polycomb group protein, Pc2, is a SUMO-ligating enzyme, indicating that sumoylation plays an important role in PcG function. Moreover, sumoylation of the Caenorhabditis elegans PcG protein SOP-2 is essential for Hox gene repression. Both Drosophila Ph- and Scm-SAM possess potential sumoylation sites, but there is still no evidence for sumoylation of these proteins (Kim, 2005)

Ph and Scm are known to bind to each other and cooperate in their repressive functions. The findings that Scm-SAM and Ph-SAM both form polymers argues that they must interact in the form of a copolymer. Measurements of binding affinities in different orientations demonstrate that one of the possible joints between the two polymers is strongly preferred. This suggests that PcG complexes involving Ph and Scm would tend to form separate domains on chromatin, by means of a single joint copolymer. If so, a domain of Ph complexes would be invisioned; these complexes are known to localize within a few kilobases around a polycomb response element, extended by Scm complexes. This hypothesis is consistent with previously reported observations. (1) The repressive function of Scm artificially tethered to a DNA binding site depends on the presence of the SAM domain and is enhanced by the presence of Ph. (2) In a Drosophila PRC1 complex, Scm co-purified with the other members including Ph and was thus originally identified as a member of the complex. Subsequent experiments, however, showed smaller amounts of Scm compared with the other members, Pc, Psc, dRING1, and Ph. From the copolymer model, variable amounts of Scm associated with a core domain of Ph complexes would be expected. (3) An analysis of regulatory DNA elements suggested the site of action of Scm is adjacent to the site of action of Psc, a member of the PRC1 complex along with Ph. Scm function extending over an adjacent site is exactly what is expected from the single junction copolymer model (Kim, 2005).

The results strongly argue that polymerization plays an important role in Ph and Scm function. The biological implications of these findings require further investigation, but it is reasonable to suggest that polymerization facilitates the spreading of PcG complexes along the chromosome. Although Ph is found localized around polycomb response elements, the location of Scm in repressed genes is not known. Scm is capable of long range repression, and the results suggest that Scm could be utilized for the extension of repression outside the immediate region of the polycomb response element (Kim, 2005).

Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro

The transcriptional status of a gene can be maintained through multiple rounds of cell division during development. This epigenetic effect is believed to reflect heritable changes in chromatin folding and histone modifications or variants at target genes, but little is known about how these chromatin features are inherited through cell division. A particular challenge for maintaining transcription states is DNA replication, which disrupts or dilutes chromatin-associated proteins and histone modifications. PRC1-class Polycomb group protein complexes, consisting of four core PcG subunits, polyhomeotic (Ph), posterior sex combs (PSC), dRING, and Polycomb (Pc), are essential for development and are thought to heritably silence transcription by altering chromatin folding and histone modifications. It is not known whether these complexes and their effects are maintained during DNA replication or subsequently re-established. When PRC1-class Polycomb complex-bound chromatin or DNA is replicated in vitro, Polycomb complexes remain bound to replicated templates. Retention of Polycomb proteins through DNA replication may contribute to maintenance of transcriptional silencing through cell division (Francis, 2009).

The data suggest that PCC is not released into solution during passage of the DNA replication fork. Furthermore, nucleosomes facilitate PCC binding to and retention on templates, but are not essential for either. The finding that PCC can be maintained on either chromatin or naked DNA is interesting in light of the finding that PREs are sites of rapid histone turnover and can be depleted of nucleosomes (Francis, 2009).

One model for the transfer of PCC during DNA replication is that the complex remains in direct contact with DNA during passage of the DNA replication fork. Contacts between PcG proteins and nucleosomes or DNA could be disrupted in front of the replication fork, but replaced by contacts with nucleosomes or DNA behind the replication fork. This mechanism has been proposed for transfers of histone-DNA contacts during replication and transcription in vitro. PCC can likely contact multiple nucleosomes or a long stretch of DNA, which may allow the complex to remain on chromatin when some template contacts are disrupted. A second model is that PCC interacts with the replication machinery, either directly or through intermediary factors. These interactions could retain PCC near DNA during replication, even if direct DNA contacts are disrupted, allowing rapid rebinding of PCC to newly replicated chromatin. Consistent with this idea, several chromatin-modifying proteins can interact with components of the DNA replication machinery (Francis, 2009).

The inhibition of DNA and chromatin replication by PCC in vitro raises the question of how PcG-bound regions are replicated if PRC1-class complexes are indeed continuously bound. If PCC inhibits replication initiation but not elongation, as the results suggest, then PRC1-class complexes would limit replication only if they were bound near replication origins (Francis, 2009).

Intriguingly, targeting of Pc to a replication origin in Drosophila that mediates developmental chorion gene amplification in follicle cells decreased gene amplification (Aggarwal, 2004) and PcG-silenced regions of polytene chromosomes (such as Hox gene clusters) are underreplicated, although this involves additional genes such as Suppressor of DNA Underreplication (Marchetti, 2003; Moshkin, 2001; Francis, 2009 and references therein).

Reduction of PcG protein levels leads to reactivation of their target genes, suggesting that these genes are continuously susceptible to transcriptional activation. It may therefore be important that PRC1-class complexes, which can directly repress transcription, maintain constant association with genes marked for silencing (Francis, 2009).

It was surprising to find that H3K27me3 is not essential for maintaining PRC1-class complexes through DNA replication in vitro. It is possible that retention of parental PRC1-class complexes and recruitment of new complexes are mechanistically distinct because no evidence was found for recruitment of new PCC during replication, and in vivo data suggest that PSC is present on newly replicated chromatin but that additional PSC is recruited after replication. This may be similar to histone proteins in that it is thought that parental histones are transferred randomly to the two daughter strands, followed by deposition of new histones by replication-coupled assembly complexes. In vivo data raise the possibility that recruitment of new PRC1 is not directly coupled to DNA replication; perhaps it involves H3K27me3 (Francis, 2009).

In these experiments, PCC interacts with chromatin through mass action, but in vivo, PRC1-class complexes are specifically targeted to PREs. It is hypothesized that the stable association of PCC with chromatin observed in this study reflects how the complex could behave once it is recruited to a PRE, but it will be important to test this mechanism in a system where PCC is targeted (Francis, 2009).

In conclusion, the ability of parental PCC to be transferred to daughter chromatin may help explain how PcG-mediated repression established by transiently acting factors can be propagated through cell generations. These data also suggest that maintenance of chromatin regulatory proteins through DNA replication might be an important mechanism of epigenetic inheritance (Francis, 2009).

The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. This study reports a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. First, this study shows that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, this finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions (Robinson, 2012).

Progressive polycomb assembly on H3K27me3 compartments generates polycomb bodies with developmentally regulated motion

Polycomb group (PcG) proteins are conserved chromatin factors that maintain silencing of key developmental genes outside of their expression domains. Recent genome-wide analyses showed a Polycomb (PC) distribution with binding to discrete PcG response elements (PREs). Within the cell nucleus, PcG proteins localize in structures called PC bodies that contain PcG-silenced genes, and it has been recently shown that PREs form local and long-range spatial networks. The nuclear distribution of two PcG proteins, PC and Polyhomeotic (PH) was examined in this study. Thanks to a combination of immunostaining, immuno-FISH, and live imaging of GFP fusion proteins, it was possible to analyze the formation and the mobility of PC bodies during fly embryogenesis as well as compare their behavior to that of the condensed fraction of euchromatin. Immuno-FISH experiments show that PC bodies mainly correspond to 3D structural counterparts of the linear genomic domains identified in genome-wide studies. During early embryogenesis, PC and PH progressively accumulate within PC bodies, which form nuclear structures localized on distinct euchromatin domains containing histone H3 tri-methylated on K27. Time-lapse analysis indicates that two types of motion influence the displacement of PC bodies and chromatin domains containing H2Av-GFP. First, chromatin domains and PC bodies coordinately undergo long-range motions that may correspond to the movement of whole chromosome territories. Second, each PC body and chromatin domain has its own fast and highly constrained motion. In this motion regime, PC bodies move within volumes slightly larger than those of condensed chromatin domains. Moreover, both types of domains move within volumes much smaller than chromosome territories, strongly restricting their possibility of interaction with other nuclear structures. The fast motion of PC bodies and chromatin domains observed during early embryogenesis strongly decreases in late developmental stages, indicating a possible contribution of chromatin dynamics in the maintenance of stable gene silencing (Cheutin, 2012).

This study showed that PC bodies co-localize with H3K27me3 and form small nuclear domains of heterogeneous intensity. Surprisingly, PC bodies are found in DAPI poor regions, often adjacent to DAPI and histone-dense euchromatic regions. This result thus indicates that PC bodies are not among the most condensed chromatin portions of the euchromatic part of the genome. This localization of PC bodies is consistent with a previous study with electron microscopy, which has shown that PC is concentrated in the perichromatin compartment of the mammalian nucleus. In contrast, these data are in apparent contrast with a series of papers reporting PcG protein-dependent chromatin condensation. PcG complexes have been shown to compact chromatin in vitro and reduce DNA accessibility in vivo. Moreover, recent works show that PcG proteins are required to maintain compaction of Hox loci in mammalian embryonic stem cells and of the mouse Kcnq1 imprinted cluster. In those studies, condensation has been addressed by measuring either the compaction of nucleosomal fibers in electron microscopy, or the distance between close genomic loci by FISH. It is difficult to relate in vitro data to the current in vivo analysis. In particular, FISH analyses do not directly distinguish between a truly dense 3D organization and other types of conformations, such as a multi-looped architecture that would not necessarily induce an increase in chromatin density. Therefore, PcG target chromatin is probably organized in higher-order 3D structures that involve nucleosome-nucleosome and protein-protein interactions, but the net density of DNA (as seen by DAPI) or histones (as seen by tagged-histone microscopy) is not particularly high in these structures (Cheutin, 2012).

Earlier studies indicated that PcG proteins rapidly exchange between the nucleoplasm and PC bodies, suggesting that PC bodies consist of a local transient accumulation of PcG proteins in the cell nucleus. Earlier studies have detected the same number of PC bodies inside the nucleus as the number of bands observed on polytene chromosomes, suggesting that PC bodies are formed by PcG proteins binding to their target chromatin. The observed colocalization of PcG target genes with PC bodies in diploid cells confirms this view. An alternative scenario posits that PC bodies could form nucleation sites onto which PcG-target genes move to become silenced. Two lines of evidence from this work suggest the first scenario to be closer to reality. Firstly, it was found that the amount of PC within a PC body depends on the linear size of the genomic region coated by PC and H3K27me3. Secondly, the higher enrichment of PC in PC bodies after homologous chromosome pairing strongly suggests that PC bodies are the nuclear counterparts of linear genomic domains identified in genome-wide studies rather than nuclear structures to which Polycomb target genes have to be localized for their silencing (Cheutin, 2012).

In the head of embryos, where the Antp and Abd-B genes are silenced, they localize in large PC bodies in all cell nuclei. In contrast, loci where PC coating is restricted to smaller genomic regions do not always localize within PC bodies in interphase cell nuclei. Interestingly, time-lapse imaging shows that large PC bodies are stable structures that can be visualized in all frames of time series, whereas small PC bodies are apparently less stable because they are not visible in all of the frames. One possible explanation for the lack of colocalization between PC target genes and PC bodies is that small genomic regions may not be coated by PC in every cell. Alternatively, the amount of PC within the PC body in which small genomic regions localize might be too small to be directly observed, and only become visible when several small PC bodies interact together. For instance a previous study showed that a transgene containing only two copies of a PRE could be detected in about 50% of cell nuclei (Cheutin, 2012).

Intense PC bodies can be visualized during entire time-lapse experiments, allowing the study of their motion. The interpretation of these time-lapse experiments is not straightforward because the MSD of PC bodies only weakly correlates with the MSC. Interestingly, tracks of PC bodies are mainly composed of narrow angles. The analysis of the motion of chromatin domains containing H2Av-GFP gave similar results, but gave unambiguous evidence for the coordinated motion of several chromatin domains. By using the Lac repressor/lac operator system, two components of chromatin motion in early G2 Drosophila spermatocyte nuclei have been reported: a short range motion which occurs in approximately 0.5 µm radius domains, and long-range motion confined to a large, chromosome-sized domain. Another study has also identified a two-regime motion of a chromatin locus inside mammalian nucleus by using a two-photon microscope, which provides high spatial and temporal resolution. This work indicated that chromatin loci undergo apparent constrained diffusion during long periods, interrupted by jumps of 150 nm lasting less than 2 s. However, none of these previous works reported any coordinated motion of adjacent chromatin domains, and therefore they both described the motion of chromatin as being consistent with a random walk (Cheutin, 2012).

In tracking experiments, it was realized that the fast regime of motion is tightly constrained within volumes much smaller than chromosome territories. This suggests that any given locus will normally explore a restricted three-dimensional environment in the cell nucleus. Since this applies generally to chromatin at all developmental stages, one can deduce that each genomic locus is likely to locate in the vicinity of neighboring loci in the three-dimensional nuclear space. The prediction is thus that each locus should most frequently contact other loci that are in its linear neighborhood along the chromosome. This behavior matches the results observed in chromosome conformation capture on chip (4C) experiments, where each 4C bait had most contacts within few hundred kb to a few Mb of surrounding chromatin. Thus, the current results provide a possible scenario for the explanation of these results obtained from large cell populations. Recent studies showed that homeotic gene clusters form an extensive network of contacts with other PcG target loci. This is consistent with the observation of multiple PC body collisions that can be stable for prolonged times in the nucleus. In contrast, the fact that PC intensity correlates with the linear extension of genomic PC and H3K27me3 domains suggests that PC-mediated associations are relatively rare, at least during embryogenesis (Cheutin, 2012).

The slower regime of long-range motion depends on coordinated large-scale chromatin movements that were not documented before. This may depend on the tools used in previous studies. Time-lapse experiments performed by using the Lac repressor/lac operator system only follow one or a few points inside the cell nucleus, limiting the probability to observe coordinated motions, especially in species containing many chromosomes. In contrast, this study followed many chromatin domains inside Drosophila nuclei and long-range coordinated motions were easily identified when at least two distinct nuclear structures moved simultaneously with a similar trajectory. This motion is directional and chromatin domains and PC bodies can cover up to 1 µm in 10 sec. Different objects having coordinated motion probably belong to the same structure, which suggests that the ensemble of chromatin domains and PC bodies displaying a similar coordinated motion forms a single higher-order nuclear structure. This kind of motion is perfectly consistent with the observation of a chromosome territory, which implies that chromosomes form distinct nuclear structures in interphase cells. A displacement of an entire chromosome, or of a chromosome arm, or a large part thereof, would induce the coordinated motion of all chromatin domains and PC bodies associated to the corresponding chromosome portion (Cheutin, 2012).

The few association and dissociation events of PC bodies observed during this work are related to long-range coordinated motion events that affect both chromatin domains and PC bodies. Therefore, gene kissing depending on PcG proteins could rely on large scale chromatin movements which lead to transient fusion of PC bodies, and may be in turn specifically stabilized by interactions among PcG proteins. Moreover, the association and dissociation of PC bodies seems to be developmentally regulated, because dynamic associations and dissociations were observed during early embryogenesis, but are strongly reduced later in development (Cheutin, 2012).

Condensed chromatin domains and PC bodies move in confined volumes much smaller than chromosome territories. This highly constrained motion prevents chromatin domains from dispersing inside the cell nucleus and can explain why chromosomes form chromosome territories in interphase cells. This movement within highly confined volumes implies that some forces prevent chromatin from diffusing within entire chromosome territories. Interestingly, it was shown before that chromatin loci localized in peri-nucleolar areas or within heterochromatin move less than the ones included in euchromatin, and it was concluded that association of chromatin loci with different nuclear compartments induces specific constraints on their motion. Another time-lapse experiment performed on one Drosophila locus flanking a large block of heterochromatin showed that random association of this locus with pericentric heterochromatin is quite stable and decreases its motion. The motion of larger chromatin structures such as heterochromatin or euchromatin domains cannot be addressed by tracking single loci. By analyzing structures larger than individual chromatin loci, the motion of both bulk chromatin domains and of PC bodies seems to be influenced by their respective local enrichment of histone and PC proteins. Therefore, one key determinant of the motion constraint is an inner property of these structures, which is coherent with the concept of self-organization (Cheutin, 2012).

The most dramatic change of PC body motion occurs during embryogenesis when nuclear volumes strongly decrease, concomitant with a decrease in bulk chromatin motion. Comparison of chromatin motion between early and late G2 Drosophila spermatocytes or between undifferentiated and differentiated cells of eye imaginal discs indicated that the volume in which chromatin loci move decreases during differentiation. However, because of the particularly rapid motion of chromatin domains and PC bodies during early embryogenesis, the slowdown of chromatin motion occurring during embryogenesis is higher than the ones previously described during differentiation. Interestingly, the reduction of the volume of constraint during developmental progression suggests a correlation between the flexibility of chromatin structures and the potential for cell differentiation (Cheutin, 2012).

It is interesting to note that the motion of PC bodies appears less sensitive to temperature than chromatin domains in late embryos, suggesting that Polycomb proteins may specifically buffer environmental effects such as temperature change. This buffering may be an important determinant of the stability of Polycomb-dependent gene silencing during development. During this work, no other fundamental difference was observed between the motion of condensed chromatin domains and of PC bodies. This apparent absence in specificity is coherent with data implying that PC bodies form molecularly specialized chromatin regions, but suggests that the molecular identity of these structures is not the main determinant of their motion. Interestingly, a previous study has shown that the artificial Mx1-YFP nuclear body exhibits a very similar mobility compared with Promyelocytic leukemia and Cajal bodies. Although being molecularly different, no specific motion of these nuclear bodies was observed, indicating that the motion of nuclear bodies mainly depends on structural issues such as their size and the nuclear volume. During fly embryogenesis, PC bodies and condensed chromatin domains move similarly, but PC bodies move in a larger volume than chromatin domains. To explain this difference, one might argue that condensed chromatin domains would form much larger structures than PC bodies. This is difficult to ascertain until the identity of these DAPI- and histone-dense regions is better understood. Genome-wide analysis of chromatin components has recently identified five different types of chromatin in Drosophila cells, among which three contained silent genes (Filion, 2010). In addition to heterochromatin and Polycomb-repressed chromatin, a third type of silent chromatin was uncovered, which is composed of very large genomic domains encompassing half of the genomic euchromatin. It is proposed that this silent chromatin portion of the genome is physically manifested as the DAPI- and histone-dense chromatin that this study has identified to be distinct from PC bodies (Cheutin, 2012).

O-GlcNAcylation prevents aggregation of the Polycomb group repressor Polyhomeotic

The glycosyltransferase Ogt adds O-linked N-Acetylglucosamine (O-GlcNAc) moieties to nuclear and cytosolic proteins. Drosophila embryos lacking Ogt protein arrest development with a remarkably specific Polycomb phenotype, arising from the failure to repress Polycomb target genes. The Polycomb protein Polyhomeotic (Ph), an Ogt substrate, forms large aggregates in the absence of O-GlcNAcylation both in vivo and in vitro. O-GlcNAcylation of a serine/threonine (S/T) stretch in Ph is critical to prevent nonproductive aggregation of both Drosophila and human Ph via their C-terminal sterile alpha motif (SAM) domains in vitro. Full Ph repressor activity in vivo requires both the SAM domain and O-GlcNAcylation of the S/T stretch. Ph mutants lacking the S/T stretch reproduce the phenotype of ogt mutants, suggesting that the S/T stretch in Ph is the key Ogt substrate in Drosophila. It is proposed that O-GlcNAcylation is needed for Ph to form functional, ordered assemblies via its SAM domain (Gambetta, 2014).

polyhomeotic: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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