polyhomeotic

DEVELOPMENTAL BIOLOGY

Embryonic

Early expression in the cellular blastoderm appears in two domains, an anterior domain extending from 40 to 50% egg length (measured from the posterior) and a posterior domain extending from 30 to 10% egg length. The region anterior to the cephalic furrow is more intensely stained than the posterior (Fauvarque, 1995).

In germ-band extended embryos, polyhomeotic is expressed in most if not all cells of the presumptive neuroectoderm and epidermis. During gastrulation, ph expression evolves rapidly to form a striped pattern, similar to that of Engrailed in germ-band extended embryos. This banded phenotype makes sense in terms of the regulation of polyhomeotic by engrailed. By stage 13 ph is limited to the CNS and disappears from the epidermis. In this case it is no longer under control by engrailed (Serrano, 1995).

The proximal and distal polyhomeotic transcription units are differentially regulated at the mRNA level during development as shown by developmental Northern analysis. The proximal mRNAs do not become abundant until 3-6 hours of development, although maternally deposited mRNA is detectable in young embryos. The 6.1 kb zygotic transcript is initially less abundant than the 6.4 kb transcript, but by 9-12 hours, the 6.1 kb transcript is more abundant. In adult females, the two transcripts are equally abundant, but in males, the smaller transcript predominates. By contrast, the distal mRNA is much more abundant in 0-1.5 hour embryos than is proximal mRNA. Distal mRNA first falls dramatically, but increases again, between 3-6 hours of development. In adult females, a new transcript is detected, just slightly smaller than the transcript found at other developmental stages. No evidence is found for differential binding of proximal and distal products to polytene chromosomes. These results show that the ph locus undergoes complex developmental regulation, and suggest that Polycomb group regulation may be more dynamic than anticipated (Hodgson, 1997).

The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins (Polycomb, Polyhomeotic and Posterior sex combs) in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression, there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. Less than 2% of Ph remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG- mediated gene repression. The findings reported in this paper do not exclude the possibillity that the minor fraction of the PcG proteins, which remains bound to mitotic chromosomes, may be associated with specific nucleation sites within the repressed genes. Repression could then be initiated in telophase from these sites via cooperative binding of previously dispersed PcG protein complexes, insuring that promoters are blocked before reassembly of functional transcription complexes. A similar marking mechanism has been proposed for active genes by residual transcription factors on mitotic chromosomes. In the BX-C, possible candidates for repression nucleation sites are regulatory elements, which are preferentially associated with PC (Buchenau, 1998).

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins Polycomb and Polyhomeotic in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). Translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes were determined in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with the experimental data. Forward and reverse rate constants for complex formation were determined. Complexes exchange within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria (Ficz, 2005).

Most FRAP studies of nuclear proteins have involved components in transcription complexes or transcriptional activators that exchange in less than 2 minutes. The only repressor protein that has previously been investigated is heterochromatin protein 1 (HP1), a protein targeted to heterochromatin in higher eukaryotes. Although HP1 is loaded directly onto the chromatin during replication, it was found by FRAP to bind only transiently to chromatin with a maximum residence time of 60 seconds. Thus, both HP1 and PcG repression complexes appear to function by dynamic competition with other chromatin-binding proteins rather than by formation of a static, higher-order chromatin structure with immobilized bound repressors. FRAP measurements on polytene chromosomes revealed differences in the dissociation rate constants between individual bands -- this implies that a flexible repression system of complexes with various compositions that influence the binding affinity of other members and whose turnover is in the order of a few minutes. It is concluded that: (1) activation and repression can be dynamically controlled by simple chemical equilibria; (2) reduction in PcG levels will facilitate epigenetic change and may explain why non-cycling cells can be reprogrammed more easily than cycling cells, and (3) PcG complexes are exchangeable protein assemblies that maintain repression over many cell cycles by simple chemical equilibria (Fitz, 2005).

Larval

In larvae, Engrailed activates polyhomeotic expression during wing morphogenesis (Serrano, 1995). Larval expression occurs in the eye-antennal disc, the metathoracic leg disc, the brain and anterior midgut (Fauvarque, 1995).

Polycomb group proteins (PcG) repress homeotic genes in cells where these genes must remain inactive during Drosophila and vertebrate development. This repression depends on cis-acting silencer sequences, called Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known sequence-specific DNA-binding PcG protein, binds to PREs, but pho mutants show only mild phenotypes compared with other PcG mutants. pho-like, a gene encoding a protein with high similarity to Pho, has been characterized. Pho-like binds to Pho-binding sites in vitro and pho-like; pho double mutants show more severe misexpression of homeotic genes than do the single mutants. These results suggest that Pho and Pho-like act redundantly to repress homeotic genes. The distribution of five PcG proteins on polytene chromosomes was examined in pho-like, pho double mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at most sites in the absence of Pho and Pho-like. At a few chromosomal locations, however, some of the PcG proteins are no longer present in the absence of Pho and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not participate in the anchoring of PcG complexes, but may be necessary for transcriptional repression mediated through PREs. In contrast to Pho and Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other DNA-binding proteins implicated in PRE function, do not cause misexpression of homeotic genes or reporter genes in imaginal discs (Brown, 2003).

The distribution of the PcG proteins Pc, Psc, Polyhomeotic (Ph), Sex combs on midleg (Scm) and Enhancer of zeste [E(z)] on polytene chromosomes was examined. Pc, Ph and Psc are all core components of the PcG protein complex called PRC1. Scm has also been reported to co-purify with PRC1. Scm and Ph may also be present in protein complexes other than PRC1. E(z) is a component of the Esc-E(z) complex, which is distinct from PRC1. The analysis focused on PcG protein binding sites on the X chromosome and on the right arm of chromosome 3, which includes the bithorax and Antennapedia gene complexes (BXC and ANTC) (Brown, 2003).

Pho, Pc, Psc, Ph and Scm all bind the same three sites in wild-type chromosomes. As expected, in phol; pho double mutants, no Pho protein is detected. Binding of Pc, Psc and Scm is lost at polytene subdivision 2D in phol; pho double mutants; binding of these proteins to all other sites on the X chromosome is unaffected. Binding of Ph is completely unaffected in phol; pho double mutants. In particular, the Ph signal at 2D is present, suggesting that Ph can bind at this site even if other PcG proteins are removed. Pc binding to 2D is not lost in either pho or phol single mutants, suggesting that the presence of either of these two proteins is sufficient for Pc to bind to this site (Brown, 2003).

Taken together, the immunolocalization data suggest that binding of PcG proteins to most sites is unaltered in the absence of Pho and Phol protein, but that two proteins are redundantly required for PcG protein binding at a few specific sites. Intriguingly, it appears that all PcG proteins tested in this study are still associated with the BXC and ANTC loci. Nevertheless, the BXC genes Ubx and Abd-B are derepressed in phol; pho double mutant wing discs. Several different explanations for this paradox are proposed. (1) Derepression of homeotic genes and binding of PcG proteins were not assayed in the same tissues. It was not possible to detect derepression of Ubx in salivary gland cells of phol, pho double mutants. (2) Pho and Phol may only be required for anchoring PcG proteins at some PREs in the BXC. Different DNA-binding proteins may provide this function at other PREs. This is supported by the finding that binding of PcG proteins is lost at some sites in phol; pho double mutants. Moreover, several different PREs have been identified in the Ubx gene. The resolution of antibody signals on polytene chromosomes is not refined enough to resolve distinct PREs in a single gene and, hence, loss of only a fraction of PcG protein complexes may not be detectable. Finally, Phol and Pho may not be necessary for the anchoring of PcG protein complexes to the DNA, but may confer the actual transcriptional repression mediated by PREs in imaginal discs, while the PcG protein complexes might function in the propagation and memory of the repression. Thus, PcG protein complexes might serve to recruit Phol and Pho or their co-repressors to the DNA (Brown, 2003).

These results show a strong requirement for the DNA-binding proteins Pho and Pho-like in homeotic gene silencing in imaginal discs. In fact, the strong misexpression of homeotic genes observed in phol; pho double mutant imaginal cells is comparable with that seen in imaginal disc clones mutant for Pc, Scm, Sce or Pcl. The loss of PcG protein binding at only a small number of sites in phol, pho polytene chromosomes is consistent with the idea that Phol and Pho are required to recruit PcG protein complexes at only a subset of PREs. Alternatively, Phol and Pho may be required for transcriptional repression mediated by PREs, but not for anchoring of PcG protein complexes (Brown, 2003).

Effects of Mutation or Deletion

Homozygous ph flies have homeotic transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes (ANTP-C, BX-C), and in addition show loss of the humerus. ph interacts with three other similar mutations: Polycomb, Polycomb-like, and extra sex comb , and acts as a dominant enhancer of Pc. The expression of ph depends on the ANTP-C and BX-C dosage. ph has no embryonic phenotype, but temperature shift studies on ph2 show that the ph+ product is required during embryogenesis and larval development. ph mutants act to disrupt the normal expression of the ANTP-C and BX-C; therefore, ph+ is needed for maintenance of segmental identity (Dura, 1985).

Viable mutations of polyhomeotic produce transformations similar to those of known gain-of-function mutants in the ANTP-C and the BX-C. These are transformations of anterior segments and structures such as wings into more posterior segments (Dura 1987).

Polyhomeotic is found in immunoprecipitates in a multimeric complex that includes Polycomb. Duplications of ph suppress homeotic transformations of Pc and Polycomb-like, supporting a mass-action model for Pc-G function. ph alleles crossed to all members of the Polycomb group show synergistic effects, suggesting that these gene products might interact directly with ph. Embryonic phenotypes of ph2 embryos that were lethal when heterozygous or homozygous for other mutations suggest that ph may perform different functions in conjunction with differing subsets of Pc-G genes (Cheng, 1994).

Mutations in the proximal and distal proteins have differing effects on regulation of a reporter under the control of a regulatory region from bithoraxoid, suggesting that ph proximal and distal proteins have different functions (Hodgson, 1997).

The ph⁴⁰⁹ mutation disrupts one of the two tandem copies of the polyhomeotic gene located on the X chromosome. Although ph⁴⁰⁹ is a hypomorphic, homozygous viable allele, it is lethal or near-lethal in combination with other PcG mutations, including alleles of Sex combs on midleg . To assess ph interaction, ph⁴⁰⁹ females were crossed to males bearing Scm mutations and viability was scored among the double mutant male progeny. All Scm alleles cause partial lethality among ph⁴⁰⁹; Scm/+ males. However, Su(z)302, R5-13, and ET50 show much higher lethality in this combination than do H1 and M56 (Bornemann, 1998).

The ph^P1 allele of Drosophila polyhomeotic encodes a chimeric P-Ph protein that contains the DNA-binding domain of the P-element transposase and the Ph protein lacking 12 amino-terminal amino acids. It has been shown that the P-Ph protein is responsible for the formation of a repressive complex on P elements inserted at the yellow locus. An enhancer element can suppress the P-Ph-mediated inhibition of yellow transcription. However, an increase of P-element copy number at the yellow locus overcomes the enhancer effect. The mobilization of P-element transposition induces the appearance with a high frequency of Su(y) mutations that partially or completely suppress the inhibitory effect of ph^P1 on yellow expression. The Su(y) mutations are localized at different sites on chromosomes. One strong Su(y) mutation, sn^eP1, was found to be induced by a 1.2-kb P-element insertion into the transcribed noncoding region of the singed locus. The Su(y) mutations result in a high level of transcription of the 1.2-kb P element that contains the sequences encoding one DNA-binding and two protein-protein interaction domains of the transposase. The effect of Su(y) mutations can be explained by the competition between the truncated transposase encoded by a 1.2-kb P element and the P-Ph protein for binding sites on P-element insertions (Biryukova, 1999).

Thus, against the ph^P1 background the P-element sequences function as Polycomb response elements (PREs). PREs are found in the regulatory regions of many homeotic genes and are responsible for transcription repression. PREs are identified by their silencing effect on a reporter gene and by the introduction of variegated or pairing-sensitive expression of the white gene in transgenic flies. DNA fragments with PRE activity are generally from several hundred to several thousand base pairs in length. The y alleles used in this study have been generated by an insertion of the 2.9-kb genomic DNA sequence from the 1A region of X chromosome located more distally with respect to the yellow gene. The genomic duplication is flanked by two identical copies of a deleted 1.2-kb P element. The 1A enhancer located in the 2.9-kb insertion is responsible for yellow activation in the body cuticle and wing blade, compensating the yellow body and wing enhancers blocked by the su(Hw) insulator. The appearance of a strong enhancer disturbs the repressive effect of the P-Ph protein. Addition of the wing and body enhancers by inactivation of the su(Hw) function further suppresses the silencing effect of the ph^P1 mutation. The assembly of a silencing complex depends both on the strength of a PRE site and on the transcriptional activity of the region involved. For example, the formation of a Pc-G complex and the binding of GAL4, which activates transcription, are mutually exclusive and the silencing state can be prevented by a strong activation of transcription (Biryukova, 1999 and references).

The level of repression induced by ph^P1 depends directly on the number of P elements but not on their localization. The ph^P1-mediated repression may act at a distance from the yellow promoter. In the y^+s32 allele the P element is located ~3 kb from the yellow promoter and is separated from the latter by the 1A enhancer. Still, in this case, the ph^P1 mutation inhibits yellow transcription. In this respect, the P elements act like PREs that are responsible in a cooperative manner for the repressive state in the adjacent genome region. The ph^P1 mutation induces only a weak repression in y alleles containing a single P-element copy upstream of the yellow gene. Thus, one copy of P element is not sufficient for the formation of a strong repression complex. Addition to a single copy of the 1.2-kb P element of 153-bp 3'-terminal P-element sequences strongly enhances ph^P1-mediated repression, leading to a complete inactivation of yellow expression. As expected, the deletion of transposase-binding sites or 11-bp inverted repeats reduces the ph^P1-mediated repression. Unexpectedly, the internal region of the 1.2-kb P element is also important for the ph^P1-mediated repression that suggests the presence of a potential site(s) for binding of the chimeric P-Ph protein (Biryukova, 1999 and references therein).

PRE-containing transposons often show a dramatic enhancement of silencing when a fly is homozygous for a transposon insertion, indicating that the homologously paired PREs interact to produce a more stable and more repressive Pc-G complex. No pairing-dependent repression is found when both paired y alleles contain a single P-element copy inducing a weak or no ph^P1-mediated repression. A pairing-dependent repression is found only if one of the y alleles in heterozygous females induces a strong repression complex in the presence of ph^P1 (Biryukova, 1999).

The inhibitory effect of the ph^P1 mutation can be partially suppressed by Su(y) mutations induced by a P-element insertion into regulatory or coding regions of different genes. The inserted 1.2-kb P element is transcribed at a high level and gives rise to a truncated transposase. For example, in the sn^eP1 mutation, the 1.2-kb P element is inserted into the 5' transcribed noncoding region leading to an enhanced P-element transcription. mRNA encoding truncated transposase is also formed as a result of the ph^P1 mutation and this may partially decrease its effect. The 1.2-kb P element has a deletion between 830 and 2560 bp and resembles the previously described KP element. The DNA-binding domain is located within the region of 98 amino-terminal amino acids. The KP protein (a zinc finger transcriptional repressor coded for by the P-element) also contains two protein-protein interaction regions, and dimerization of the KP protein is essential for high-affinity DNA binding. A putative leucine zipper is located between 101 and 122 amino acids of the KP protein . The second protein-protein interaction region is present within a segment of 69 carboxy-terminal amino acids of the KP protein, that is, still in the amino-terminal part of the intact P element . All these sequences are also present in the protein encoded by the 1.2-kb P element (Biryukova, 1999 and references therein).

The KP protein binds to multiple sites on the P-element termini with a higher affinity than the full-length transposase. The binding sites include the high-affinity transposase binding sites, an 11-bp transpositional enhancer, and the terminal 31-bp inverted repeats. Both protein-protein interaction regions are important for this binding. The 1.15-kb P element in sn^e, a derivative of the sn^w mutation, is inserted in almost the same place and has the same orientation as the P element in sn^eP1. The protein product of the 1.15-kb P element contains the DNA-binding domain and a leucine zipper, but not the second protein-protein interaction region. This explains why the sn^eP1 mutation strongly suppresses the ph^P1 inhibitory effect, while sn^e does not. The Delta2-3 construct generating a full length transposase has a very weak suppression effect on the ph^P1 mutation. This may also be explained by its lower affinity to the P-element DNA. The defective transposase with the DNA-binding domain and two protein-protein interaction domains is most efficient for realization of the suppression effect. Thus, the suppression of ph^P1-induced inhibition is a result of the competition for binding sites on the P elements between the KP-like protein and the P-Ph protein. The presence of a strong enhancer in the chimeric element helps the KP-like protein in blocking the assembly of a P-Ph-mediated Pc-G complex on the P element. However, the Su(y) mutations, even expressing the truncated P-element transposase at high levels at all stages of Drosophila development, fail to affect the ph^P1-mediated inhibition of y^2s alleles induced by a double P element in the absence of the 1A enhancer. The formation of a strong repression complex prevents binding of the KP protein to transposase-binding sites on the P elements. Thus, the general low level of transcription activity facilitates P-Ph protein binding to DNA and the formation of a repressive complex (Biryukova, 1999).

Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. An investigation was carried out to see whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. Topoisomerase II (TopoII) and Barren (Barr: a subunit of the condensin complex - see Gluon) are shown to interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, the PcG protein Polyhomeotic (Ph) interacts physically with TopoII and Barr and Barr is required for Fab-7-regulated homeotic gene expression. Conversely, defects in chromosome segregation have been found associated with ph mutations. It is proposed that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression (Lupo, 2001).

PcG genes have been proposed to act as chromosomal components maintaining transcriptional repression by 'heterochromatinizing' their target sites. However, the molecular mechanisms underlying chromosomal silencing by the PcG, heterochromatin formation, and the transmission of the silenced state through mitosis are not known. It was reasoned that chromosome condensation machineries could provide an important functional link between the regulation of chromosome domain structure, gene silencing, and mitotic inheritance. Thus, the interaction of the PcG with the machinery involved in orchestrating chromosome dynamics has been investigated and in particular with those machines enabling mitotic chromosome condensation. The in vivo formaldehyde-fixed chromatin immunoprecipitation (X-ChIP) method was used to analyze the distribution in the BX-C locus of two proteins: TopoII, an enzyme involved in the regulation of DNA supercoiling, chromosome condensation, and segregation, and Barr. Barr is the homolog of the Xenopus XCAP-H and C. elegans DPY26 proteins, a TopoII-interacting protein associated with the SMC2/4 condensins complexes, known to be involved in mitotic chromosome condensation (Lupo, 2001).

A striking colocalization of TopoII and Barr with previously mapped PC binding sites was found, suggesting that the two groups of functions are at least acting on the same DNA regions. A clear colocalization was found at major PREs (Fab-7, Mcp, iab-3, bxd, and bx). In particular, the Fab-7 element appears to be a major TopoII/Barr binding site. Strong association of PC to Fab-7 is found. The expression of the major BX-C genes was examined by RT-PCR, and it was found that the AbdB gene is expressed, whereas Ubx and abdA are silent. No Barr/TopoII binding site was found at the Fab-8 PRE, which might define the border between the repressed and active BX-C domains in SL-2 cells (Lupo, 2001).

In iab-2 and iab-3, large fragments (11.0 and 11.5 kb, respectively) have PRE activity. Here specific Barr and TopoII sites are also found. These sites do not match the PC/GAGA peaks previously described. Yet, since these regions show considerable levels of PC, it is suggested that minor PC binding sites adjacent to the reported 'peaks' may also be functionally relevant. Another important aspect of PcG function is the interaction with promoters; major PC binding sites include core promoters, and it is known that PREs perform better when combined with their natural target promoters. Interestingly, a striking colocalization of TopoII and Barr is also found at promoters (AbdB gamma, abdA II, and Ubx) (Lupo, 2001).

Based on the mitotic phenotype and previous immunolocalization data, a direct association of TopoII and Barr with chromosomes mostly at mitosis is expected. In this context, the colocalization of TopoII and Barr in regulatory regions of the BX-C is striking. Although asyncronous tissue culture cells were used, it is believed that the association of Barr and TopoII with the regulatory regions of the BX-C occurs not only at mitosis but also in interphase. In particular, in X-ChIP experiments, the number of mitotic cells at the time of formaldehyde fixation is around 5%, thus, if only mitotic cells contributed to the overall precipitated DNA, this approach would have been below the detectable limit. Hence, it is proposed that TopoII and Barr are associated with their target sites throughout the cell cycle (Lupo, 2001).

The short proximal isoform of Ph (Ph 140p) can be copurified from nuclear extracts with TopoII and Barr. This isoform is not found coimmunoprecipitated with Pc and Psc, and neither Barr nor TopoII copurified with Pc and Psc. The three PcG members Pc, Psc, and the long proximal product Ph 170p have been shown to coimmunopurify from nuclear extracts with antibodies against one of the three. Due to the absence of Ph 140p signals in the Pc/Psc immunoprecipitations, these results might be taken to indicate that there is no functional connection between the presumptive TopoII/Barr/Ph 140p complex and the Pc/Psc/Ph 170p complex. For three reasons this is thought to be unlikely. (1) Both the 170p and the 140p isoforms of Ph are derived from the same transcript by posttranscriptional regulation and differ by a 244 N-terminal stretch of amino acids present only in the 170p isoform. Functional domains of Ph (zinc finger, coiled-coil region, GTP binding site, serine/threonine-rich region, and SAM/SPM domain) are all contained in both isoforms, suggesting that both proteins can fulfill related functions. (2) X-ChIP data, obtained with the same Ph antibodies used in this study, show an extended overlap of Pc and Ph binding regions in the BX-C. Together with the finding of a colocalization of TopoII and Barr with PcG binding sites in regulative regions of the BX-C, this suggests that these proteins act on the same DNA regions. (3) The data show that a reduction of the amount of Barren protein in barren heterozygotes parallels PcG-negative effects on the silencing function of the Fab-7 PRE (Lupo, 2001).

An additional finding supports the conclusion that Ph protein(s) are involved both in PcG function and mitotic chromosome condensation. ph null embryos show defects in chromosome segregation, the same phenotype observed for barren mutant embryos. Conversely, the results of Barren haplo-insufficiency on Fab-7 silencing are suggestive of a role for Barr in early embryogenesis. Since in early embryogenesis Ph 140p is the only Ph product made, these defects are diagnostic of a specific role of Ph 140p in mitosis. These results with regard to Barren protein and Fab-7 silencing are reminiscent of another previously documented role for SMCs in gene regulation. In C. elegans, the DPY27 protein, a homolog of the Xenopus XCAP-C (SMC4), has been shown to bind the X chromosome in females, whereas its absence results in lethality due to abnormally high gene expression levels from the X chromosome. Thus, it is concluded that Ph 140p shares an important role with the Barr/TopoII condensin complexes in mitosis and cell memory processes (Lupo, 2001).

In order to further study interactions between barren and the PcG the null alleles ph⁵⁰² and ph⁶⁰² were used for genetic analysis, and strains heterozygous for ph and barren mutations were crossed. Surprisingly, no effect was found. PcG genes, in contrast, show dosage effects, suggesting that the interaction between PcG and Barr/TopoII may imply a different, more dosage-insensitive regulation. However, it has been shown that barren mutations affect PRE silencing in the same way as mutations in PcG genes do. Taken together, these results may indicate a nonstoichiometric relationship between PcG and Barr/TopoII protein complexes. It is proposed that major PcG and condensin proteins belong to distinct protein complexes, but that they nevertheless cooperate at PREs and promoters to maintain the silenced state of homeotic genes. From the SMC standpoint, these results are intriguing because they show that proteins involved in chromosome condensation and segregation processes bind to regulatory elements in chromosomal domains responsible for the inheritance of transcription states. This would suggest that the 'structural maintenance of chromosome' function could also affect epigenetic control of gene expression (Lupo, 2001).

These data reveal novel molecular aspects of BX-C regulation. The distribution of PC and TopoII/Barr sites in the BX-C appears as a reiterated array suggestive of heterochromatic hallmarks, perhaps providing in cis information for higher-order organization of the BX-C chromosomal domain. In particular, TopoII oligomerizes in a DNA-dependent manner. Similar interactions in trans are proposed to occur between PcG proteins in vivo. According to this ability, spaced molecules at distant sites on the DNA could come into contact, giving rise to more condensed domains. A model has been proposed to explain how condensin proteins and Topoisomerases may act together in condensation. In this model, the size of the condensin complex (perhaps 1000 Å) could introduce (+) supercoils by affecting the global writhe of DNA, thus creating a more condensed state. In this study, Barr is found only at discrete sites, whereas PC and other PcG proteins are associated also with large chromosomal regions. Possibly, one aspect of PcG protein function and binding to chromatin in interphase is to stabilize and expand the condensed state by topological effects (Lupo, 2001 and references therein).

The positioning of TopoII at complex regulatory regions (e.g., abx/bx and iab-3-iab-8) may indicate the existence of minidomains providing tight control on the chromatin structure of intervening regulatory DNA sequences by localized changes of DNA superhelicity. The activity of TopoII could be locally regulated by the association with other proteins like Barr and perhaps some PcG and trxG members [e.g., Ph 140p, CCF, E(z), and Gaga]. Interestingly, Barr has been found to stimulate TopoII activity. It has to be pointed out that these data show, in a direct way, where in vivo TopoII binds to single-copy genes but they cannot tell if these sites correspond to TopoII cutting sites. However, it is likely that a tight association with DNA corresponds to enzymatic activity. Thus, it is proposed that in vivo TopoII activity may be enhanced at specific sites, whereas at others it could be reduced, resulting in local differences in chromatin condensation states controlled by DNA topology (Lupo, 2001).

The presence of multiple Barr and TopoII sites within the BX-C could thus provide a powerful way to fine-tune the structure of each of the parasegment-specific chromosomal subdomains. As a direct consequence of controlled condensation of specific parts of the BX-C, determined states could be fixed by enabling or not enabling specific interactions between cis elements. The mechanism by which Fab-7 regulates the AbdB promoters is, in fact, not known. It has been proposed that a combination of 'chromatin effects' and insulating activity may regulate enhancer-promoter interactions. It is proposed that the homeotic loss-of-function phenotypes observed in Fab-7 or Mcp deletions could be due to a change in local DNA topology altering the communication of segment-specific enhancers with the AbdB promoters. In this way, local differences in chromosome domain topology may contribute to stabilize or interfere with correct phasing between regulatory elements and promoters. If topological effects are at least part of Fab-7 function, this may also help to explain distance-dependent effects on enhancer-promoter interactions. Interestingly, in Drosophila, mutations in the Nipped-B gene facilitate enhancer-promoter interactions by overcoming the action of ectopic insulator elements in the Ubx domain. Nipped-B is the homolog of the yeast SMC-associated protein Scc2 (sister chromatid cohesion 2), suggesting that adherins may have a broader role in chromosomal domain organization and gene regulation. It is proposed that chromatin condensation proteins may be involved in a pathway acting also in interphase that regulates chromosome domain structure by DNA topology and is essential for maintenance of gene expression (Lupo, 2001).

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, since bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repress white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC is stronger than ZnF-SCM, which is stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression is more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produce silencing, indicating that these effects are long range. Repression by ZnF-SCM requires a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo (Roseman, 2001).

Biochemical studies indicate that PcG repression involves multiple, distinct PcG complexes. Thus, an underlying assumption of the assay system that was used is that gene silencing by the tethered ZnF-PcG protein involves assembly with endogenous PcG proteins at the reporter site. This hypothesis leads to the prediction that repression by a tethered ZnF-PcG protein should be compromised by loss of function for an endogenous PcG partner. It was difficult to test this prediction for the comprehensive set of endogenous PcG proteins because the basic assay system involved generating a very complex genotype. Nevertheless, the requirement was tested for endogenous PH protein, which is encoded by an X-linked gene and for which a hemizygous viable allele is available (Roseman, 2001).

A reporter integration site was identified that normally lacks PH binding, as scored on polytene chromosomes: it was repressed by all three ZnF-PcG proteins. Genetic tests show that reduction in PH dosage relieves tether-based repression by PC and SCM at this site. These results can be reconciled with the known PC-PH and SCM-PH molecular interactions. Surprisingly, ZnF-ESC repression is sensitive to PH dosage. This result was not expected since ESC-PH interactions have not been reported and there is evidence that ESC and PH are in separate complexes in embryos (Roseman, 2001).

Several explanations may account for the effect of PH dosage upon ZnF-ESC repression. (1) Since only a single reporter site was investigated, the PH dependency at this site may not be a general property at other genomic sites. It is noted, however, that this reporter site was chosen for analysis because polytene chromosome immunostaining studies indicate that it is not pre-equipped with endogenous PH. (2) Alternatively, it is possible that the functions of biochemically separable PcG complexes are interdependent in vivo, at least at certain loci. This could also explain the basic observation that PC and ESC are both required for repression at homeotic loci even though they sort into distinct complexes. An excellent example of the interplay between distinct chromatin complexes at a single locus is provided by regulation of the HO gene in yeast. Both the SWI/SNF nucleosome remodeling complex and the SAGA histone acetyltransferase complex are required for HO activation in vivo. These complexes cooperate in an ordered series of events, wherein SWI/SNF action is a prerequisite for SAGA activity upon HO chromatin. (3) Similarly, loci that require multiple PcG complexes for transcriptional repression may use a multistep mechanism where one PcG complex alters the chromatin template to 'pave the way' for binding or action of another PcG complex. Indeed, this type of interplay could explain the observation that E(Z) function is required for association of the PRC1 components PSC and PH at many chromosomal sites (Roseman, 2001).

The carboxyl-terminal SPM domain of SCM is highly conserved in mammalian SCM homologs. Analyses of Scm mutant alleles that remove the SPM domain, together with site-directed mutational analysis, have shown that the SPM domain is required for SCM function in vivo. Although in vitro studies indicate that the SPM domain is a protein interaction module, the functional contribution of this domain to SCM repression in vivo is not known. One possible role for the SPM domain would be to recruit SCM to target sites in chromatin, by analogy to the role that the conserved chromodomain plays in chromatin targeting of PC. Deletion of the SPM domain in ZnF-SCM^DeltaSPM abolishes silencing in the tethering system. Although ZnF-SCM^DeltaSPM accumulates to a level similar to that of wild-type ZnF-SCM, its repression activity is indistinguishable from ZnF alone. These results imply that the SPM domain does not solely provide interactions that target SCM to chromosomes, since the Su(Hw)-binding domain circumvents this targeting function. Instead, it is suggested that the SPM domain is more directly involved in the repression mechanism or in maintaining integrity of SCM complexes (Roseman, 2001).

Telomeric associated sequences of Drosophila recruit Polycomb-group proteins in vivo and can induce pairing-sensitive repression

In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. This study presents the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes reveal a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE) (Boivin, 2003).

Among the 50 mutant alleles of PcG and trxG genes tested, <10 behave as dominant modifiers of TPE. By contrast, combination analyses reveal that 10 alleles that have no effect alone have synergistic effects on TPE. Interestingly, the subgroup of dominant suppressors that act alone on TPE (Pc, ph, Psc, and Scm) are members of the PRC1 complex that has been purified from embryonic nuclear extracts. Some other PcG mutations, such as Asx, E(z), Pcl, or Sce, act as suppressors in combination, suggesting that the products of these genes participate with a specific telomeric PcG complex. Strikingly, this subgroup of eight PcG genes was already highlighted in a genetic interaction study showing that Pc, Scm, Psc, Pcl, Sce, and Asx are lethal when heterozygous with ph², a temperature-sensitive mutation, all combinations leading to similar phenotypes in the dying embryos (Boivin, 2003).

It has been shown that telomeric inserts are less accessible than euchromatic inserts to restriction enzymes and to DAM methylase. In addition, the accessibility of telomeric inserts to DAM methylase increases in a ph⁴¹⁰ background and this is correlated to derepression of the white gene. This result is similar to that obtained with the ph PRE-mini-white transgenes suggesting that PcG products adopt a similar chromatin-based mechanism to repress their euchromatic and telomeric targets (Boivin, 2003).

PREs were initially identified by their ability to prevent ectopic activation of a Hox reporter gene construct. This capacity depends on the dose of the PcG proteins. Placed in a transgene, PREs can also induce mosaic expression of the flanking reporter gene, a phenotype resembling that of PEV and TPE. Moreover, PRE-mediated repression often exhibits pairing sensitivity, defined as the lower expression of the flanking reporter gene in a homozygous state than in a heterozygous one. This study shows that a 1.2-kb fragment of the 1.8-kb X-chromosome TAS induces variegation or pairing-sensitive repression in an orientation-dependent manner and creates new binding sites for the PcG proteins as detected by immunostaining on polytene chromosomes. These results demonstrate that this TAS fragment mimics some properties of a PRE and thus reinforce the parallels that can be made between telomeric silencing and PcG-mediated euchromatic repression. TASs from the left tip of chromosome 2 (2L-TAS) retain aspects of telomeric silencing in ectopic positions. At this telomere, TASs are composed of repeats of 457 bp that present only limited homology with TASs present at the X, 2R, and 3R telomeres. Analysis of the sequence of one repeat (457 bp) revealed nine GAF-binding sites but no PHO-binding site. Several transgenic lines have been establised carrying different constructs made up of 6 kb of 2L-TAS (~13 repeats) adjacent to the mini-white reporter gene and flanked by Su(Hw) insulator sequences. Depending on the orientation of the TASs inside the transgene, some lines present reduced expression of the mini-white gene when compared to lines carrying a similar transgene without TASs or with TASs in the opposite orientation. Such orientation-dependent silencing has been described for the Fab7 PRE of the Ubx gene, but does not appear to be a general property of PREs since another PRE from Ubx (Mcp) has been shown to function in both orientations . From this study, the more efficient orientation for the 1.2-kb X-TAS-induced repression appears to be the same as that described for the 2L-TASs: repression appears to be stronger from the centromere-proximal side (Boivin, 2003).

Repression induced by the 2L-TAS when inserted within a transgene is weakly sensitive to Su(z)2⁵. Surprisingly, no effect of PcG mutations on the repression induced by the 1.2-kb X-TASs could be detected, except a slight suppressor effect of Su(z)2⁵ on P-CoT-1 in a homozygous state. At the moment, no explanation is available for why the repression induced by the 1.2-kb X-TASs in a euchromatic environment is not sensitive to modification of the dose of PcG proteins that could otherwise affect TPE (Boivin, 2003).

Increasing the distance between the 2L-TAS and the mini-white gene with 2.4 kb of unrelated DNA in another transgene did not change the silencing capacity of 2L-TAS. In this study, the 1.2 kb of X-TAS is located >5 kb away from the mini-white gene, thus showing the silencing capability of TASs over an extended distance. Similar results were obtained with transgenes containing the Fab7 PRE. According to chromatin-immunoprecipitation experiments, PcG products can spread as far as 10-15 kb from PREs and repression could be expected to occur over such a distance (Boivin, 2003).

In fact, what was observed with the 1.2 kb of X-TAS in the pCoT- transgenes resembles what has been observed with PREs from the Bithorax complex. Using Fab7-mini-white transgenes, it has been shown that some insertion sites present pairing sensitivity (as observed with P-CoT-2 and P-CoT-3), while others present variegation with darker spots (as observed with P-CoT-1). The Fab7 PRE has been shown to convey a derepressed state through meiosis after being activated in the embryonic stage by the UAS/GAL4 system. In the case of TPE, the derepressed state observed in a PcG mutant background is not transmitted to the next generation. A fundamental difference between these studies is that the suppressor effect observed in the case of TPE is due to the lack of one PcG partner. It is hyperactivation (forced activation) induced by GAL4 via the UAS sequences that abolishes the repressor capacity of the Fab7 PRE. This activation likely involves fundamental changes in chromatin conformation and/or epigenetic marks (such as hyperacetylation) that may be different from the effect of a decrease in the dosage of a repressor. To compare TPE and the Fab7 PRE it would thus be interesting to test transmission through meiosis of the derepressed state of the UAS-Fab7 transgene induced by a PcG mutation rather than upon activation by GAL4. Different PREs thus share properties but also present particularities that likely depend on their sequence. Indeed, the dissection of Mcp, another PRE from the Bithorax complex, revealed that repression in cis and pairing-sensitive repression could be separated. This shows that PREs may combine several regulatory properties and future dissection of the different TASs will tell which functions telomeric PREs combine (Boivin, 2003).

polyhomeotic is required for somatic cell proliferation and differentiation during ovarian follicle formation

The polyhomeotic (ph) gene of Drosophila is a member of the Polycomb group (Pc-G) genes, which are required for maintenance of a repressed state of homeotic gene transcription, which stabilizes cell identity throughout development. The ph gene was recovered in the course of a gain-of-function screen aimed at identifying genes with a role during ovarian follicle formation in Drosophila, a process that involves coordinated proliferation and differentiation of two cell lineages, somatic and germline. Subsequent analysis revealed that ph loss-of-function mutations led to production of follicles with greater or fewer than the normal number of germ cells associated with reduced proliferation of somatic prefollicular cells, abnormal prefollicular cell encapsulation of germline cysts and an excess of both interfollicular stalk cells and polar cells. Clonal analysis showed that ph function for follicle formation resides specifically in somatic cells and not in the germline. This is thus the first time that a role has been shown for a Pc-G gene during Drosophila folliculogenesis. In addition, mutations in a number of other Pc-G genes were tested, and two of them, Sex combs extra (Sce) and Sex comb on midleg (Scm), also display ovarian defects similar to those observed for ph. These results provide a new model system, the Drosophila ovary, in which the function of Pc-G genes, distinct from that of control of homeotic gene expression, can be explored (Narbonne, 2004).

A new role for ph was first revealed by the reduced fecundity and associated ovarian anomalies observed upon analysis of a P{y+}UAS insertion in the first exon of the ph-p locus (4061 line). That the ovarian phenotypes characterized for this line are due to overexpression of ph is supported by three lines of evidence: (1) the ovarian phenotypes produced depend on the presence of a GAL4 driver (da-GAL4); (2) the 4061/w; da-GAL4/+ flies also present a ph gain-of-function haltere-to-wing transformation; and (3) flip-out clone overexpression in ovarian somatic cells of several UAS-ph cDNA transgenes gave very similar ovarian phenotypes. In particular, overexpression of ph is associated with production of multicyst follicles in which several (two to four) germline cysts develop within a single follicular epithelium. Importantly, each multicyst follicle contains several pairs of polar cells corresponding to the number of cysts present in the follicle. Here, therefore, unlike other mutants (notch and hedgehog) for which inclusion of several cysts in one follicle has been attributed to a problem in polar cell specification, this does not seem to be the case. Interestingly, multicyst follicles produced by ph overexpression are covered by a follicular epithelium that is not completely regular, showing indentations that appear to mark boundaries between cysts as evidenced by the presence of polar cells at the level of the indentations. This suggests that earlier, cyst individualization may have begun and been subsequently aborted. In support of this, analysis of the associated germaria shows an abnormally long region 3, with adjacent mature germline cysts between which prefollicular cells fail to complete centripetal migration (visualized by specific anti-Fas III antibody staining of prefollicular cells). Overexpression of ph may thus specifically affect the expression of proteins necessary for recognition and/or adhesion between prefollicular cells and germline cysts for encapsulation. These effects seem to be specific to this stage since later interactions between these two cell lineages, for instance between follicular epithelial cells and the nurse cells and oocyte, are not perturbed by overexpression of ph (Narbonne, 2004).

Surprisingly, a similar phenotype as that observed in the ph overexpression study, multicyst follicles (several cysts within one follicle), was observed with loss-of-function ph alleles. Importantly, in contrast to ph overexpression, multicyst follicles in ph loss-of-function mutant ovaries always have only two groups of polar cells, one at each pole. Therefore, it seems that, unlike for overexpression of ph, delayed or deficient polar cell specification in ph mutants contributes to inclusion of several cysts within a single follicle. Thus, ph overexpression and loss-of-function phenotypes are distinguishable, indicating that the origin of the phenotypes is probably different (Narbonne, 2004).

The implication of the ph gene in ovarian somatic cells was also studied using two different loss-of-function mutations: the hypomorphic ph^lac mutation, which consists of a PlacW transposon inserted in the first intron of ph-p; and via clonal analysis of the amorphic ph⁵⁰⁴ (noted ph⁰) allele, which eliminates the functions of both ph-p and phd. The origin of the multicyst phenotype caused by ph loss-of-function mutations was characterized more precisely by analysis of the process of follicle formation in the germarium. This study showed that several early aspects of the somatic cell developmental program (including proliferation, morphogenesis and differentiation) are perturbed by these ph mutations (Narbonne, 2004).

In contrast, the rate of division of germarial somatic cells is reduced in a ph hypomorphic mutant background, as assayed by immunohistochemical analysis of the mitosis-specific PH3. This probably contributes to delayed follicle encapsulation and budding, evidenced by the accumulation of mature germline cysts in germarial region 3 of ph mutant ovarioles, and, consequently, by the formation of multicyst follicles. Although the same type of analysis was not possible upon induction of clones of the ph⁰ amorphic mutation in somatic ovarian cells, the fact that these clones are very small or absent compared with control clones suggests that a proliferation defect may also be associated with this ph mutation (Narbonne, 2004).

In contrast, the morphogenetic properties of prefollicular cells and their differentiation into polar cells, interfollicular stalks and follicular epithelia are also specifically perturbed in ph mutants, which also probably contributes to formation of multicyst follicles. ph⁰ clonal analysis shows that ph function is necessary specifically in somatic cells, and not in the germline, for proper follicle formation. Almost all the phenotypes observed in ph mutant ovaries were reproduced upon induction of ph⁰ clones in prefollicular cells and their descendants (Narbonne, 2004).

In one observed phenotype, prefollicular cell individualization of germline cysts was shown to be compromised in germarial regions 2a and 2b of ph mutant ovarioles. Fas III, which is specifically upregulated in prefollicular cells in wild-type germaria, is expressed normally in ph mutant prefollicular cells (ph^lac and ph⁰), but these cells remain at the periphery of the germarium and fail to undergo normal morphological changes necessary for germline cyst encapsulation, allowing multiple cysts, not individualized by somatic cells, to accumulate in region 3. In other ph mutant germaria in which prefollicular cells have migrated between germline cysts, encapsulation is disorganized, and germline cysts can be split and follicle budding significantly delayed. Therefore, in the germarium, interaction between prefollicular cells and the germline is defective (Narbonne, 2004).

In a second observed phenotype, prefollicular cell differentiation into polar cells, interfollicular stalk cells and follicular epithelial cells was delayed and/or incomplete in the presence of ph mutations. Using a polar cell specific marker (A101/neu-lacZ), specification of polar cells, normally appearing by stage 2 in wild-type follicles, was shown to be delayed in ph mutant ovarioles until stage 3. In addition, multicyst follicles produced in ph mutant ovarioles contained two pairs of polar cells, one at each extremity of the anterior/posterior axis. Therefore, polar cell specification, which is necessary for individualization of germline cysts, is perturbed by ph mutations. Interfollicular stalk cell differentiation was assayed by expression of the stalk-specific marker, alpha-Spectrin, and by loss of Fas III, which is normally expressed at high levels in precursor prefollicular cells. In stalks of both ph mutant and ph⁰/ph⁺ mosaic ovarioles, although alpha-Spectrin expression was normally upregulated, Fas III was also present at high levels, indicating an ambiguous state of differentiation. Poor differentiation of stalk cells was further substantiated by the abnormally long and disorganized interfollicular stalks that showed intercalation defects. Abnormal perdurance of the early prefollicular cell marker Fas III was also observed at the level of the follicular epithelium in very affected ph mutant ovarioles, as well as in ph⁰ epithelial cell clones. Taken together, these results suggest that ph mutations result in the prolongation of a precursor state for polar, stalk and follicular epithelial cells (Narbonne, 2004).

In a third observed phenotype, ph mutant ovarioles exhibited an excess of polar cells (up to 11) at both the anterior and posterior poles of follicles, which persisted beyond stage 9, accompanied by an excess of adjacent interfollicular stalk cells (from 10 to 50). ph⁰ clones induced in the polar cell lineage also produced an excess of polar cells after stage 5, and the presence of ph⁰ cells was also associated with abnormally long stalks. Overproliferation of the pool of precursor cells common to both polar and stalk cells would result in an excess of these cells. However, the mitotic activity of germarial somatic cells was assayed by PH3 staining and a reduction compared to wild-type was observed in regions 2a, 2b and 3. It has recently been shown that an excess of polar cells is normally present in early stage follicles in wild-type females, and that the final pair of polar cells is selected from this group via apoptosis-induced cell death. However, in that study, a maximum of six such pre-polar cells was observed when apoptosis was specifically blocked. Since ph mutant ovarioles exhibit up to 11 polar cells, it would seem that apoptosis of pre-polar cells is probably not the only aspect of polar cell development affected. Finally, it is possible that the process by which the pool of polar and stalk cell precursors, distinct from the progenitors of the epithelial follicle cells, is set aside may be affected by ph mutations, leading to both problems in their number and differentiation. Determination of this pool probably involves several cell-cell signaling pathways in region 2b of the germarium, implicating Delta/Notch and EGFR signaling initiating from the germline and Hedgehog signaling from anterior terminal filament somatic cells. ph may participate, in parallel or within one (or several) of these signaling pathways, to the regulation of the somatic cell differentiation program in the germarium. So far, attempts to uncover genetic or molecular interactions between ph and genes of these signaling pathways have proven unfruitful (Narbonne, 2004).

The ph gene was first characterized as one of the Drosophila Pc-G genes, which encode transcriptional repressors required for maintaining the spatial pattern of homeotic gene expression during embryonic and larval development. ph has additional functions during development, since it has also been implicated in restriction of anterior compartment expression of engrailed and hedgehog in the wing imaginal disc. The present results, which implicate ph function and that of two other Pc-G genes (Sce and Scm) in somatic cell development during early oogenesis, thus suggest that Pc-G function may be more generalized than previously thought. One other study reported ovarian defects associated with two temperature-sensitive alleles (pco^ox736hs and pco^my939hs) of the E(z) gene, but the defects observed do not resemble those of ph (degeneration of nurse cells and little growth in the size of the follicle beyond stage 3 or 4) (Narbonne, 2004).

Ovarian phenotypes associated with mutations in several other Pc-G genes were also examined in this study. The product of the Scm gene interacts directly with Ph, and, with Ph, forms part of the same complex, PRC1, in Drosophila embryos. The presence of large somatic cell clones of Scm^D1 (amorphic mutation) leads to similar, though not completely overlapping, phenotypes than those observed for mutations in the ph gene. In particular, the results suggest that somatic cells are poorly differentiated and this leads to formation of multicyst follicles and abnormal interfollicular stalks with an excess number of cells. Other Pc-G members were examined; some also belong to the PRC1 complex (Sce and Psc), while others do not [Asx, Su(z)2 and Pcl]. Ovarian defects were only observed with a mutation in the Sce gene, and these defects closely resembled those obtained with Scm^D1. Since follicle cell clones mutant for Scm^D1 and Sce¹ covered large areas of the follicular epithelium, like wild-type clones, it is concluded that (unlike ph⁰, Scm^D1 and Sce¹) somatic cells are not affected in their proliferative property and/or viability. In addition, Scm^D1 and Sce¹ mutations did not affect polar cell number or differentiation. These results indicate that these anomalies are specific to mutations in the ph gene. Thus, several components of the PRC1 complex, but not all, seem to be implicated in follicle formation and their functions do not seem to overlap fully. In addition, none of the genetic interactions between Pc-G genes known to exist for embryonic segment identity were reproduced in the ovary system. Two hypotheses can be made concerning the role of these Pc-G genes in ovarian folliculogenesis: (1) either each Pc-G gene acts specifically on its own specific subset of target genes in somatic cells of the ovary, possibly regulating the transcriptional machinery directly rather than forming particular Pc-G complexes that alter chromatin structure, or (2) repression of target genes in somatic cells of the ovary occurs via Pc-G complexes in a chromatin-dependent manner, but the complexes involved differ markedly in composition from those identified for embryonic cell identity. Further experiments will be needed to distinguish between these two possibilities (Narbonne, 2004).

Polycomb group mutants exhibit mitotic defects in syncytial cell cycles of Drosophila embryos

The Polycomb Group (PcG) of epigenetic regulators maintains the repressed state of Hox genes during development of Drosophila, thereby maintaining the correct patterning of the anteroposterior axis. PcG-mediated inheritance of gene expression patterns must be stable to mitosis to ensure faithful transmission of repressed Hox states during cell division. Previously, two PcG mutants, polyhomeotic and Enhancer of zeste, were shown to exhibit mitotic segregation defects in embryos, and condensation defects in imaginal discs, respectively. polyhomeotic^proximal but not polyhomeotic^distal is necessary for mitosis. To test if other PcG genes have roles in mitosis, embryos derived from heterozygous PcG mutant females were examined for mitotic defects. Severe defects in sister chromatid segregation and nuclear fallout, but not condensation are exhibited by Polycomb, Posterior sex combs and Additional sex combs. By contrast, mutations in Enhancer of zeste (which encodes the histone methyltransferase subunit of the Polycomb Repressive Complex 2) exhibit condensation but not segregation defects. It is proposed that these mitotic defects in PcG mutants delay cell cycle progression. Possible mitotic roles for PcG proteins are discussed, and suggest that delays in cell cycle progression might lead to failure of maintenance (O'Dor, 2006).

The data for ph mutations confirm the original observation that ph⁵⁰³ mutations exhibit mitotic defects. These original observations have been confirmed in several ways. First, the observation that strains out-crossed to wild-type flies show similar frequencies of defects compared to heterozygous mutants show that the phenotypes arise from ph mutations rather than background effects. Second, embryos derived from homozygous ph⁴⁰⁹ mothers show similar frequencies of mitotic defects to those derived from heterozygous mothers. These results suggest that for ph, the severity of the phenotype reaches a plateau when the amount of Ph is reduced below a threshold which must be greater than 50% of the wild-type amount. Third, only ph^p (ph⁴⁰⁹) is necessary for normal mitosis, because mutations in ph^d (ph⁴⁰¹) have no effect on mitosis. This observation is consistent with data that has shown that only one isoform of Ph-P coimmunoprecipitates with Barren or Topoisomerase II. This observation supports the conclusion that Ph-P and Ph-D have different functions. Fourth, because homozygous ph⁴⁰⁹ flies are viable, the ph phenotypes reported here represent those of maternal germline nulls (O'Dor, 2006).

The results show that early embryos of PcG and Asx mutants exhibit highly penetrant and expressive mitotic phenotypes in syncytial embryos, consistent with problems in cell cycle progression. Two classes of phenotypes are observed: segregation defects and condensation defects, but no mutant exhibits both phenotypes. In these experiments, with the exception of ph, embryos were scored derived from heterozygous mothers, in which 50% of the wild-type product remain. Therefore, the possibility cannot be ruled out that more severe mitotic phenotypes would be observed in embryos derived from homozygous mothers, resulting in less distinct differences between E(z) and other mutants. Consistent with this caveat, when homozygous E(z)⁵ (l(3)1902) mutant imaginal disks were examined, both condensation defects and chromosome breakage consistent with problems in segregation were observed, so E(z) may function in both condensation and segregation. The data show that embryos derived from homozygous ph-proximal mutants do not have condensation defects, so at a minimum, E(z) has at least one role in mitosis different from that of ph. To accurately compare the roles of different PcG and ETP genes in mitosis, it will be necessary to examine mutations derived from homozygous mutant mothers, or from germline clones (O'Dor, 2006).

Mutations in the PcG genes Sex combs extra and Sex combs on midleg reduce proliferation of ovarian follicle cells in Drosophila, suggesting that other PcG members are also required for cell cycle progression. It is predicted that other PcG and ETP group mutants not tested here will also exhibit significant mitotic defects (O'Dor, 2006).

Given the high penetrance and expressivity of the chromatin bridge phenotype in PcG mutant embryos, what becomes of the embryos with severe chromatin bridges, and more specifically, what happens to chromatin bridges themselves? Four observations suggest that most chromatin bridges are resolved in PcG mutants. First, nuclear fallout should remove unresolved nuclei, but relatively few fallout nuclei were observed in any embryo. Second, anaphase and telophase embryos together made up 7–9% of the total developed embryos, a proportion that is consistent with the short duration of those mitotic phases. This low proportion of embryos in anaphase or telophase argues that the embryos that exhibited severe chromatin bridges were not developmentally arrested or dead. Third, only a few embryos out of all mutants tested appeared to have bridged prometaphase nuclei. If chromatin bridges did not resolve, one would expect a higher proportion of these prometaphase bridges. Fourth, unresolved chromatin bridges should break. However, fragmented chromosomes, evidence of chromosome breakage and all low-penetrant mitotic defects accounted for only 4–10% of the total mitotic defects observed in mutant embryos (O'Dor, 2006).

C(2)EN embryos carrying an abnormally long second chromosome exhibited chromatin bridges between some nuclei since the extra-long chromosomes were not able to fully segregate during anaphase. These bridged nuclei lagged behind neighboring nuclei and were subsequently removed from the cortex by the fallout mechanism once they reached telophase. Therefore, the fallout nuclei observed in PcG mutants are likely previously-bridged nuclei not able to resolve in time to maintain overall mitotic synchrony. Interestingly, the fallout nuclei were never joined by chromatin bridges. This may be because the delay is only detected once the bridges are resolved, or the bridged nuclei “snap-back” and fuse with each other, as has been observed for bridged nuclei in embryos mutant for grapes, a checkpoint gene required at several cell cycle stages (O'Dor, 2006).

Occasionally, the fallout mechanism may be unable to detect or remove delayed nuclei. If resolved, these nuclei may appear as asynchronous to neighboring nuclei, or, if bridged, they appear as prometaphase bridges, polyploid, giant nuclei or chromosome breaks. The embryos of polyhomeotic mutants develop at a slower rate than those of wild-type flies as judged by timed embryo collections. The slower developmental rate may reflect delays in the mitotic cycles due to segregation defects. In other cases, the most extreme segregation defects overwhelm the fallout mechanisms and continue with the mitotic program until the segregation failures reach a critical point and the embryo dies. In some embryos, the cortex is completely disorganized with very large amorphous nuclei and extensive chromosome breakage. These embryos are probably dead and appear to be the result of cumulative effects of several rounds of segregation defects (O'Dor, 2006).

It remains to be determined whether the length of cell cycle stages in PcG mutants is altered by a checkpoint pathway. In syncytial embryos, the metaphase to anaphase transition is delayed in response to damaged DNA, improper spindle assembly, or faulty centrosome activation. Activation of the spindle checkpoint also delays mitotic progression. It is possible that the mitotic defects of PcG embryos also delay the mitotic cycle by activating a pre-mitotic checkpoint (O'Dor, 2006).

PcG proteins could have a direct structural or enzymatic role in mitosis, separate from their role in silencing. PcG proteins associate with chromatin in a cell cycle-dependent manner. In Drosophila embryos, Polyhomeotic (PH), Polycomb (PC), and Posterior sex combs (PSC) proteins associate with chromatin at S phase, almost completely dissociate by metaphase and reassociate at telophase. BMI1, the human homologue of PSC, shows a similar pattern of association and dissociation in primary and tumor cell lines. Therefore, PcG proteins are present during the key events of mitosis that occur prior to metaphase. An interesting recent report shows that Set1, the yeast homolog of the MP Trithorax, methylates a component of the kinetochore, consistent with the possibility that the methyltransferase activity of E(z) could directly modify proteins needed for mitosis (O'Dor, 2006).

The presence of anaphase bridges in ph, Pc, Psc, and Asx mutants does not necessarily imply that PcG proteins act at anaphase. Mitotic defects may arise at other cell cycle stages but carry forward to manifest as a segregation phenotype. Many different Drosophila cell cycle genes regulating every stage of the cell cycle also have chromatin bridge phenotypes. Some examples include kinesin-like enzymes, a variety of regulatory kinases such as polo kinase and aurora-like kinases, replication checkpoint regulators such as grapes, chk2, and mei-41, and genes involved in sister chromatid segregation such as pimples and three rows (O'Dor, 2006).

PcG proteins could be required at DNA synthesis. Cramped colocalizes with PCNA, which is required for DNA synthesis. Therefore, Cramped, and by extension, other PcG proteins could have a role in elongation of replication forks. RAE28, the homolog of PH, interacts and colocalizes with GEMININ, a replication licensing factor. There have been suggestions in the literature that silenced genes are late-replicating, and this observation has been supported in Drosophila. Therefore, PcG mutations, by interfering with silencing or chromatin structure, could affect replication timing in mitosis. Consistent with this idea, PC tethered near an origin interferes with origin activity. However, it has been reported that an E(z) mutation does not affect replication timing in polytene chromosomes. This may be a reflection of the differences in possible mitotic roles between E(z) and other PcG members. Interestingly, in mammals, heritable gene silencing delays chromatid resolution without affecting timing of DNA replication (O'Dor, 2006).

PcG proteins could also be required for association of sister chromatids. Interaction between PcG Response Elements (PREs), presumably mediated by PcG proteins, is important for repression of PcG targets. Interaction between PcG proteins has been proposed to account for the high likelihood of insertion of PRE-containing transgenes in genomic regions that already contain a PRE. By analogy, PcG proteins could have roles in sister chromatid adhesion or resolution (O'Dor, 2006).

Finally, PcG proteins could be required for chromatin condensation prior to metaphase. This hypothesis is consistent with the E(z) phenotype, in which mitotic chromosomes fail to condense. E(z) is a histone methyltransferase. Histone modifications, notably hypoacetylation and methylation of histone H3 lysine 9 and 27, have been associated with heterochromatin and silencing, consistent with the idea that E(z) might have a role in chromosome condensation. In support of this idea, PH-P coimmunoprecipitates with Barren and Topoisomerase II. Though Barren is a member of the condensin complex, it is not essential for condensation, but is required for sister chromatid resolution. It is speculated that E(z) has a specific role in condensation separate from the role of other PcG proteins, perhaps because its role as a methyltransferase might be required for targets other than histones. It will be interesting to determine if all PRC2 members exhibit condensation defects (O'Dor, 2006).

PcG genes could have indirect effects on mitosis if they are required for regulation of genes that are themselves important for mitosis, or to prevent expression of genes that disrupt mitosis. There are two clear precedents for this possibility. Bmi1, the mammalian Psc homolog originally identified as an oncogene, is also required for regulating lymphoid cell proliferation via repression of the ink4a tumor suppressor locus. Mel18, another mammalian Psc homologue, was originally identified as a tumor suppressor and inhibits cell cycle progression likely via repression of c-myc, leading to downregulation of cyclins and CDKs. If PcG-mediated regulation of proteins important for the cell cycle accounts for the mitotic phenotypes observed in embryos, then this challenges the assumption that maintenance proteins are required only to propagate expression states of genes between cell cycles (O'Dor, 2006).

Steroid hormone-dependent transformation of polyhomeotic mutant neurons in the Drosophila brain

Polyhomeotic (Ph), which forms complexes with other Polycomb-group (PcG) proteins, is widely required for maintenance of cell identity by ensuring differential gene expression patterns in distinct types of cells. Genetic mosaic screens in adult fly brains allow for recovery of a mutation that simultaneously disrupts the tandemly duplicated Drosophila ph transcriptional units. Distinct clones of neurons normally acquire different characteristic projection patterns and can be differentially labeled using various subtype-specific drivers in mosaic brains. Such neuronal diversity is lost without Ph. In response to ecdysone, ph mutant neurons are transformed into cells with unidentifiable projection patterns and indistinguishable gene expression profiles during early metamorphosis. Some subtype-specific neuronal drivers become constitutively activated, while others are constantly suppressed. By contrast, loss of other PcG proteins, including Pc and E(z), causes different neuronal developmental defects; and, consistent with these phenomena, distinct Hox genes are differentially misexpressed in different PcG mutant clones. Taken together, Drosophila Ph is essential for governing neuronal diversity, especially during steroid hormone signaling (Wang, 2006).

Ph is well implicated in maintaining cell fates via controlling transcription of genes in distinct cell type-characteristic manners. Deregulation of multiple genes aberrantly occurs in ph mutant tissues. A similar mechanism probably underlies most of the abnormalities in ph mutant neurons. In particular, there are multiple lines of evidence suggesting mal-expression of various subtype-specific GAL4 drivers in ph mutant clones. First, with respect to GAL4-OK107, GAL4-NP225 and elav-GAL4, the use of various GAL4 drivers results in labeling of similar numbers of clones. Second, clones were induced in the central brain versus the optic lobe, depending on when mitotic recombination was induced; the result is the same as in wild-type mosaic brains. Third, ato-GAL4 and GAL4-EB1 fail to label any clone, arguing against constitutive expression of UAS-transgenes in mutant clones. Finally, examining clones through development reveals no evidence for derivation of some clones from other clones; and, instead, sudden labeling of full-sized clones was constantly observed shortly after a big ecdysone pulse. Apparently, loss of Ph function alone is short of causing the full spectrum of abnormalities. Mass ecdysone is required for the pathological transformation of ph mutant neurons in the Drosophila brain, raising several interesting possibilities about mutual involvement between the epigenetic function of PcG and the global nuclear signaling of steroid hormones (Wang, 2006).

Distinct wild-type cells respond differentially to ecdysone, but ph mutant neurons of distinct origins become no longer distinguishable after ecdysone signaling. Ecdysone mediates diverse biological activities partially via binding to different heterodimeric receptors. Its conventional receptors consist of the nuclear receptor superfamily members ecdysone receptor (EcR) and Ultraspiracle (USP; the Drosophila RXR). There are three documented EcR isoforms; and cells that express different EcR isoforms have been shown to undergo different changes in response to the prepupal ecdysone peak. For example, abundant EcR-B1 exists selectively in the neurons that remodel projections during early metamorphosis. Since no change was observed in EcR expression patterns in ph mutant neurons, it is unlikely that the aberrant responses of ph mutant neurons to the prepupal ecdysone peak occur as a result of derepression of specific EcR isoforms. In addition, derepression of multiple Hox genes appears not to be involved either. Nevertheless, given the involvement of Ph in silencing transcription, it remains possible that derepression of other unidentified genes directly re-programs ecdysone-induced transcriptional hierarchies, leading to transformation of ph mutant neurons. Alternatively, it is possible that loss of the epigenetic function of Ph may permit diffuse activation of prohibited genes by normal transcriptional hierarchies. Moreover, massive steroid hormone signaling might directly modify genomic imprinting when PcG functions are compromised (Wang, 2006).

Ecdysone-dependent transformation of ph mutant neurons provides a possible model system for characterizing the epigenetic functions of steroid hormones. In addition, the demonstration of the unusual potent epigenetic effects of ecdysone in ph mutant neurons suggests complex mechanisms may underlie pathogenesis of other documented PcG loss-of-function phenotypes (Wang, 2006).

Both derepression and inactivation of genes occur in transformed ph mutant neurons, characterization of which offers some molecular insights into this status of transformation. First, the fine-tuning of gene expression in transformed cells was no longer detected; and all the examined drivers appeared either fully on or completely off. Second, on or off could not be simply attributed to the genomic locations of drivers, as evidenced by constitutive silencing of the multiple independently inserted atonal-GAL4 transgenes. Third, transformed cells retained neuron-type morphologies and remained positive for the neuron-specific gene elav; and ph mutant neurons had been earlier reported to acquire normal-looking neurites in culture. Taken together, the transformation leads to loss of subtype identity without affecting basic neuronal fates, abolishes the genomic imprints governing fine controls over gene expression, and locks gene expression in 'on' or 'off' possibly in a promoter-autonomous manner (largely independent of its chromatin environment) (Wang, 2006).

Finally, loss of Ph, Pc, versus E(z) results in distinct phenotypes in the developing fly brain. Differences in their underlying pathological mechanisms are well exemplified by differential derepression of distinct Hox genes in different PcG clones. In addition, for a given PcG mutation, patterns of Hox gene derepression vary from neural clones to wing disc clones and visceral mesoderm. It remains to be elucidated how distinct PcG functions are governed in diverse cell type-characteristic manners (Wang, 2006).

Alkema, M. J., et al. (1997). Identification of Bmi1-interacting proteins as constituents of a multimeric mammalian Polycomb complex. Genes Dev. 11: 226-240

Beuchle, D., Struhl, G., and M¸ller, J. (2001). Polycomb group proteins and heritable silencing of Drosophila Hox genes. Development 128: 993-1004. 11222153

Biryukova, I., et al. (1999). The P-Ph protein-mediated repression of yellow expression depends on different cis- and trans-factors in Drosophila melanogaster . Genetics 152: 1641-1652.

Bloyer, S., et al. (2003). Identification and characterization of polyhomeotic PREs and TREs. Dev. Biol. 261: 426-442. 14499651

Boivin, A., et al. (2003). Telomeric associated sequences of Drosophila recruit Polycomb-group proteins in vivo and can induce pairing-sensitive repression. Genetics 164: 195-208. 12750332

Bornemann, D., Miller, E. and Simon J. (1998). Expression and properties of wild-type and mutant forms of the Drosophila Sex Comb on Midleg (SCM) repressor protein. Genetics 150: 675-686.

Breiling, A., et al. (2001). General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412: 651-655. 11493924

Brown, J. L., Fritsch, C., Mueller, J. and Kassis, J. A. (2003). The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing. Development 130: 285-294. 12466196

Buchenau, P., et al. (1998). The distribution of polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing. J. Cell Biol. 141(2): 469-481 98215719

Cheng, N.N., et al. (1994). Interactions of polyhomeotic with polycomb group genes of Drosophila melanogaster. Genetics 138: 1151-1162

Comet, I., et al. (2006). PRE-mediated bypass of Two Su(Hw) insulators targets PcG proteins to a downstream promoter. Dev. Cell 11: 117-124. 16824958

Deatrick, J. and Brock, H.W. (1991). The complex genetic locus of polyhomeotic in Drosophila melanogaster potentially encodes two homologous zinc fingers. Gene 105: 185-195

DeCamillis, M., Cheng, N. S., Pierre, D. and Brock, H. W. (1992). The polyhomeotic gene of Drosophila encodes a chromatin protein that shares polytene chromosome-binding sites with Polycomb. Genes Dev 6: 223-32

Dura, J-M., Brock, W. H. and Santamaria, P. (1985). Polyhomeotic: a gene of Drosophila melanogaster required for correct expression of segmental identity. Mol. Gen. Genet. 198: 213-20

Dura, J-M., et al. (1987). A complex genetic locus, polyhomeotic, is required for segmental specification and epidermal development in D. melanogaster. Cell 51: 829-839

Dura, J-M., et al. (1988). Maternal and zygotic requirements for the polyhomeotic complex genetic locus in Drosophila. Roux's Arch Dev Biol 197: 239-246

Faucheux, M., et al. (2003). batman interacts with Polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor. Mol. Cell. Biol. 23: 1181-1195. 12556479

Fauvarque, M.O., Zuber, V and Dura, J-M. (1995). Regulation of polyhomeotic transcription may involve local changes in chromatin activity in Drosophila. Mech Dev 52: 343-355

Ficz, G., Heintzmann, R. and Arndt-Jovin, D. J. (2005). Polycomb group protein complexes exchange rapidly in living Drosophila. Development 132(17): 3963-76. 16079157

Francis, N. J., et al. (2001) Reconstitution of a functional core Polycomb repressive complex. Mol. Cell 8: 545-556. 11583617

Franke, A., et al. (1992). Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster. EMBO J 11: 2941-50

Franke, A. Messmer, S. and Paro, R. (1995). Mapping functional domains of the polycomb protein of Drosophila melanogaster. Chromosome Res 3: 351-360

Gunster, M. J., et al. (1997). Identification and characterization of interactions between the vertebrate polycomb-group protein BMI1 and human homologs of polyhomeotic. Mol. Cell. Biol. 17: 2326-35

Hasegawa, M., et al. (1998). Mammalian Polycomb group genes are categorized as a new type of early response gene induced by B-cell receptor cross-linking. Mol. Immunol. 35(9): 559-63.

Hodgson, J. W., et al. (1997). The polyhomeotic locus of Drosophila melanogaster is transcriptionally and post-transcriptionally regulated during embryogenesis. Mech. Dev. 66(1-2): 69-81.

Hodgson, J. W., Argiropoulos, B. and Brock, H. W. (2001). Site-specific recognition of a 70-base-pair element containing d(GA)n repeats mediates bithoraxoid Polycomb group response element-dependent silencing. Mol. Cell. Biol. 21: 4528-4543. 11416132

Isono, K., Fujimura, Y., Shinga, J., Yamaki, M., O-Wang, J., Takihara, Y., Murahashi, Y., Takada, Y., Mizutani-Koseki, Y. and Koseki, H. (2005). Mammalian polyhomeotic homologues Phc2 and Phc1 act in synergy to mediate Polycomb-repression of Hox genes. Mol. Cell. Biol. 25: 6694-6706. Medline abstract: 16024804

Kim, C. A., Sawaya, M. R., Cascio, D., Kim, W. and Bowie, J. U. (2005). Structural organization of a sex-comb-on-midleg/polyhomeotic copolymer. J. Biol. Chem. 280(30): 27769-75. 15905166

King, I. F., Francis, N. J., and Kingston, R. E. (2002). Native and recombinant polycomb group complexes establish a selective block to template accessibility to repress transcription in vitro. Mol. Cell. Biol. 22: 7919-7928. 12391159

Korenjak, M., Taylor-Harding, B., Binne, U.K., Satterlee, J.S., Stevaux, O., Aasland, R., White-Cooper, H., Dyson, N., and Brehm, A. (2004). Native E2F/RBF complexes contain Myb-interacting proteins and repress transcription of developmentally controlled E2F target genes. Cell 119: 181-193. 15479636

Kyba, M. and Brock, H. W. (1998a). The SAM domain of polyhomeotic, RAE28, and scm mediates specific interactions through conserved residues. Dev. Genet. 22(1): 74-84.

Kyba, M. and Brock, H. W. (1998b). The Drosophila polycomb group protein Psc contacts ph and Pc through specific conserved domains. Mol. Cell. Biol. (5): 2712-2720.

Lonie, A., D'Andrea, R. Paro, R. and Saint, R. (1994). Molecular characterization of the Polycomb-like gene of Drosophila melanogaster, a trans-acting negative regulator of homeotic gene expression. Development 120: 2629-36

Lupo, R., et al. (2001). Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalize with Polycomb and Maintain Fab-7 PRE silencing. Mol. Cell 7: 127-136. 11172718

Martinez, A. M., Colomb, S., Dejardin, J., Bantignies, F. and Cavalli G. (2006). Polycomb group-dependent Cyclin A repression in Drosophila. Genes Dev. 20(4): 501-13. 16481477

Maschat, F., et al. (1998). engrailed and polyhomeotic interactions are required to maintain the A/P boundary of the Drosophila developing wing. Development 125: 2771-2780.

Maurange, C. and Paro, R. (2002). A cellular memory module conveys epigenetic inheritance of hedgehog expression during Drosophila wing imaginal disc development. Genes Dev. 20: 2672-2683. 12381666

McKeon, J. and Brock, H. (1991). Interaction of the Polycomb group of genes with homeotic loci of Drosophila. Roux's Arch Dev Biol 199: 387-396

McKeon, J., et al. (1994). Mutations in some Polycomb group genes of Drosophila interfere with regulation of segmentation genes. Mol Gen Genet 244: 474-483

Mishra, K., et al. (2003). Trl-GAGA directly interacts with lola like and both are part of the repressive complex of Polycomb group of genes. Mech. Dev. 120: 681-689. 12834867

Montini, E., et al. (1999). Identification of SCML2, a second human gene homologous to the Drosophila sex comb on midleg (Scm): A new gene cluster on Xp22. Genomics 58(1): 65-72.

Muller, H., et al. (2001). E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev. 15: 267-285. 11159908

Narbonne, K., et al. (2004). polyhomeotic is required for somatic cell proliferation and differentiation during ovarian follicle formation in Drosophila. Development 131: 1389-1400. 14993188

Nomura, M., Takihara, Y. and Shimada, K. (1994). Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells: one of the early inducible clones encodes a novel protein sharing several highly homologous regions with a Drosophila polyhomeotic protein. Differentiation 57: 39-50

Nomura, M., et al. (1998). Sequence-specific DNA binding activity in the RAE28 protein, a mouse homologue of the Drosophila polyhomeotic protein. Biochem. Mol. Biol. Int. 46(5): 905-12.

O'Dor, E., Beck, S. A. and Brock, H. W. (2006). Polycomb group mutants exhibit mitotic defects in syncytial cell cycles of Drosophila embryos. Dev. Biol. 290(2): 312-22. 16388795

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (1997). Cosuppression in Drosophila: gene silencing of Alcohol dehydrogenase by white-Adh transgenes is Polycomb dependent. Cell 90(3): 479-490.

Peterson, A. J., et al. (1997). A domain shared by the Polycomb group proteins Scm and Ph mediates heterotypic and homotypic interactions. Mol. Cell. Biol. 17(11): 6683-6692.

Ponting, C. P. (1995). SAM: a novel motif in yeast sterile and Drosophila polyhomeotic proteins. Protein Sci. 4: 1928-1930

Poux, S., McCabe, D. and Pirrotta, V. (2001). Recruitment of components of Polycomb Group chromatin complexes in Drosophila. Development 128: 75-85

Randsholt, N. B., Maschat, F. and Santamaria, P. (2000). polyhomeotic controls engrailed expression and the hedgehog signaling pathway in imaginal discs. Mech. Dev. 95(1-2): 89-99. 10906453

Roseman, R. R., et al. (2001). Long-range repression by multiple Polycomb Group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila. Genetics. 158: 291-307. 11333237

Rusch, D. B. and Kaufman, T. C. (2000). Regulation of proboscipedia in Drosophila by homeotic selector genes. Genetics 156: 183-194.

Satijn, D. P., et al. (1997). RING1 is associated with the polycomb group protein complex and acts as a transcriptional repressor. Mol. Cell. Biol. 17(7): 4105-4113.

Saurin. A. J., et al. (2001). A Drosophila Polycomb group complex includes Zeste and dTAFII proteins. Nature 412: 655-660. 11493925

Serrano, N., et al. (1995). polyhomeotic appears to be a target of engrailed regulation in Drosophila. Development 121: 1691-1703

Serrano, N. and Maschat, F. (1998). Molecular mechanism of polyhomeotic activation by Engrailed. EMBO J. 17(13): 3704-3713.

Shao, Z., et al. (1999). Stabilization of chromatin structure by PRC1, a polycomb complex. Cell 98: 37-46.

Strutt, H. and Paro, R. (1997). The polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes. Mol. Cell. Biol. 17(12): 6773-6783.

Takada, Y., et al. (2007). Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes. Development 134(3): 579-90. Medline abstract: 17215307

Takihara, Y., et al. (1997). Targeted disruption of the mouse homologue of the Drosophila polyhomeotic gene leads to altered anteroposterior patterning and neural crest defects. Development 124: 3673-3682.

Trimarchi, J. M., et al. (2001). The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex. Proc. Natl. Acad. Sci. 98: 1519-1524. 11171983

van de Vosse, E., et al. (1998). Characterization of SCML1, a new gene in Xp22, with homology to developmental polycomb genes. Genomics 49(1): 96-102.

Wang, Y.-J., et al. (2003). Polyhomeotic stably associates with molecular chaperones Hsc4 and Droj2 in Drosophila Kc1 cells. Dev. Biol. 262: 350-360. 14550797

Wang, J., Lee, C. H., Lin, S. and Lee, T. (2006). Steroid hormone-dependent transformation of polyhomeotic mutant neurons in the Drosophila brain. Development 133(7): 1231-40. 16495309

Yoshitake, Y., et al. (1999). Misexpression of Polycomb-group proteins in Xenopus alters anterior neural development and represses neural target genes. Dev. Biol. 215(2): 375-387

date revised: 25 July 2007


 

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.