Posterior sexcombs and Suppressor two of zeste

DEVELOPMENTAL BIOLOGY

Embryonic

PSC mRNA accumulates in developing oocytes. This maternal RNA is presumably responsible for the maternal rescue. PSC protein is expressed ubiquitously. Levels are highest in the brain and ventral ganglia. PSCmRNA is found in pole cell nuclei from the time the nuclei bud off from the posterior pole through their migration to the gonad.

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).

PSC has been found on larval polytene chromosomes at 45 chromosomal loci where two other Pc-G proteins are also present. Major binding sites include dorsal/bicaudal-D, Psc, ANTP-C, and Bx-C. Minor sites include polyhomeotic, odd-paired, Krüppel/Gooseberry and Pox-meso (Martin, 1993).

Effects of Mutation or Deletion

Flies mutant in both maternal and zygotic Psc have severe defects including head involution problems, missing denticle bands, segmental transformation and twisting of embryos. Suppressor of zeste 2 maternal/zygotic mutants show no obvious segmental transformations. Less than half the animals have head abnormalities. Zygotic deletion of both genes shows strong mis-expression of Abd-B. ABD-A and UBX protein patterns are also mis-expressed, with ectopic expression of ABD-A ahead of the wild-type anterior boundary (Soto, 1995).

A distal breakpoint divides the Posterior sex combs-Suppressor 2 of zeste region into two parts. Posterior sex combs is proximal to this breakpoint. Suppressor 2 of zeste gene lies distal to this breakpoint. Since the breakpoint does not cause a loss of function in either gene, no essential sequences are shared by these two neighboring genes. There are three dominant gain of function mutations in this region that result in abnormal bristle development and increased accumulation of the Su(z)2 mRNA (Brunk 1991a).

Three dominant second-chromosome rearrangement mutations result in developmental abnormalities of the bristle sense organs on the notum, abdomen, legs, and wing margin. Similar bristle abnormalities are associated with overexpression of Suppressor 2 of zeste and Posterior sex combs . The bristle abnormalities are reminiscent of those seen with reduced function of Notch, a neurogenic gene. Su(z)2 overexpression, mimicing the second-chromosome rearrangements, reduces the expression of the neurogenic gene neuralized. Previous experiments found that loss of function mutations in Su(z)2 resulted in no bristle abnormalities. There is no essential function of Psc in bristle development. Thus the bristle abnormalities may result from altered expression of genes involved in bristle sense organ development, such as Notch, that are not normal regulatory targets of these genes (Sharp, 1994).

Although genetic interactions indicate that Psc functions as a Pc-G gene, the zygotic mutant phenotype of Psc shows little evidence of segmental transformations. Mutant embryos derived from a mutant maternal germ line give a stronger mutant phenotype, indicating that the weak zygotic phenotype of Psc is due to maternal rescue (Martin, 1993).

The genes of the Polycomb group repress the genes of the bithorax and Antennapedia complexes, among others. To observe a null phenotype for a Pc-G gene, one must remove its maternal as well as zygotic contribution to the embryo. Five members of the Pc-G group were compared: Enhancer of Polycomb [E(Pc)], Additional sex combs (Asx), Posterior sex combs (Psc), Suppressor of zeste 2 [Su (z) 2] and Polycomblike (Pcl). E(Pc) and Su(z) 2 mutations have only subtle effects on the target genes, even when the maternal contributions are removed. Asx and Pcl mutants show derepression of the targets only in specific cell types. Psc shows unusual effects on two of the targets, Ultrabithorax and abdominal-A. Thus the PC-G genes do not act only in a common complex or pathway; they must have some independent functions (Soto, 1995).

The zeste1 (z1) mutation of the zeste gene produces a mutant yellow eye color instead of the wild-type red. Genetic and molecular data suggest that z1 achieves this change by altering expression of the wild-type white gene in a manner that exhibits transvection effects. The alleles of Psc define two overlapping phenotypic classes, the hopeful and the hapless. In the former class, flies can survive as homozygotic mutants with defects, while the latter class is lethal. The distinctions between these two classes and the intragenic complementation seen among some of the Psc alleles are consistent with a multidomain structure for the product of Psc (Wu, 1995).

Drosophila Mi-2 protein binds to a domain in the gap protein Hunchback which is specifically required for the repression of HOX genes. dMi-2 protein was tested to see if it participates in PcG repression. As in the case of dMi-2, maternally deposited PcG product often rescues homozygous mutant PcG embryos to a considerable extent. Extensive derepression of HOX genes can be observed if such homozygous embryos are also mutant for another PcG gene. Thus embryos homozygous for the PcG gene Posterior sex combs (Psc) and dMi-2 were examined and it was found that Ubx and Abd-B are derepressed more extensively in this double mutant than in Psc homozygotes alone. A similar result was found if dMi-2 is combined with other PcG mutations; these double mutants consistently lead to much enhanced homeotic transformations compared with the single PcG mutants. Thus, there is a synergy between dMi-2 and PcG genes. dMi-2 behaves like the PcG mutations Enhancer of Polycomb and Suppressor 2 of zeste, neither of which on their own cause a homeotic phenotype but do so in combination with other PcG mutations. This suggests that dMi-2 functions in PcG repression (Kehle, 1998).

Imaginal discs were examined for derepression of HOX genes as well as the phenotypes of their adult derivatives. Clonal analysis suggests that dMi-2 is required for the survival of somatic cells. Do dMi-2 mutations exhibit gene-dosage interactions with PcG mutations? While larvae heterozygous for Polycomb (Pc) mutations show slight derepression of Ubx, larvae transheterozygous for both Pc and dMi-2 mutations show more extensive derepression. Furthermore, derepression of the HOX gene Sex combs reduced (Scr) in the second and third leg discs of Pc heterozygotes results in the formation of a first leg structure, the sex comb, on the second and third legs. The extent of this homeotic transformation reflects the number of cells that misexpress Scr protein. This homeotic transformation is far stronger in dMi-2/Pc transheterozygotes than in adults heterozygous for Pc alone, which is consistent with more extensive derepression of Scr in the double mutant. These results are further evidence that dMi-2 acts together with PcG proteins to repress HOX genes (Kehle, 1998).

It has been proposed that Hb directly or indirectly recruits PcG proteins to DNA to establish PcG silencing of homeotic genes. The present data suggest that dMi-2 might function as a link between Hb and PcG repressors. Although dMi-2 contains two motifs with similarity to DNA-binding domains (the myb and HMG domains), dMi-2 does not seem to bind to DNA on its own. Therefore, Hb may recruit dMi-2 to DNA. Xenopus Mi-2 was recently purified as a subunit of a histone deacetylase complex with nucleosome remodeling activity. In yeast and in vertebrates, several transcription factors repress transcription by recruiting histone deacetylases. It is possible that in Drosophila, nucleosome remodeling and deacetylase activities of a dMi-2 complex, recruited to homeotic genes by Hb, may result in local chromatin changes that allow binding of PcG proteins to the nucleosomal template. Alternatively, the proposed Hb-dMi-2 complex might directly bind a PcG protein and recruit it to DNA. Finally, the involvement of dMi-2 in PcG silencing suggests that this process may involve deacetylation of histones (Kehle, 1998 and references).

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allows for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV (Cryderman, 1999).

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. The expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing were examined by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate the described methods for expression profiling as a way to analyze developmental programs, and they show that modifications to chromatin structure are key to changes in cell fate. These findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells (Klebes, 2005).

When prothoracic (1st) leg discs are fragmented and cultivated in vivo, cells in a proximodorsal region known as the 'weak point' can switch fate and transdetermine. These 'weak point' cells give rise to cuticular wing structures. The leg-to-wing switch is regulated, in part, by the expression of the vestigial (vg) gene, which encodes a transcriptional activator that is a key regulator of wing development. vg is not expressed during normal leg development, but it is expressed during normal wing development and in 'weak point' cells that transdetermine from leg to wing. Activation of vg gene expression marks leg-to-wing transdetermination (Klebes, 2005).

Sustained proliferation appears to be a prerequisite for fate change, and conditions that stimulate growth increase the frequency and enlarge the area of transdetermined tissue. Transdetermination was discovered when fragments of discs were allowed to grow for an extensive period of in vivo culture. More recently, ways to express Wg ectopically have been used to stimulate cell division and cell cycle changes in 'weak point' cells (Sustar, 2005), and have been shown to induce transdetermination very efficiently. Experiments were performed to characterize the genes involved in or responsible for transdetermination that is induced by ectopic Wg. Focus was placed on leg-to-wing transdetermination because it is well characterized, it can be efficiently induced and it can be monitored by the expression of a real-time GFP reporter. These attributes make it possible to isolate transdetermining cells as a group distinct from dorsal leg cells, which regenerate, and ventral leg cells in the same disc, which do not regenerate; and, in this work, to directly define their expression profiles. This analysis identified unique expression properties for each of these cell populations. It also identified a number of genes whose change in expression levels may be significant to understanding transdetermination and the factors that influence developmental plasticity. One is lamina ancestor (lama), whose expression correlates with undifferentiated cells and is shown to control the area of transdetermination. Another has sequence similarity to the mammalian augmenter of liver regeneration (Alr; Gfer -- Mouse Genome Informatics), which controls regenerative capacity in the liver and is upregulated in mammalian stem cells. Fifteen regulators of chromatin structure [e.g. members of the Polycomb group (PcG) and trithorax group (trxG)] are differentially regulated in transdetermining cells, and mutants in seven of these genes have significant effects on transdetermination. These studies identify two types of functions that transdetermination requires -- functions that promote an undifferentiated cell state and functions that re-set chromatin structure (Klebes, 2005).

The importance of chromatin structure to the transcriptional state of determined cells makes it reasonable to assume that re-programming cells to different fates entails reorganization of the Polycomb group (PcG) and trithorax group (trxG) protein complexes that bind to regulatory elements. Although altering the distribution of proteins that mediate chromatin states for transcriptional repression and activation need not involve changes in the levels of expression of the PcG and trxG proteins, the array hybridization data was examined to determine if they do. The PcG Suppressor of zeste 2 [Su(z)2] gene had a median fold repression of 2.1 in eight TD to D_Wg/V_Wg comparisons, but the cut-off settings did not detect significant enrichment or repression of most of the other PcG or trxG protein genes with either clustering analysis or the method of ranking median ratios. Since criteria for assigning biological significance to levels of change are purely subjective, the transdetermination expression data was re-analyzed to identify genes whose median ratio changes within a 95% confidence level. Fourteen percent of the genes satisfied these conditions. Among these genes, 15/32 PcG and trxG genes (47%) had such statistically significant changes. Identification of these 15 genes with differential expression suggests that transdetermination may be correlated with large-scale remodeling of chromatin structure (Klebes, 2005).

To test if the small but statistically significant changes in the expression of PcG and trxG genes are indicative of a functional role in determination, discs from wild-type, Polycomb (Pc), Enhancer of Polycomb [E(Pc)], Sex comb on midleg (Scm), Enhancer of zeste [E(z)], Su(z)2, brahma (brm) and osa (osa) larvae were examined. The level of Wg induction was adjested to reduce the frequency of transdetermination and both frequency of transdetermination and area of transdetermined cells was determined. The frequency of leg discs expressing vg increased significantly in E(z), Pc, E(Pc), brm and osa mutants, and the frequency of leg to wing transdetermination in adult cuticle increased in Scm, E(z), Pc, E(Pc) and osa mutants. Remarkably, Su(z)2 heterozygous discs had no vg expression, suggesting that the loss of Su(z)2 function limits vg expression (Klebes, 2005).

Members of the PcG and trxG are known to act as heteromeric complexes by binding to cellular memory modules (CMMs). The functional tests demonstrate that mutant alleles for members of both groups have the same functional consequence (they increase transdetermination frequency). The findings are consistent with recent observations that the traditional view of PcG members as repressors and trxG factors as activators might be an oversimplification, and that a more complex interplay of a varying composition of PcG and trxG proteins takes place at individual CMMs. Furthermore the opposing effects of Pc and Su(z)2 functions are consistent with the proposal that Su(z)2 is one of a subset of PcG genes that is required to activate as well as to suppress gene expression. In addition to measuring the frequency of transdetermination, the relative area of vg expression was examined in the various PcG and trxG heterozyogous mutant discs. The relative area decreased in E(Pc), brm and osa mutant discs, despite the increased frequency of transdetermination in these mutants. There is no evidence to explain these contrasting effects, but the roles in transdetermination of seven PcG and trxG genes that were identified by these results support the proposition that the transcriptional state of determined cells is implemented through the controls imposed by the regulators of chromatin structure (Klebes, 2005).

The determined states that direct cells to particular fates or lineages can be remarkably stable and can persist after many cell divisions in alien environments, but they are not immune to change. In Drosophila, three experimental systems have provided opportunities to investigate the mechanisms that lead to switches of determined states. These are: (1) the classic homeotic mutants; (2) the PcG and trxG mutants that affect the capacity of cells to maintain homeotic gene expression, and (3) transdetermination. During normal development, the homeotic genes are expressed in spatially restricted regions, and cells that lose (or gain) homeotic gene function presumably change the transcriptional profiles characteristic of the particular body part. In the work reported here, techniques of micro-dissection, RNA amplification and array hybridization were used to monitor the transcription profiles of cells in normal leg and wing imaginal discs, in leg disc cells that regenerate and in cells that transdetermine from leg to wing. The results validate the idea that changing determined states involves global changes in gene expression. They also identify genes whose function may be unrelated to the specific fates of the cells characterized, but instead may correlate with developmental plasticity (Klebes, 2005).

Overlap between the transcriptional profiles in the wing and transdetermination lists (15 genes) and with genes in subcluster IV (high expression in wing discs) is extensive. The overlap is sufficient to indicate that the TD leg disc cells have changed to a wing-like program of development, but interestingly, not all wing-specific genes are activated in the TD cells. The reasons could be related to the incomplete inventory of wing structures produced (only ventral wing) or to the altered state of the TD cells. During normal development, vg expression is activated in the embryo and continues through the 3rd instar. Although the regulatory sequences responsible for activation in the embryo have not been identified, in 2nd instar wing discs, vg expression is dependent upon the vgBE enhancer, and in 3rd instar wing discs expression is dependent upon the vgQE enhancer. Expression of vg in TD cells depends on activation by the vgBE enhancer, indicating that cells that respond to Wg-induction do not revert to an embryonic state. Recent studies of the cell cycle characteristics of TD cells support this conclusion (Sustar, 2005), but the role of the vgBE enhancer in TD cells and the incomplete inventory of 'wing-specific genes' in their expression profile probably indicates as well the stage at which the TD cells were analyzed: they were not equivalent to the cells of late 3rd instar wing discs (Klebes, 2005).

Investigations into the molecular basis of transdetermination have led to a model in which inputs from the Wg, Dpp and Hh signaling pathways alter the chromatin state of key selector genes to activate the transdetermination pathway. The analyses were limited to a period 2-3 days after the cells switched fate, because several cell doublings were necessary to produce sufficient numbers of marked TD cells. As a consequence, these studies did not analyze the initial stages. Despite this technical limitation, this study identified several genes that are interesting novel markers of transdetermination (e.g., ap, CG12534, CG14059 and CG4914), as well as several genes that function in the transdetermination process (e.g., lama and the PcG genes). The results from transcriptional profiling add significant detail to a general model proposed for transdetermination (Klebes, 2005).

(1) It is reported that ectopic wg expression results in statistically significant changes in the expression of 15 PcG and trxG genes. Moreover, although the magnitudes of these changes were very small for most of these genes, functional assays with seven of these genes revealed remarkably large effects on the metrics used to monitor transdetermination -- the fraction of discs with TD cells, the proportion of disc epithelium that TD cells represent, and the fraction of adult legs with wing cuticle. These effects strongly implicate PcG and trxG genes in the process of transdetermination and suggest that the changes in determined states manifested by transdetermination are either driven by or are enabled by changes in chromatin structure. This conclusion is consistent with the demonstrated roles of PcG and trxG genes in the self-renewing capacity of mouse hematopoietic stem cells, in Wg signaling and in the maintenance of determined states. The results now show that the PcG and trxG functions are also crucial to pluripotency in imaginal disc cells, namely that pluripotency by 'weak point' cells is dependent upon precisely regulated levels of PcG and trxG proteins, and is exquisitely sensitive to reductions in gene dose (Klebes, 2005).

The data do not suggest how the PcG and trxG genes affect transdetermination, but several possible mechanisms deserve consideration. A recent study (Sustar, 2005) reported that transdetermination correlates with an extension of the S phase of the cell cycle. Several proteins involved in cell cycle regulation physically associate with PcG and trxG proteins, and Brahma, one of the proteins that affects the metrics of transdetermination, has been shown to dissociate from chromatin in late S-phase and to reassociate in G1. It is possible that changes in the S-phase of TD cells are a consequence of changes in PcG/trxG protein composition (Klebes, 2005).

Another generic explanation is that transdetermination is dependent or sensitive to expression of specific targets of PcG and trxG genes. Among the 167 Pc/Trx response elements (PRE/TREs) predicted to exist in the Drosophila genome, one is in direct proximity to the vg gene. It is possible that upregulation of vg in TD cells is mediated through this element. Another factor may be the contribution of targets of Wg signaling, since targets of Wg signaling have been shown to be upregulated in osa and brm mutants. These are among a number of likely possible targets, and identifying the sites at which the PcG and trxG proteins function will be necessary if an understand is to be gained of how transdetermination is regulated. Importantly, understanding the roles of such targets and establishing whether these roles are direct will be essential to rationalize how expression levels of individual PcG and trxG genes correlate with the effects of PcG and trxG mutants on transdetermination (Klebes, 2005).

(2) The requirement for lama suggests that proliferation of TD cells involves functions that suppress differentiation. lama expression has been correlated with neural and glial progenitors prior to, but not after, differentiation, and it is observed that lama is expressed in imaginal progenitor cells and in early but not late 3rd instar discs. lama expression is re-activated in leg cells that transdetermine. The upregulation of unpaired in TD cells may be relevant in this context, since the JAK/STAT pathway functions to suppress differentiation and to promote self-renewal of stem cells in the Drosophila testis. It is suggested that it has a similar role in TD cells (Klebes, 2005).

(3) A role for Notch is implied by the expression profiles of several Notch pathway genes. Notch may contribute directly to transdetermination through the activation of the vgBE enhancer [which has a binding site for Su(H)] and of similarly configured sequences that were found to be present in the regulatory regions of 45 other TD genes. It will be important to test whether Notch signaling is required to activate these co-expressed genes, and if it is, to learn what cell-cell interactions and 'community effects' regulate activation of the Notch pathway in TD cells (Klebes, 2005).

(4) The upregulation in TD cells of many genes involved in growth and division, and the identification of DNA replication element (DRE) sites in the regulatory region of many of these genes supports the observation that TD cells become re-programmed after passing through a novel proliferative state (Sustar, 2005), and suggests that this change is in part implemented through DRE-dependent regulation (Klebes, 2005).

There was an interesting correlation between transdetermination induced by Wg mis-expression and the role of Wg/Wnt signaling for stem cells. Wg/Wnt signaling functions as a mitogen and maintains both somatic and germline stem cells in the Drosophila ovary, and mammalian hematopoetic stem cells. Although the 'weak point' cells in the Drosophila leg disc might lack the self-renewing capacity that characterizes stem cells, they respond to Wg mis-expression by manifesting a latent potential for growth and transdetermination. It seems likely that many of the genes are conserved that are involved in regulating stem cells and that lead to disease states when relevant regulatory networks lose their effectiveness (Klebes, 2005).

The prevalence of transcription factors among the genes whose relative expression levels differed most in the tissue comparisons was intriguing. It is commonly assumed that transcription factors function catalytically and that they greatly amplify the production of their targets, so the expectation was that the targets of tissue-specific transcription factors would have the highest degree of tissue-specific expression. In these studies, tissue-specific expression of 15 transcription factors among the 40 top-ranking genes in the wing and leg data sets (38%) is consistent with the large number of differentially expressed genes in these tissues, but these rankings suggest that the targets of these transcription factors are expressed at lower relative levels than the transcription factors that regulate their expression. One possible explanation is that the targets are expressed in both wing and leg disc cells, but the transcription factors that regulate them are not. This would imply that the importance of position-specific regulation lies with the regulator, not the level of expression of the target. Another possibility is that these transcription factors do not act catalytically to amplify the levels of their targets, or do so very inefficiently and require a high concentration of transcription factor to regulate the production of a small number of transcripts. Further analysis will be required to distinguish between these or other explanations, but it is noted that the prevalence of transcription factors in such data sets is neither unique to wing-leg comparisons nor universal (Klebes, 2005).

Polycomb group genes Psc and Su(z)2 restrict follicle stem cell self-renewal and extrusion by controlling canonical and noncanonical Wnt signaling

Stem cells are critical for maintaining tissue homeostasis and are commonly governed by their niche microenvironment, although the intrinsic mechanisms controlling their multipotency are poorly understood. Polycomb group (PcG) genes are epigenetic silencers, and have emerged as important players in maintaining stem cell multipotency by preventing the initiation of differentiation programs. This paper describes an unexpected role of specific PcG genes in allowing adult stem cell differentiation and preventing stem cell-derived tumor development. Posterior sex combs (Psc), which encodes a core Polycomb-repressive complex 1 (PRC1) component, functions redundantly with a similar gene, Suppressor of zeste two [Su(z)2], to restrict follicle stem cell (FSC) self-renewal in the Drosophila ovary. FSCs carrying deletion mutations of both genes extrude basally from the epithelium and continue to self-propagate at ectopic sites, leading to the development of FSC-like tumors. Furthermore, it was shown that the propagation of the mutant cells is driven by sustained activation of the canonical Wnt signaling pathway, which is essential for FSC self-renewal, whereas the epithelial extrusion is mediated through the planar cell polarity pathway. This study reveals a novel mechanism of epithelial extrusion, and indicates a novel role of polycomb function in allowing adult stem cell differentiation by antagonizing self-renewal programs. Given evolutionary conservation of PcG genes from Drosophila to mammals, they could have similar functions in mammalian stem cells and cancer (Li, 2010).

In murine and human ES cells, PcG complexes bind to many genes that are involved in differentiation, indicating that they could be essential for maintaining ES cell pluripotency by preventing differentiation. Surprisingly, ES cells lacking PRC2 components such as Eed or SuZ12 can be maintained stably, although they simultaneously express key pluripotency factors as well as differentiation genes. However, when SuZ12 mutant ES cells are induced to differentiate, key pluripotency genes cannot be turned off, resulting in defective differentiation. These observations indicate that PcG genes could serve as both positive and negative modulators of stem cell self-renewal. This study found that specific PcG genes play critical roles in allowing adult stem cells to differentiate. Deletion mutations of Psc and Su(z)2 render FSCs incapable of further differentiation. Instead, they self-propagate continuously and develop into tumors. Thus, contrary to the proposed differentiation-preventing activity of PcG genes in stem cells, Psc and Su(z)2 have unexpected essential role for FSC differentiation in the Drosophila ovary. Cellular and genetic analyses further demonstrate that derepression of Wg self-renewal signaling drives the stem cell-like tumor development in Psc Su(z)2-deficient FSCs, as inhibiting Wg signaling activity efficiently prevents tumorigenesis, suggesting that specific PcG genes are required for stem cell differentiation by inhibiting self-renewal programs. A critical role for Wnt signaling has been implicated in multiple types of stem cells and cancer, and the latter one might be due to its ability to promote self-renewal of cancer stem cells. Previous genome-wide mapping of Polycomb targets in Drosophila demonstrates that PcG genes not only target genes that are important for cellular differentiation, but also target genes known as self-renewal signals for many types of adult stem cells, including hh and wg. Therefore, it is possible that PcG proteins could target different sets of genes in different tissues or at different developmental stages, and, within a specific type of stem cell, they could control both directions of stem cell fate by regulating both self-renewal and differentiation programs (Li, 2010).

An appealing explanation for the seemingly opposite stem cell functions of PcG genes is that different polycomb components or complexes may target different sets of genes that regulate either self-renewal or differentiation. Consistently, the differentiation-promoting activity of Psc and Su(z)2 in FSCs is independent of canonical PRC1 complex function, since mutations in other core PRC1 components, including Pc and Sce, do not lead to the same phenotype. It was further demonstrated that the difference is due, at least in part, to the differential regulation of wg repression in the follicle cell lineage. Mutation in ph, which encodes another PRC component, causes underproliferation of follicle cells, yet produces a similar, but weak, follicle cell morphology phenotyp, indicating that Ph might partner with Psc or Su(z)2 in regulating FSC differentiation and extrusion. Notably, although Psc is an essential component for PRC1 complex function, Su(z)2 has not been demonstrated to be associated with the PRC1 complex. Thus, it is also possible that Su(z)2 and Psc may form complexes with other unknown proteins for this novel function in FSCs. It is proposed that PcG genes may be central players in orchestrating both self-renewal and differentiation of stem cells, two opposite functions that could be achieved by different PcG protein components and/or complexes. To do so, they might function to modulate both self-renewal and differentiation programs and to maintain specific chromatin states in order to facilitate either self-renewal or differentiation. Differential requirements for epigenetic regulators in different types of stem cells have been demonstrated previously in the Drosophila ovary. This study shows that Psc and Su(z)2 are also specific for FSC maintenance, and are dispensable in GSCs. These observations further suggest that different epigenetic regulators may be used to maintain specific self-renewal programs and chromatin states in various tissue-specific stem cells (Li, 2010).

In general, epithelial stem cell maintenance defects can be explained by two possible mechanisms: Cells could be eliminated by cell death, or they could undergo differentiation with or without transit amplification to form epithelium. The latter case is best seen in niche signaling pathway (Hh, Wg, or BMP)-compromised FSCs in the Drosophila ovary, in which the mutant FSCs move away from the niche and differentiate. The maintenance defects of Psc Su(z)2-deficient FSCs, however, cannot be explained by either mechanism. Instead, the mutant cells show a series of morphological changes that have not been observed previously, including apical membrane retraction and basal extrusion from the epithelium. Thus, this study reveals cell extrusion as a novel process of FSC loss from their normal location. It was also demonstrated that the extrusion is mediated through the PCP pathway, which has not been implicated previously in the epithelial extrusion process. It is not clear how PCP controls epithelial extrusion, but because it has also been implicated in regulating directed cell movement, a similar molecular machinery might be used in the epithelial extrusion process observed in this study. The PCP components include core Fz and Dsh as well as Fat-Dachsous (FT-DS) PCP factors, although whether those two pathways function linearly or in parallel is still not clear. Notably, it was found that both Fz and Dsh and FT-DS PCP factors are required for tumor cell extrusion, indicating that the two pathways function nonredundantly in this process. Because tumor cells do not have apicobasal polarity, an intriguing question arises as to whether loss of apicobasal polarity could lead to basal extrusion. Disrupting the apicobasal polarity of FSCs, however, although disrupting the epithelial organization, does not cause cell extrusion. Instead, the mutant follicle cell clones usually invade apically into germline cysts, suggesting that the extrusion phenotype cannot be reproduced by apicobasal polarity mutants. A similar epithelial cell extrusion process has been reported in Drosophila wing imaginal discs, where BMP pathway-compromised epithelial cells extrude from the monolayer epithelium. BMP pathway cascade is also required for the maintenance of FSCs in the Drosophila ovary, but the mutant FSCs and their daughters do not display the extrusion phenotype. In addition, BMP pathway-compromised imaginal disc cells, although extruded from the epithelium, still maintain apicobasal polarity, further suggesting that the process of epithelial extrusion of polycomb mutant FSCs occurs via a distinct mechanism. Because the mutant FSCs are able to initiate tumor formation, cell extrusion may be a novel mechanism for tumor cells to leave their original tissue context and migrate to ectopic sites. It is proposed that similar mechanisms could be used in mammalian cancer cells to promote their migration (Li, 2010).

This study has shown that, although Psc Su(z)2-deficient FSCs initiated tumor formation, Psc Su(z)2-deficient differentiating follicle cells outside the germarium could not initiate tumorigenesis, demonstrating that, in this case, the stem cells and possibly early progenitors were more prone to initiate tumorigenesis than the downstream differentiating cells upon oncogenic mutations. Increasing evidence supports the cancer stem cell theory, at least for certain types of cancers. But it is not clear what the origin of these cancer stem cells is. They could be derived from differentiated cells or stem cells. Although somewhat conflicting, studies of murine hematopoiesis showed that stem cells and multipotent progenitors harboring oncogenic mutations are generally more efficient in initiating leukemia. The stem cell origin of tumors is also supported by a recent study showing that APC mutation in intestinal stem cells, not differentiating progenitors, initiates tumorigenesis. The establishment of this Drosophila model of stem cell-derived tumor formation may help to further understand the underlying mechanisms governing a cell's tumorigenic potential (Li, 2010).

Potential roles of PcG genes in tumorigenesis have been suggested in mammals and humans. PcG proteins such as EZH2 and Su(z)12 are frequently up-regulated in several types of human cancers. In addition, the mammalian bmi-1 gene, which is homologous to Psc and Su(z)2, is considered to be a proto-oncogene because up-regulated bmi-1 functions synergistically with c-myc to cause B or T lymphomas. Furthermore, loss of bmi-1 function causes cell quiescence of both hematopoietic and neural stem cells, partially caused by the derepression of p16^ink4 and p19^ARF cell senescence genes. Contrary to the oncogenic function of bmi-1, Psc and Su(z)2 have tumor-suppressive activity in the Drosophila ovary. It was also observed previously that Psc and Su(z)2-deficient epithelial cells in imaginal discs develop tumorous growths, which could be a consequence of cellular overgrowth caused by derepression of cell cycle genes, as well as the activation of JAK/STAT and Notch signaling pathways. Interestingly, there is no obvious up-regulation of JAK/STAT or Notch signaling activities in Su(z)2^1.b8 mutant FSC clones, and inhibiting the activity of either pathway could not prevent tumor development from the mutant FSCs, indicating that there are diverse mechanisms underlying the tumor-suppressive activity of Psc and Su(z)2 in different tissues. Further studies should reveal whether Psc and Su(z)2 have a common role in other epithelial stem cell types. Interestingly, mel-18, a mammalian gene closely related to bmi-1, has been reported to have tumor-suppressive activity in cultured breast cancer cells and in NIH 3T3 cells when injected subcutaneously into nude mice. Thus, the tumor-suppressive activity of this RING finger family of PcG genes reported in this stud might be conserved in mammals (Li, 2010).

Taken together, this study reveals a novel mechanism of epithelial extrusion and a novel role of Drosophila PcG genes in suppressing self-renewal programs in epithelial stem cells to allow lineage differentiation. Dysfunction of these genes may lead to tumorigenesis in these tissues. Given evolutionary conservation of polycomb genes from Drosophila to mammals, it would be interesting to investigate whether these genes play a similar role in mammalian stem cells and cancer (Li, 2010).

The role of the histone H2A ubiquitinase Sce in Polycomb repression

Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRing) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. This study created an Sce knockout allele and showed that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression (Gutiérrez, 2012).

This study analyzed how PRC1 regulates target genes in Drosophila to investigate how the distinct chromatin-modifying activities of this complex repress transcription in vivo. Because H2A monoubiquitylation is thought to be central to the repression mechanism of PRC1-type complexes, focus was placed on the role of Sce. The following main conclusions can be drawn from the work reported in this study. First, in the absence of Sce, bulk levels of H2A-K118ub1 are drastically reduced but the levels of the PRC1 subunits Psc and Ph are undiminished. Sce is therefore the major E3 ligase for H2A monoubiquitylation in developing Drosophila but is not required for the stability of other PRC1 subunits. Second, PRC1-bound genes fall into two classes. Class I target genes are misexpressed if any of the PRC1 subunits is removed. Class II target genes are misexpressed in the absence of Ph or Psc-Su(z)2 but remain stably repressed in the absence of Sce or Pc. At class II target genes, Ph and the Psc-Su(z)2 proteins work together to repress transcription by a mechanism that does not require Sce and Pc and is therefore independent of H2A monoubiquitylation. Third, removal of the Ph, Psc-Su(z)2 or Pc proteins results in imaginal disc tumors that are characterized by unrestricted cell proliferation. However, removal of Sce does not cause this phenotype, suggesting that this tumor suppressor activity by the PcG system does not require H2A monoubiquitylation. Finally, these analyses reveal that PRC1 subunits are essential for repressing the elB, noc, dac and pros genes outside of their normal expression domains in developing Drosophila. This expands the inventory of developmental regulator genes in Drosophila for which PcG repression has been demonstrated in a functional assay (Gutiérrez, 2012).

In the Sce^33M2 allele Arg65 is mutated to Cys, but this mutant Sce protein is undetectable and therefore does not appear to be stable in vivo. The crystal structure of the Ring1B-Bmi1 complex provides a molecular explanation for this observation: the Arg70 residue in Ring1B that corresponds to Arg65 in Sce is thought to be critical for interaction with Bmi1. A likely scenario therefore is that the Sce^Arg65Cys protein in Drosophila is unstable and is degraded because it is unable to associate with Psc or its paralog Su(z)2. Interestingly, removal of Sce protein has no detectable effect on the levels of the Psc and Ph proteins. Psc is therefore stable in the absence of its binding partner Sce. This is in contrast to the situation in mice in which Ring1B mutant ES cells show a drastic reduction in the levels of the Ring1B partner protein Bmi1 and its paralog Mel18 (Pcgf62) and also a reduction in the levels of Mph2 (Phc2) and Mpc2 (Cbx4) (Leeb, 2007). The interdependence between PRC1 subunits for protein stability is therefore different in mammals and Drosophila (Gutiérrez, 2012).

Reconstitution of the Drosophila PRC1 core complex in a baculovirus expression system suggests that Sce is important for complex stability. At present, it is not know whether the Psc, Ph and Pc proteins still form a complex in vivo in the absence of Sce. It is currently unknown whether Psc, Ph and Pc are still bound to all PRC1 target genes in the absence of Sce. However, the finding that class II genes remain repressed in the absence of Sce, even though their repression depends on Psc-Su(z)2 and Ph, argues against a crucial role of Sce in the targeting of these other PRC1 subunits to these genes. Interestingly, the repression of all class II target genes analyzed in this study always requires both the Ph and the Psc-Su(z)2 proteins. A possible explanation for this observation is that Ph and Psc-Su(2) still form a PRC1 subcomplex in the absence of Sce and that this complex is fully functional to repress class II target genes. Alternatively, it is possible that Ph and Psc-Su(z)2 repress class II target genes as components of as yet uncharacterized complexes that are distinct from PRC1 and Drosophila dRing-associated factors (dRAF) complex (Gutiérrez, 2012).

In vitro, Psc and Su(z)2 proteins compact nucleosome templates, inhibit nucleosome remodeling by SWI/SNF complexes and repress transcription on chromatin templates. The observation that repression of class II target genes requires Psc-Su(z)2 and Ph but not Pc and Sce supports the idea that the chromatin-modifying activities of Psc-Su(z)2 identified in vitro are the main mechanism by which PRC1 represses these genes. Previous structure/function analyses in Drosophila showed that the same domains of the Psc protein responsible for chromatin compaction and remodeling inhibition in vitro are crucial for HOX gene repression in vivo. Chromatin modification by Psc and Su(z)2 is therefore also crucial for repression of class I target genes. Regulation of the class I target gene en further illustrates this point. In some tissues (e.g. in the dorsal hinge region of the wing imaginal disc) repression of en requires all PRC1 core subunits, but in other tissues (e.g. in the pouch of the wing imaginal disc) en remains repressed in the absence of Sce and Pc, and only Psc-Su(z)2 and Ph seem to be crucial to keep the gene inactive. At present, the molecular mechanism of Ph is not well understood. In vitro, Ph protein has the capacity to inhibit chromatin remodeling and transcription but it does so less effectively than Psc. At the target genes analyzed in this study, Ph is required for transcriptional repression wherever Psc-Su(z)2 is required, suggesting that Ph and Psc-Su(z)2 act in concert in this repression. Nevertheless, it is possible that repression of other PRC1 target genes requires a different subset of PRC1 subunits, or that, as in the case of en, the subunit requirement for repression changes depending on the cell type (Gutiérrez, 2012).

In mammals, Ring1B and Ring1A are responsible for the bulk of H2A-K119 monoubiquitylation. Similarly, Sce generates the bulk of H2A-K118 monoubiquitylation in Drosophila, both in tissue culture cells (Lagarou, 2008) and in the developing organism (this study). The requirement for Sce at class I target genes is consistent with the idea that H2A monoubiquitylation of their chromatin is part of the repression mechanism. Repression of a subset of class I genes, namely the HOX genes, also requires the H2A deubiquitinase PR-DUB (Gaytán de Ayala Alonso, 2007; Scheuermann, 2010). Moreover, PR-DUB and Sce strongly synergize to repress HOX genes. Specifically, the phenotype of Sce PR-DUB double mutants suggests that H2A monoubiquitylation becomes ineffective for HOX gene repression if PR-DUB is absent. However, embryos that lack PR-DUB alone show a 10-fold increase in the bulk levels of H2A-K118ub1 and it is estimated that ~10% of all H2A molecules become monoubiquitylated in these animals. How could this conundrum be explained? One possibility is that H2A monoubiquitylation and deubiquitylation at HOX gene chromatin need to be regulated in a precisely balanced manner. However, an alternative explanation considers H2A-K118ub1 levels in the context of ubiquitin homeostasis. In particular, the high H2A-K118ub1 levels in PR-DUB mutants suggest that Sce generates widespread H2A monoubiquitylation at most Sce-bound genes and possibly also elsewhere in the genome, but that in wild-type animals PR-DUB continuously deubiquitylates H2A-K118ub1 at these locations and thereby recycles ubiquitin. The observation that PR-DUB is widely co-bound with Sce, not only at HOX but also at many other class I and class II target genes, is consistent with this idea. It is tempting to speculate that the widespread H2A monoubiquitylation in PR-DUB mutants sequesters a substantial fraction of the pool of free ubiquitin. It is therefore possible that removal of PR-DUB effectively depletes the pool of free ubiquitin in the nucleus to an extent that H2A monoubiquitylation at HOX target genes becomes inefficient and, consequently, their repression can no longer be maintained. According to this model, the crucial function of PR-DUB would not be the deubiquitylation of H2A-K118ub1 at HOX genes but rather at class II target genes and elsewhere in the genome where Sce 'wastefully' monoubiquitylates H2A (Gutiérrez, 2012).

Aggarwal, B. D. and Calvi, B. R. (2004). Chromatin regulates origin activity in Drosophila follicle cells. Nature 430: 372-376. PubMed Citation: 15254542

Akasaka, T. M., et al. (1996). A role for mel-18, a Polycomb group-related vertebrate gene, during the anteroposterior specification of the axial skeleton. Development 122(5): 1513-1522. PubMed Citation: 8625838

Akasaka, T., et al. (2001). Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression. Development 128: 1587-1597. 11290297

Alkema, M. J., et al. (1995). Transformation of axial skeleton due to overexpression of bmi-1 in transgenic mice. Nature 374: 724-7. PubMed Citation: 7715727

Alkema, M. J., et al. (1997). Identification of Bmi1-interacting proteins as constituents of a multimeric mammalian Polycomb complex. Genes Dev. 11: 226-240. PubMed Citation: 9009205

Beh, L. Y., Colwell, L. J. and Francis, N. J. (2012). A core subunit of Polycomb repressive complex 1 is broadly conserved in function but not primary sequence. Proc Natl Acad Sci U S A 109: E1063-1071. PubMed ID: 22517748

Bel, S., et al. (1998). Genetic interactions and dosage effects of Polycomb group genes in mice. Development 125(18): 3543-3551. PubMed Citation: 9716520

Boukarabila, H., et al. (2009). The PRC1 Polycomb group complex interacts with PLZF/RARA to mediate leukemic transformation. Genes Dev. 23(10): 1195-206. PubMed Citation: 19451220

Bruggeman, S. W., et al. (2005). Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice. Genes Dev. 19(12): 1438-43. 15964995

Brunk, B. P., Martin, E. C. and & Adler, P. N. (1991a). Molecular genetics of the Posterior sex combs/Suppressor 2 of zeste region of Drosophila: aberrant expression of the Suppressor 2 of zeste gene results in abnormal bristle development. Genetics 128: 119-32. PubMed Citation: 1905661

Brunk, B. P., Martin, E. C. and Adler, P. N. (1991b). Drosophila genes Posterior Sex Combs and Suppressor two of zeste encode proteins with homology to the murine bmi-1 oncogene. Nature 353: 351-3. PubMed Citation: 1833647

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

Chang, Y.-L., et al. (2001). Essential role of Drosophila Hdac1 in homeotic gene silencing. Proc. Natl. Acad. Sci. Vol. 98: 9730-9735. 11493709

Cohen, K. J., et al. (1996). Transformation by the Bmi-1 oncoprotein correlates with its subnuclear localization but not its transcriptional suppression activity. Mol. Cell. Biol. 16 (10): 5527-5535. PubMed Citation: 8816465

Cryderman, D. E., et al. (1999). Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles. EMBO J. 18(13): 3724-35. PubMed Citation: 10393187

Dhawan, S., Tschen, S. I. and Bhushan, A. (2009). Bmi-1 regulates the Ink4a/Arf locus to control pancreatic β-cell proliferation. Genes Dev. 23(8): 906-11. PubMed Citation: 19390085

Fasano, C. A., et al. (2009). Bmi-1 cooperates with Foxg1 to maintain neural stem cell self-renewal in the forebrain. Genes Dev. 23(5): 561-74. PubMed Citation: 19270157

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

Francis, N. J., et al. (2009). Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro. Cell 137(1): 110-22. PubMed Citation: 19303136

Gaytán de Ayala Alonso, A., et al. (2007). A genetic screen identifies novel polycomb group genes in Drosophila. Genetics 176: 2099-2108. PubMed Citation: 17717194

Gilde, J. J., Lopez, R. and Shearn, A. (2000). A screen for new Trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2. Genetics 156: 645-663. PubMed Citation: 11014813

Grau, D. J., et al. (2011). Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge. Genes Dev. 25(20): 2210-21. PubMed Citation: 22012622

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. PubMed Citation: 9121482

Gutiérrez, L., et al. (2012). The role of the histone H2A ubiquitinase Sce in Polycomb repression. Development 139(1): 117-27. PubMed Citation: 22096074

Hanson, R. D., et al. (1999). Mammalian Trithorax and Polycomb-group homologues are antagonistic regulators of homeotic development. Proc. Natl. Acad. Sci. 96: 14372-14377. PubMed Citation: 10588712

Jacobs, J. J. L., et al. (1999). Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF. Genes Dev. 13: 2678-2690. PubMed Citation: 10541554

Kahn, T. G., Schwartz, Y. B., Dellino, G. I. and Pirrotta, V. (2006). Polycomb complexes and the propagation of the methylation mark at the Drosophila Ubx gene. J. Biol. Chem. 281(39): 29064-75. Medline abstract: 16887811

Kanno, M., et al (1995). mel-18, a Polycomb group-related mammalian gene, encodes a transcriptional negative regulator with tumor suppressive activity. EMBO J 14: 5672-5678. PubMed Citation: 8521824

Kehle, J., et al. (1998). dMi-2, a Hunchback-interacting protein that functions in Polycomb repression. Science 282(5395): 1897-900. PubMed Citation: 9836641

Kim, S. N., Jung, K. I., Chung, H. M., Kim, S. H. and Jeon, S. H. (2008). The pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila melanogaster. Dev. Biol. 319(1): 121-9. PubMed Citation: 18495104

Kim, S. Y., Paylor, S.W., Magnuson, T. and Schumacher, A. (2006). Juxtaposed Polycomb complexes co-regulate vertebral identity. Development 133(24): 4957-68. Medline abstract: 17107999

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

Klebes, A., et al. (2005). Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes. Development 132: 3753-3765. 16077094

Kotake, Y., et al. (2007). pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16^INK4a tumor suppressor gene. Genes Dev. 21: 49-54. Medline abstract: 17210787

Kyba, M. and Brock, H. W. (1998). The Drosophila polycomb group protein Psc contacts ph and Pc through specific conserved domains. Mol. Cell. Biol. (5): 2712-2720. PubMed Citation: 9566890

Lagarou, A., et al. (2008). dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 22: 2799-2810. PubMed Citation: 18923078

Leeb, M. and Wutz, A. (2007). Ring1B is crucial for the regulation of developmental control genes and PRC1 proteins but not X inactivation in embryonic cells. J. Cell Biol. 178: 219-229. PubMed Citation: 17620408

Lessard, J., et al. (1999). Functional antagonism of the Polycomb-Group genes eed and Bmi1 in hemopoietic cell proliferation. Genes Dev. 13: 2691-2703. PubMed Citation: 10541555

Lessard, J. and Sauvageau, G. (2003). Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 423(6937): 255-60. 12714970

Li, X., Han, Y, and Xi, R. (2010). Polycomb group genes Psc and Su(z)2 restrict follicle stem cell self-renewal and extrusion by controlling canonical and noncanonical Wnt signaling. Genes Dev. 24(9): 933-46. PubMed Citation: 20439432

Marchetti, M., et al. (2003). Differential expression of the Drosophila BX-C in polytene chromosomes in cells of larval fat bodies: a cytological approach to identifying in vivo targets of the homeotic Ubx, Abd-A and Abd-B proteins. Development 130: 3683-3689. PubMed Citation: 12835385

Martin, E. C. and Adler, P. N. (1993). The Polycomb group gene Posterior Sex Combs encodes a chromosomal protein. Development 117: 641-55. PubMed Citation: 7687213

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. PubMed Citation: 7915818

Molofsky, A. V., et al. (2005). Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16^Ink4a and p19^Arf senescence pathways. Genes Dev. 19(12): 1432-7. 15964994

Mohd-Sarip, A., Lagarou, A., Doyen, C. M., van der Knaap, J. A., Aslan, U., Bezstarosti, K., Yassin, Y., Brock, H. W., Demmers, J. A. and Verrijzer, C. P. (2012). Transcription-independent function of Polycomb group protein PSC in cell cycle control. Science 336: 744-747. PubMed ID: 22491092

Moshkin, Y. M., et al. (2001). The Bithorax complex of Drosophile melanogaster: underreplication and morphology in polytene chromosomes. Proc. Natl. Acad. Sci. 98: 570-574. PubMed Citation: 11136231

Park, S. Y., Schwartz, Y. B., Kahn, T. G., Asker, D. and Pirrotta, V. (2012). Regulation of Polycomb group genes Psc and Su(z)2 in Drosophila melanogaster. Mech. Dev. 128(11-12): 536-47. PubMed Citation: 22289633

Pelegri, F. and Lehmann, R. (1994) A role of Polycomb group genes in the regulation of Gap gene expression in Drosophila. Genetics 136: 1341-1353. PubMed Citation: 8013911

Poux, S., McCabe, D. and Pirrotta, V. (2001). Recruitment of components of Polycomb Group chromatin complexes in Drosophila. Development 128: 75-85. PubMed Citation: 11092813

Rastelli, L., Chan, C. S., and Pirrotta, V. (1993). Related chromosome binding sites for zeste, suppressors of zeste and Polycomb group proteins in Drosophila and their dependence on Enhancer of zeste function. EMBO J 12: 1513-22. PubMed Citation: 8467801

Reijnen, M. J., et al. (1995). Polycomb and bmi-1 homologs are expressed in overlapping patterns in Xenopus embryos and are able to interact with each other. Mech. Dev. 53: 35-46. PubMed Citation: 8555110

Ringrose, L., Rehmsmeier, M., Dura, J. M. and Paro, R. (2003). Genome-wide prediction of Polycomb/Trithorax response elements in Drosophila melanogaster. Dev. Cell 5: 759-771. 14602076

Rusch, D. B. and Kaufman, T. C. (2000). Regulation of proboscipedia in Drosophila by homeotic selector genes. Genetics 156: 183-194. PubMed Citation: 10978284

Saget, O., et al. (1998). Needs and targets for the multi sex combs gene product in Drosophila melanogaster. Genetics 149(4): 1823-1838. PubMed Citation: 9691040

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

Scheuermann, J. C., et al. (2010). Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB. Nature 465: 243-247. PubMed Citation: 20436459

Schwartz, Y. B., et al. (2006). Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nat. Genet. 38(6): 700-5. 16732288

Sharp, E. J, Martin, E. C. and Adler, P. N. (1994). Directed overexpression of suppressor 2 of zeste and Posterior sex combs results in bristle abnormalities in Drosophila melanogaster. Dev. Biol. 161: 379-92. PubMed Citation: 8313990

Shao, Z., et al. (1999). Stabilization of chromatin structure by PRC1, a polycomb complex. Cell 98: 37-46. PubMed Citation: 10412979

Simon, J., Chiang, A. and Gender, W. (1992). Ten different Polycomb group genes are required for spatial control of the abd-A and Abd-B homeotic products. Development 114: 493-505. PubMed Citation: 1350533

Sing, A., et al. (2009). A vertebrate Polycomb response element governs segmentation of the posterior hindbrain. Cell 138(5): 885-97. PubMed Citation: 19737517

Soto, M. C., Chou, T. B. and Bender, W. (1995). Comparison of germline mosaics of genes in the Polycomb group of Drosophila melanogaster. Genetics 140: 231-243. PubMed Citation: 7635288

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. PubMed Citation: 9372908

Sustar, A. and Schubiger, G. (2005). A transient cell cycle shift in Drosophila imaginal disc cells precedes multipotency. Cell 120: 383-393. 15707896

van der Lugt, N. M. T., et al. (1996). The Polycomb-group homolog Bmi-1 is a regulator of murine Hox gene expression. Mech. Dev. 58: 153-164. PubMed Citation: 8887324

van der Vlag, J., et al. (2000). Transcriptional repression mediated by Polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators. J. Biol. Chem. 275: 697-704. PubMed Citation: 10617669

van Lohuizen, M., et al. (1991). Sequence similarity between the mammalian bmi-1 proto-oncogene and the Drosophila regulatory genes Psc and Su(z)2. Nature 353: 353-5. PubMed Citation: 1922340

Woo, C. J., et al. (2010). A region of the human HOXD cluster that confers polycomb-group responsiveness. Cell 140(1): 99-110. PubMed Citation: 20085705

Wu, C. T. and Howe, M. (1995). A genetic analysis of the Suppressor 2 of zeste complex of Drosophila melanogaster. Genetics 140: 139-181. PubMed Citation: 7635282

date revised: 15 April 2013 

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