extra sexcombs

DEVELOPMENTAL BIOLOGY

Embryonic

ESC maternal mRNA is present in embryos up to 8 hours after fertilization. ESC RNA is uniformly distributed in early embryos but absent from pole cells. It becomes concentrated in the germ band during germ band extention, representing zygotic expression. The RNA is absent from the anterior dorsal region. The pattern is undistinguishable from that of Polycomb and polyhomeotic. Later expression in the esophageal ganglion is transient (Sathe, 1995a).

Alternative ESC and ESC-like subunits of a Polycomb group histone methyltransferase complex are differentially deployed during Drosophila development

The Extra sex combs (ESC) protein is a Polycomb group (PcG) repressor that is a key noncatalytic subunit in the ESC-Enhancer of zeste [E(Z)] histone methyltransferase complex. Survival of esc homozygotes to adulthood, based solely on maternal product and peak ESC expression during embryonic stages, indicates that ESC is most critical during early development. In contrast, two other PcG repressors in the same complex, E(Z) and Suppressor of zeste-12 [SU(Z)12], are required throughout development for viability and Hox gene repression. A novel fly PcG repressor, called ESC-Like (ESCL), is described whose biochemical, molecular, and genetic properties can explain the long-standing paradox of ESC dispensability during postembryonic times. Developmental Western blots show that ESCL, which is 60% identical to ESC, is expressed with peak abundance during postembryonic stages. Recombinant complexes containing ESCL in place of ESC can methylate histone H3 with activity levels, and lysine specificity for K27, similar to that of the ESC-containing complex. Coimmunoprecipitations show that ESCL associates with E(Z) in postembryonic cells and chromatin immunoprecipitations show that ESCL tracks closely with E(Z) on Ubx regulatory DNA in wing discs. Furthermore, reduced escl⁺ dosage enhances esc loss-of-function phenotypes and double RNA interference knockdown of ESC/ESCL in wing disc-derived cells causes Ubx derepression. These results suggest that ESCL and ESC have similar functions in E(Z) methyltransferase complexes but are differentially deployed as development proceeds (Wang, 2006).

ESC and E(Z), and their homologs, are functional partners in the chromatin of plants, invertebrates, and mammals. Working together, they control a diverse array of developmental processes, including flower and seed differentiation in Arabidopsis thaliana, germ line development in Caenorhabditis elegans, X chromosome inactivation in mice, and Hox gene repression in flies and mammals. Recent studies show that this partnership reflects a requirement for ESC in potentiating the histone methyltransferase activity of E(Z) (Wang, 2006).

In light of this functional interdependence, a paradox is presented by developmental studies in Drosophila melanogaster, which show that ESC is primarily needed during early embryogenesis, whereas E(Z) is required throughout embryonic, larval, and pupal development. Analysis of ESCL, which can replace ESC in E(Z) HMTase complexes in vitro, provides a plausible solution to this puzzle. ESCL expression is largely complementary to that of ESC, peaking during later developmental stages, and functional studies show that ESCL is partially redundant with ESC in imaginal tissues. These results, together with prior genetic data that address esc time of action, indicate that ESC predominates in embryos, whereas both ESCL and ESC make functional contributions during postembryonic development (Wang, 2006).

Phenotypic analyses of esc loss-of-function mutants provided the original evidence that the primary time of ESC action is during embryogenesis. Although complete loss of esc⁺ product is embryonic lethal and yields wholesale misexpression of Hox genes, it was shown that maternally provided esc⁺ product provides sufficient function during embryogenesis to enable zygotically null esc^– animals to survive to adulthood. These esc^– adults are fertile, healthy, and phenotypically normal except for minor homeotic transformations such as extra sex combs on the meso- and meta-thoracic legs. In contrast, animals that are zygotically null for any other PcG subunit of the ESC-E(Z) complex or PRC1 fail to survive beyond early pupal stages, with most dying by the embryonic/L1 stage (Wang, 2006).

Additional experiments with a conditional esc allele further delimited the main time of ESC function to a period of mid-embryogenesis extending from about the onset of gastrulation (about 3 h at 25°C) until germ band shortening (approximately 9 to 12 h). An independent study that measured phenotypic rescue by a heat-inducible esc⁺ transgene confirmed that the time of ESC action begins at about 3 h of embryogenesis. These genetically determined times of esc⁺ function coincide with the accumulation of ESC protein, which peaks during mid-embryogenesis and declines by the end of embryogenesis (Wang, 2006).

However, full consideration of the genetic evidence also indicates that ESC does contribute to postembryonic PcG repression, particularly in imaginal tissues. Analysis of esc^– larvae showed modest defects in Hox gene repression in imaginal discs as well as in the central nervous system. In particular, this study attributed the extra sex combs phenotype of esc^– larvae to misexpression of the Scr Hox gene in the T2 and T3 leg discs. In addition, production of extra sex combs from patches of esc^– tissue generated by somatic recombination during larval development indicates that the time of ESC action extends into the larval period, at least in leg discs (Wang, 2006).

A postembryonic role is consistent with the detection of ESC on the Ubx gene in wing discs and with the overlapping roles of ESC and ESCL in Ubx repression in disc-derived MCW12 cells. This result might explain why esc^– wing discs did not produce homeotic phenotypes even after sufficient passage to ensure depletion of maternal esc⁺ product; presumably, both ESC and ESCL would need to be disrupted in this tissue to yield robust Hox misexpression. Finally, although it is much less abundant at late developmental times than in embryos, ESC is detected by Western blotting in larval and pupal extracts. Thus, the genetic and molecular data together indicate that ESC does function during postembryonic stages, albeit with a more modest overall contribution than its critical role in embryos (Wang, 2006).

These considerations imply that the developmental division of labor between ESC and ESCL is not simply that ESC functions only in embryos and ESCL takes over for subsequent stages. Rather, although ESC does predominate early, as evidenced by the global loss of H3 K27 methylation in esc^– embryos, postembryonic development appears to involve both ESC and ESCL. It was originally hypothesized that late developmental functions of the esc locus might be executed by an esc⁺ isoform distinct from the embryonic version. The current data confirm that multiple ESC-related proteins do operate during fly development, with a late-acting version supplied by a second copy of the esc gene (Wang, 2006).

The functional context for ESCL during postembryonic development is presumably as a subunit in E(Z)-containing complexes with histone methyltransferase activity. The fact that ESCL can assemble in place of ESC and restore HMTase activity to a reconstituted E(Z) complex indicates that the biochemical roles of ESCL and ESC are similar. ESCL/ESC functional overlap could reflect a mixture of postembryonic E(Z) complexes, with some containing ESCL and others containing ESC. The simplest version of this scenario would entail four-subunit postembryonic HMTase complexes similar to the embryonic core complex of E(Z), SU(Z)12, NURF-55, and ESCL or ESC. However, postembryonic E(Z) complexes have yet to be purified, so their molecular compositions are not yet known. In fact, there is evidence that larval E(Z) complexes may differ from embryonic E(Z) complexes in features besides the ESCL/ESC subunit. For example, the SIR2 histone deacetylase has been reported to associate with larval but not embryonic E(Z) complexes. Much remains to be determined about postembryonic E(Z) complexes, including subunit compositions and characterization of presumed HMTase activity (Wang, 2006).

Although the catalytic subunit E(Z) contains the conserved SET domain, studies on fly, worm, and mammalian homologs reveal that the ESC subunit is also critical for HMTase function. The single loss of ESC from the fly complex or loss of its homolog, MES-6, from the C. elegans complex yields subcomplexes with little or no HMTase activity in vitro. In agreement with this, genetic removal of ESC eliminates most or all methyl-H3 K27 in fly embryos, loss of MES-6 eliminates most or all methyl-H3 K27 in worm germ lines and early embryos, and loss of EED removes most or all methyl-H3 K27 from embryonic mouse cells. The mechanism by which ESC and its relatives potentiate the activity of HMTase complexes is not known. An in vitro study argues against a role for fly ESC in mediating stable contacts with nucleosome substrate. In contrast, loss of ESC by RNA interference in fly S2 cells leads to dissociation of E(Z) from chromatin targets (Wang, 2006).

A biochemical analysis of the human EED-EZH2 complex (also called PRC2) has revealed an intriguing difference in the HMTase depending upon the subtype of EED subunit present in the complex. Multiple isoforms of EED are expressed in HeLa cells that differ in the extents of their N-terminal tails through use of alternative translation start sites. Incorporation of particular EED isoforms into EZH2 complexes can shift the enzyme specificity so that K26 of histone H1 is methylated in addition to H3 K27. Taken together with other studies, this suggests that EED is a regulatory subunit that can influence both substrate specificity and catalytic efficiency of the HMTase (Wang, 2006).

In light of this finding, it seems possible that ESCL-E(Z) complexes might also have HMTase activity with altered lysine specificity. However, both ESCL-E(Z) and ESC-E(Z) recombinant complexes showed similar specificities for H3 K27 in H3/H4 tetramers and no methylation of mammalian histone H1 by either of these recombinant complexes was detected in vitro. It is noted that the human H1 K26 methylation site is embedded in an ARKS sequence, which is also present surrounding H3 K27. This sequence is not conserved in Drosophila histone H1, suggesting that the ability of certain EZH2 complexes to methylate H1 may not be conserved in the fly system. However, there may well be other relevant methylation substrates besides histone H3 and it remains possible that alternative ESC isoforms could alter lysine specificities for these other substrates (Wang, 2006).

Based upon their temporal expression profiles, it seems clear that esc and escl have distinct functions in a developmental context. Their temporal division of labor is most clearly demonstrated by esc^– escl⁺ embryos, which show extreme homeotic transformations accompanied by dramatically reduced levels of methylated H3 K27. This division could be entirely a consequence of differential transcriptional controls built into their divergent promoters. That is, ESC and ESCL could be functionally identical proteins that are just expressed at peak levels at different times. Alternatively, the two proteins may possess intrinsic differences that are also important during development but are not revealed by the assays applied so far. One possibility is that ESC and/or ESCL may play a role in methylation of nonhistone proteins. The only nonhistone proteins yet identified that fly E(Z) complexes can methylate are two subunits of the core complex itself, E(Z) and SU(Z)12. It is not clear if this self-methylation is functionally relevant and, in any case, it occurs at comparable levels with the ESC- and ESCL-containing recombinant complexes (Wang, 2006).

It is also possible that ESC and ESCL could differ in contributions to E(Z) complexes besides HMTase activity. These other functions could include interacting with and recruiting histone deacetylases, mediating physical interactions with PRC1 components, recruiting E(Z) complexes to target loci, and influencing the way E(Z) complexes interact with other (non-K27) histone tail modifications. Indeed, there is evidence for differential association of histone deacetylases with E(Z) complexes at embryonic versus larval stages, which parallels temporal changes in ESC and ESCL abundance. At the same time, ESC and ESCL functions must overlap enough to account for the sufficiency of either one to maintain Ubx repression in at least some postembryonic cells (Wang, 2006).

Definitive answers will require promoter swap experiments in which ESCL is placed under control of the ESC promoter and vice versa, to determine which combinations provide genetic rescue of esc and escl mutations in vivo. Along with this approach, a complete understanding of the developmental role of ESCL will require generation of escl mutant alleles and systematic analysis of the phenotypic consequences of escl loss of function (Wang, 2006).

Effects of Mutation or Deletion

The esc+ gene product is required during a discrete period of embryogenesis. If the gene product is absent or inactive during this period, most segments develop like the eighth abdominal segment. In contrast, absence of active gene product before or after this period has relatively little effect on segmental development. It has therefore been suggested that the esc+ gene product has a discrete early function in the initiation of segmental determination (Struhl, 1982)

Phenotypes of adult flies heterozygous for every pairwise combination of Polycomb group genes have been examined in an attempt to functionally subdivide the Pc-G. It appears that all these genes have similar functions in some imaginal tissues. There are genetic interactions among extra sexcombs and Enhancer of Pc, Pc-like, Enhancer of zeste (E(z), and super sexcombs. The idea that most Pc-G genes function independently of extra sexcombs needs to be reassessed. Most duplications of Pc-G genes neither suppress the extra sexcombs phenotype of Pc-G mutations nor exhibit anterior transformations, suggesting that not all Pc-G genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of extra sexcombs mutants. Flies with increasing doses of esc+ do exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes (Campbell, 1995).

In the leg discs of extra sexcombs mutants, both Ubx and Scr are expressed at increased levels or in new locations. Ubx also is expressed in new locations in the posterior wing disc and in small groups of cells in the antenna disc. Antp is expressed ectopically in the eye-antenna disc; however, obvious abnormal expression of Antp was not found in the thoracic imaginal discs. Particularly striking is the fact that a single disc, such as the mesothoracic leg, can show increased expression of both a more "anterior" homeotic gene (Scr) and a more "posterior" gene (Ubx). Ectopic expression of Ubx and Antp, but not of Scr, is seen in the central nervous system of mutant larvae (Glicksman, 1988).

Esc was identified in a genome-wide analyses for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4²²¹) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4²²¹-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

RNAi of the Polycomb group (PcG) genes Su(z)12, E(z), esc, or Caf1 similarly caused an increase in branch number and an expansion of the receptive field of class I neurons. Consistent with the similar RNAi phenotypes for these genes, Su(z)12, E(z), esc, and Caf1 are components of the multiprotein esc/E(z) polycomb repressor complex. One critical role for PcG-mediated gene silencing is the regulation of hox gene expression. Therefore, Polycomb-mediated regulation of hox gene expression likely contributes to arborization of class I neurons (Parrish, 2006).

Polycomb genes interact with the tumor suppressor genes hippo and warts in the maintenance of Drosophila sensory neuron dendrites

Dendritic fields are important determinants of neuronal function. However, how neurons establish and then maintain their dendritic fields is not well understood. Polycomb group (PcG) genes are required for maintenance of complete and nonoverlapping dendritic coverage of the larval body wall by Drosophila class IV dendrite arborization (da) neurons. In esc, Su(z)12, or Pc mutants, dendritic fields are established normally, but class IV neurons display a gradual loss of dendritic coverage, while axons remain normal in appearance, demonstrating that PcG genes are specifically required for dendrite maintenance. Both multiprotein Polycomb repressor complexes (PRCs) involved in transcriptional silencing are implicated in regulation of dendrite arborization in class IV da neurons, likely through regulation of homeobox (Hox) transcription factors. Genetic interactions and association between PcG proteins and the tumor suppressor kinase Warts (Wts) is demonstrated, providing evidence for their cooperation in multiple developmental processes including dendrite maintenance (Parrish, 2007).

Dendrite arborization patterns are a hallmark of neuronal type; yet how dendritic arbors are maintained after they initially cover their receptive field is an important question that has received relatively little attention. The Drosophila PNS contains different classes of sensory neurons, each of which has a characteristic dendrite arborization pattern, providing a system for analysis of signals required to achieve specific dendrite arborization patterns. Class IV neurons are notable among sensory neurons because they are the only neurons whose dendrites provide a complete, nonredundant coverage of the body wall. This study found tha the function of Polycomb group genes is required specifically in class IV da neurons to regulate dendrite development. In the absence of PcG gene function, class IV dendrites initially cover the proper receptive field but subsequently fail to maintain their coverage of the field. Time-lapse analysis of dendrite development in esc or Pc mutants suggests that a combination of reduced terminal dendrite growth and increased dendrite retraction likely accounts for the gradual loss of dendritic coverage in these mutants. Maintenance of axonal terminals in class IV da neurons is apparently unaffected by loss of PcG gene function, suggesting that PcG genes function as part of a program that specifically regulates dendrite stability (Parrish, 2007).

Establishment of dendritic territories in class IV neurons is regulated by homotypic repulsion, and this process proceeds normally in the absence of PcG function. In PcG mutants, class IV neurons tile the body wall by 48 h AEL, similar to wild-type controls. However, beginning at 48 h AEL, likely as a result of reduced dendritic growth and increased terminal dendrite retraction, class IV neurons of PcG mutants gradually lose their dendritic coverage. In contrast, the axon projections and terminal axonal arbors of PcG mutants show no obvious defects. Although an early role for PcG genes in regulating axon development cannot be ruled out, MARCM studies showed that PcG genes are required for the maintenance of dendrites but not axons in late larval development. Thus, different genetic programs appear to be responsible for the establishment and maintenance of dendritic fields, and for the maintenance of axons and dendrites (Parrish, 2007).

It is well established that PcG genes participate in regulating several important developmental processes including expression of Hox genes for the specification of segmental identity. In comparison, much less is known about the function of PcG genes in neuronal development. Studies of the expression patterns of PcG genes and the consequences of overexpression of PcG genes suggest that PcG genes may affect the patterning of the vertebrate CNS along the anterior-posterior (AP) axis, analogous to their functions in specifying the body plan. A recent study demonstrates that the PcG gene Polyhomeotic regulates aspects of neuronal diversity in the Drosophila CNS. The current study now links the function of PcG genes to maintenance of dendritic coverage of class IV sensory neurons. Thus it will be interesting to determine whether PcG genes play a conserved role in the regulation of dendrite maintenance (Parrish, 2007).

Since Hox genes function in late aspects of neuronal specification and axon morphogenesis, it seems possible that regulation of Hox genes by PcG genes may be important for aspects of post-mitotic neuronal morphogenesis, including dendrite development. The PcG genes esc and E(z) are required for proper down-regulation of BX-C Hox gene expression in class IV neurons. The timing of this change in BX-C expression corresponds to the time frame during which PcG genes are required for dendritic maintenance. Furthermore, post-mitotic overexpression of BX-C genes in class IV da neurons, but not other classes of da neurons, is sufficient to cause defects in dendrite arborization, thus phenocopying the mutant effects of PcG genes. Finally, it was found that Hox genes are required cell-autonomously for dendrite development in class IV neurons, and loss of Hox gene function causes defects in terminal dendrite dynamics that are opposite to the defects caused by loss of PcG genes. Therefore, it seems likely that PcG genes regulate dendrite maintenance in part by temporally regulating BX-C Hox gene expression (Parrish, 2007).

Several recent studies have focused on the identification of direct targets of PcG-mediated silencing, demonstrating that PcG genes regulate expression of distinct classes of genes in different cellular contexts. During Drosophila development, PRC proteins likely associate with >100 distinct loci, and the chromosome-associate profile of PRC proteins appears dynamic. Therefore, identifying the targets of PcG-mediated silencing in a given developmental process has proven difficult. Thus far, alleles of >20 predicted targets of PcG-mediated silencing have been analyzed for roles in establishment or maintenance of dendritic tiling and a potential role has been found for only Hox genes. Future studies will be required to identify additional targets of PcG-mediated silencing in regulation of dendrite maintenance (Parrish, 2007).

PcG genes are broadly expressed, so it seems likely that interactions with other factors or post-translational mechanisms may be responsible for the cell type-specific activity of PcG genes. Indeed, PcG genes genetically interact with components of the Wts signaling pathway to regulate dendrite development specifically in class IV neurons. Based on the observations that wts mutants also show derepression of Ubx in class IV neurons and that Wts can physically associate with PcG components, it seems likely that Wts may directly or indirectly influence the activity of PcG components. In proliferating cells, Wts phosphorylates the transcriptional coactivator Yorkie to regulate cell cycle progression and apoptosis, demonstrating that Wts can directly influence the activity of transcription factors. In support of a possible role for Wts directly modulating PcG function, several recent reports have documented roles for phosphorylation in regulating PcG function both in Drosophila and in vertebrates. Thus, it is possible that some of the components involved in PcG-mediated silencing are regulated by Wts phosphorylation. Alternatively, association of Wts with PcG proteins may facilitate Wts-mediated phosphorylation of chromatin substrates (Parrish, 2007).

The tumor suppressor kinase Hpo regulates both establishment and maintenance of dendritic tiling in class IV neurons through its interactions with Trc and Wts, respectively, but how Hpo coordinately regulates these downstream signaling pathways is currently unknown. Similar to mutations in wts, mutations in PcG genes interact with mutations in hpo to regulate dendrite maintenance but show no obvious interaction with trc, consistent with the observation that PcG gene function is dispensable for establishment of dendritic tiling. Although it is possible that different upstream signals control Hpo-mediated regulation of establishment and maintenance of dendritic tiling, the nature of such signals remain to be determined. Another possibility is that the activity of the Wts/PcG pathway could be antagonized by additional unknown factors that promote establishment of dendritic tiling (Parrish, 2007).

In addition to their interaction in regulating dendrite maintenance, PcG genes and wts interact to regulate expression of the Hox gene Scr during leg development. This finding suggests that the Hpo/Wts pathway may play a general role in contributing to PcG-mediated regulation of Hox gene expression. The presence of ectopic sex combs provides a very simple and sensitive readout of wts/PcG gene interactions and should form the basis for conducting large-scale genetic screens to identify other genes that interact with wts or PcG genes and participate in this genetic pathway (Parrish, 2007).

ESC, ESCL and their roles in Polycomb Group mechanisms

The Drosophila esc gene is a Polycomb Group (PcG) gene whose product is essential for histone H3 K27 methylation and PcG silencing yet genetic analysis indicated that its product was needed only in the very early embryo. escl, a close homologue of esc exists in the Drosophila genome. In contrast with earlier studies, both esc and escl were found to be expressed at all stages of development. Three major differences between the two genes are in the transcriptional control, which allows esc to make a much stronger maternal contribution; in the splicing efficiency, which makes a major difference in the early escl function; and in the lower participation of ESCL in the PRC2 complex and lower enzymatic activity of the resulting complex. Both genes can sustain normal development in the absence of the other except for the critical role provided by maternal esc product in early embryonic development. Finally, using zygotic mutations in both genes, it was shown that the gradual loss of function of PRC2 activity leads first to a loss of histone H3 K27 methylation and only at a later stage to a gradual loss of PRC1 binding to chromatin (Ohno, 2008).

The results reported here make it clear that the escl gene is redundant for viability and essential functions. At the same time, the results show that, although maternal esc function is necessary to supply the embryo with the massive amounts of esc product that ensure early establishment of PcG silencing, a function not adequately provided by escl, esc and escl are each capable of supporting later development. It is remarkable that the escl paralogue is conserved in D. pseudoobscura and in the 10 other Drosophila species that have been sequenced to date. Thus both genes are present in the Drosophilid radiation although in D. melanogaster, no physical or developmental defects are detectable its absence. A BLAST analysis of the related species shows, however, that both Anopheles and Tribolium have only one esc homologue that is overall slightly more similar to escl at the amino acid level (in Anopheles, 291/419 similarities with escl and 265/472 with esc). In detail, some regions resemble more esc and others escl, consistent with the idea that a single esc gene was duplicated and then diversified in a dipteran ancestor of Drosophilids (Ohno, 2008).

The results show that the esc and escl gene functions differ in transcriptional control, splicing efficiency, participation in the PRC2 complex and the relative enzymatic activity of the resulting complexes. The esc promoter is responsible for the massive maternal deposits of esc mRNA during oogenesis that enable the embryo to establish PcG repressive states. The escl transgenes driven by the esc promoter can provide this maternal contribution while the endogenous escl gene cannot. In addition, there are differences in the profile of esc and escl RNA accumulation during development: esc RNA decreases substantially during larval stages while escl RNA appears to remain relatively higher. This, however does not seem to have a major developmental effect (Ohno, 2008).

Both esc and escl primary transcripts appear to be inefficiently spliced in the early embryo, accumulating a substantial fraction that retains intron 3 of esc or the corresponding intron 2 of escl. This makes very little difference in the case of esc since in the early embryo there is still a massive amount of maternal RNA which is fully spliced. While maternal escl RNA is also fully spliced, it is much less abundant and disappears rapidly. The inefficient splicing of zygotic escl RNA therefore means that insufficient escl product is available in the early embryo. Combined with the lower maternal contribution, this explains why escl cannot substitute for esc in the critical stages of early embryonic development. The transgene experiments show however that a fully spliced escl RNA can provide both the maternal component and the early embryonic function when expressed from either the esc or escl promoter while the incompletely spliced RNA is insufficient regardless of the promoter. This leads to the conclusion that the inefficient splicing of escl compared to esc is a critical factor during embryonic development. However, promoter function is also important. Even the fully spliced escl gene cannot rescue the sex comb phenotype when driven by the escl promoter but does rescue when driven by the esc promoter, suggesting that the esc promoter is more active in leg discs. Both genes have short promoter regions, about 700 bp for esc but only 100 bp for escl to the end of the preceding transcript. In addition, in the case of escl, some transcripts from the preceding gene may run into escl and arguably affect its expression. Sequence similarity between esc and escl in the 100 bp region preceding the transcription start is confined to two or three pentanucleotide motifs (Ohno, 2008).

The in vitro reconstitution experiments show that ESCL can be assembled in vitro in a functional PRC2 complex. Differences in the assembly efficiency were not detected and may not be noticeable at the concentrations used in the in vitro experiments. The reconstituted complex, however, has a fivefold lower enzymatic activity in methylating recombinant histone H3. While it is difficult to prove that the in vitro results have a direct bearing on the in vivo behavior of the escl product, the most straightforward conclusion would be that this lower activity is likely to contribute to the lower functionality of the escl gene in comparison with esc. The lower activity, for example, may explain the fact that loss of esc function, leaving only escl activity, results in the eponymous phenotype of extra sex combs on posterior legs. Despite the lower activity, the massive amounts of product supplied by the esc > Flag-ESCLsp transgene in the early embryonic stages rescues both early development and the extra sex comb phenotype. This suggests that the ESCL protein can support all ESC functions and that its lower activity can be compensated by higher levels of expression (Ohno, 2008).

Mammalian complexes containing different isoforms of EED, the mammalian ESC homologue, have been reported to have in vitro different target specificities. The EED isoforms are produced by alternative translation start sites, resulting in different lengths of the N-terminal region. In Drosophila, however, despite the differences between ESC and ESCL in the N-terminal tail and some of the loop regions, both mediate di- and tri-methylation of H3 K27 and do not display qualitative functional differences (Ohno, 2008).

The mutation analysis and the RNAi analysis agree in showing that esc is the predominant function throughout development, at least in all functions whose absence produces a detectable phenotype. In tissue culture cells, in imaginal discs and in nervous system development, escl appears to play a back-up role under laboratory conditions and phenotypically and enzymatically it normally makes very little contribution to the total function. Yet in the absence of a functional esc gene, escl can take over and support virtually normal development, except for the extra sex comb phenotype. The substantial decrease in H3 K27 methylation in esc mutant larvae indicates that, in most stages and tissues, a considerably lower level of K27 methylation is sufficient for normal function and adequate PcG repression. Only at critical stages and in critical tissues such as early embryogenesis and in the notorious case of the sex comb, which gives their name to many PcG genes, is a higher level of methylation required (Ohno, 2008).

A second explanation for the ability of escl to supply adequate function is that competition between ESC and ESCL products normally excludes much ESCL from participation in the PRC2 complex. The results suggest that the excluded ESCL might be degraded, leading to an apparently low level of accumulation and of contribution to overall H3 K27 methylation. However, ESCL becomes important when ESC is unavailable for incorporation in the PRC2 complex. The supply of esc function is therefore buffered, probably at the post-translational level by degradation of excess protein that does not find employment in a PRC2 complex. The effect of this buffering action is that in the absence of ESC, ESCL is incorporated in the PRC2 complex and the resulting stabilization increases its accumulation several fold. This explains why the escl mutation (or RNAi knockdown) seems to make a contribution to the double mutant (or double RNAi) out of proportion to the effect of the single escl mutant or RNAi. It is not yet known if excess ESC is also rapidly degraded. Attempts to alter the ESCL accumulation by changing the levels of E(Z) failed to show an effect. However, mammalian EZH2 is said to be lost in eed mutants and in Suz12 mutants suggesting that a similar effect governs the accumulation of EZH2 (Ohno, 2008).

The polytene chromosomes clearly show that in the double mutant larvae, the loss of H3 K27 methylation does not immediately lead to the loss of PcG complexes from chromatin. In chromosomes in which no H3 K27me3 is detectable, staining for PSC protein appears normal in intensity and distribution. Loss of PSC from the majority of sites occurs eventually in the double mutant larvae, as has been reported for a E(z) temperature sensitive mutant, but much later than the loss of K27 methylation. It is true that antibody staining of polytene chromosomes is a far from quantitative method and the relative strengths of the three antibodies used for H3 K27me2, H3 K27me3 and PSC could be very different. These results, however, are in agreement with the idea that PcG complex binding is not directly dependent on K27 methylation. Chromatin immunoprecipitation experiments show that the core binding sites of PcG complexes are devoid of histones. In clones mutant for E(z), derepression of homeotic genes does not occur until 96 h after the loss of E(z) function has been induced. Genomic tiling microarray analysis shows that the distribution of H3 K27me3 is much more extensive than that of PcG proteins, covering a domain of many tens of kilobases surrounding the PREs. These results provide compelling evidence against the argument that K27me3 is responsible for recruiting PRC1 complexes through its interaction of the PC chromodomain. Clearly, K27 methylation is not sufficient to recruit PcG complexes but it is equally clear that loss of methylation eventually results in loss of PcG complex binding. The processes that lead to this loss remain to be investigated (Ohno, 2008).

Polycomb repressive complex 2 and Trithorax modulate Drosophila longevity and stress resistance

Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are key epigenetic regulators of global transcription programs. Their antagonistic chromatin-modifying activities modulate the expression of many genes and affect many biological processes. This study reports that heterozygous mutations in two core subunits of Polycomb Repressive Complex 2 (PRC2), the histone H3 lysine 27 (H3K27)-specific methyltransferase E(Z) and its partner, the H3 binding protein ESC, increase longevity and reduce adult levels of trimethylated H3K27 (H3K27me3). Mutations in trithorax (trx), a well known antagonist of Polycomb silencing, elevate the H3K27me3 level of E(z) mutants and suppress their increased longevity. Like many long-lived mutants, E(z) and esc mutants exhibit increased resistance to oxidative stress and starvation, and these phenotypes are also suppressed by trx mutations. This suppression strongly suggests that both the longevity and stress resistance phenotypes of PRC2 mutants are specifically due to their reduced levels of H3K27me3 and the consequent perturbation of Polycomb silencing. Consistent with this, long-lived E(z) mutants exhibit derepression of Abd-B, a well-characterized direct target of Polycomb silencing, and Odc1, a putative direct target implicated in stress resistance. These findings establish a role for PRC2 and TRX in the modulation of organismal longevity and stress resistance and indicate that moderate perturbation of Polycomb silencing can increase longevity (Siebold, 2009).

The evidence presented in this study establishes a role for PRC2 and TRX in the modulation of life span and stress resistance. Using multiple alleles of several PRC2 subunits, evidence is provided that heterozygous mutations in the PRC2 subunits E(z) and esc extend life span and increase resistance to oxidative stress and starvation in Drosophila. Consistent with the enzymatic function of PRC2 in the methylation of H3K27, long-lived E(z) and esc mutants have reduced H3K27me3 levels. Furthermore, mutations in trx suppress the increased longevity and stress resistance phenotypes of E(z) mutants, while concomitantly increasing their reduced H3K27me3. The moderate reduction of H3K27me3 in long-lived E(z) mutants is sufficient to partially derepress some direct targets of Polycomb silencing, and this is also counteracted by mutations in trx. These results provide strong evidence that derepression of one or more Polycomb target genes is likely to be responsible for their increased longevity. Interestingly, E(z) was also recently identified as one of a number genes whose mRNA expression levels were significantly associated with variation in longevity in a large set of wild-type derived inbred lines (Siebold, 2009).

The counterbalancing effects of PRC2 and TRX on H3K27me3 levels suggest a simple model for their modulation of longevity. Although complete loss of PRC2 activity results in preadult lethality, moderately reducing H3K27me3 destabilizes Polycomb silencing sufficiently to cause partial derepression of some Polycomb target genes that can increase life span and stress resistance. Simultaneously reducing TRX and E(Z) exerts a compensatory effect, reestablishing more normal levels of H3K27me3 and Polycomb target gene expression. Based on this model, it is expected that heterozygous trx mutations would decrease longevity. However, the modestly elevated H3K27me3 level (13%) of the heterozygous trxB11 null mutant may simply be insufficient to cause this effect in a wild-type background. It will be interesting to see whether increased TRX levels, which decrease H3K27me3 levels (much like PRC2 mutants) by elevating CBP-mediated H3K27 acetylation, will promote increased longevity, as the model would predict. The evolutionary conservation of PRC2 components in metazoans and their conserved function in epigenetic silencing raises the possibility that they may play a conserved role in modulating life span in other organisms. Although histone methyl-transferases have not been previously implicated in modulating organismal longevity, several other highly conserved chromatin- modifying enzymes have been. In addition to SIR2 and RPD3, the histone H3K4 demethylase LSD-1 has also recently been implicated in modulating longevity in C. elegans. Given the roles of these enzymes in the epigenetic maintenance of transcriptional states, it seems likely that additional chromatin modifying enzymes will be found to modulate longevity. The most well-characterized targets of Polycomb silencing are the homeotic genes of the Bithorax and Antennapedia complexes (Siebold, 2009).

Although heterozygous PRC2 mutants exhibit no overt homeotic phenotypes, the elevated level of Abd-B expression in E(z) heterozygotes demonstrates that their moderately reduced H3K27me3 level is sufficient to partially derepress Polycomb target genes. Could modest derepression of one or more of the homeotic genes be responsible for the increased longevity? Given that they encode transcription factors, their potential for regulating expression of many other genes leaves this possibility open. PRC2 mutants exhibit increased resistance to oxidative stress and starvation. The elevated expression level of Odc1, a putative direct target of Polycomb silencing may contribute to this as it has been shown to mediate resistance to oxidative stress and a variety of other chemical and environmental stresses. Dietary supplementation with the polyamine spermidine was also recently shown to increase longevity in yeast, C. elegans, Drosophila, and mice, consistent with the possibility that Odc1 overexpression may contribute to the increased longevity of PRC2 mutants. Recent evidence suggests that other changes in metabolism and adult physiology might also contribute to the increased longevity of PRC2 mutants. YY1, the mammalian homolog of Drosophila PHO (a DNA-binding PcG protein involved in recruiting PRC2 to chromatin), directly regulates many genes required for mitochondrial oxidative metabolism. It will be interesting to determine whether transcriptional regulation of metabolic genes is a broader theme in the adult function of PcG proteins. PRC2 and TRX play key roles in promoting epigenetically stable transcriptional states through their mutually antagonistic effects on H3K27me3 levels. Recent work has revealed a growing number of biological processes in which they play an important role. The results presented here now point to a role for these epigenetic transcriptional regulators in modulating life span (Siebold, 2009).

Barnett, M. W., Seville, R. A., Nijjar, S., Old, R. W., and Jones, E. A. (2001). Xenopus Enhancer of zeste (XEZ); an anteriorly restricted polycomb gene with a role in neural patterning. Mech. Dev. 102: 157-167. 11287189

Boyer, L. A., et al. (2006). Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441: 349-353. Medline abstract: 16625203

Campbell, R. B., et al. (1995). Genetic interactions and dosage effects of Polycomb group genes of Drosophila. Mol. Gen. Genet. 246: 291-300. PubMed Citation: 7854314

Cao, R., et al. (2002). Role of Histone H3 lysine 27 methylation in Polycomb-Group silencing. Science 298: 1039-1043. 12351676

Ciferri, C., Lander, G. C., Maiolica, A., Herzog, F., Aebersold, R. and Nogales, E. (2012). Molecular architecture of human polycomb repressive complex 2. Elife 1: e00005. PubMed ID: 23110252

Cooper, J. P., Roth, S. Y. and Simpson, R. T. (1994). The global transcriptional regulators, SSN6 and TUP1, play distinct roles in the establishment of a repressive chromatin structure. Genes Dev 8: 1400-1410. PubMed Citation: 7926740

Davidovich, C., Goodrich, K. J., Gooding, A. R. and Cech, T. R. (2014). A dimeric state for PRC2. Nucleic Acids Res 42(14): 9236-48. PubMed ID: 24992961

Denisenko, O. N. and Bomsztyk, K. (1997). The product of the murine homolog of the Drosophila extra sex combs gene displays transcriptional repressor activity. Mol. Cell. Biol. 17(8): 4707-4717. PubMed Citation: 9234727

Denisenko, O., et al. (1998). Point mutations in the WD40 domain of Eed block its interaction with Ezh2. Mol. Cell. Biol. 18(10): 5634-5642. PubMed Citation: 9742080

Faust, C., et al. (1998). The Polycomb-group gene eed is required for normal morphogenetic movements during gastrulation in the mouse embryo. Development 125(22): 4495-4506. PubMed Citation: 9778508

Frei, E., Baumgartner, S., Edstrom, J.E. and Noll, M. (1985a). Cloning of the extra sex combs gene of Drosophila and its identification by P-element mediated gene transfer. EMBO J. 4: 979-987. PubMed Citation: 16453608

Frei, E., et al. (1985b). Isolation and structural analysis of the extra sex combs gene of Drosophila. Cold Spring Harb Symp Quant Biol 50: 127-34. PubMed Citation: 3006985

Furuyama, T., Tie, F. and Harte, P. J. (2003). Polycomb group proteins ESC and E(Z) are present in multiple distinct complexes that undergo dynamic changes during development. Genesis 35(2): 114-24. 12533794

Glicksman, M. A. and Brower, D. L. (1988). Misregulation of homeotic gene expression in Drosophila larvae resulting from mutations at the extra sex combs locus. Dev. Biol. 126: 219-27. PubMed Citation: 2895027

Gutjahr, T., et al. (1995). The Polycomb-group gene, extra sex combs, encodes a nuclear member of the WD-40 repeat family. EMBO J. 14: 4296-4306. PubMed Citation: 7556071

Hannah-Alava, A. (1958). Developmental genetics of the posterior legs in Drosophila Melanogaster. Genetics 43: 878-905. PubMed Citation: 17247802

Hansen, K. H. et al. (2008). A model for transmission of the H3K27me3 epigenetic mark. Nature Cell Biol. 10: 1291-1300. PubMed Citation: 18931660

Jones, C. A., et al. (1998). The Drosophila Esc and E(z) proteins are direct partners in polycomb group-mediated repression. Mol. Cell. Biol. 18(5): 2825-2834. PubMed Citation: 9566901

Kelly, W. G. and Fire, A. (1998). Chromatin silencing and the maintenance of a functional germline in Caenorhabditis elegans. Development 125: 2451-2456. PubMed Citation: 9609828

Ketel, C. S., et al. (2005). Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes. Mol. Cell. Biol. 25: 6857-6868. 16055700

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

Kirmizis, A., et al. (2004). Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27. Genes Dev. 18: 1592-1605. 15231737

Komachi, K., Redd, M.J. and Johnson, A.D. (1994). The WD repeats of Tup1 interact with the homeo domain protein alpha 2. Genes Dev 8: 2857-67. PubMed Citation: 7995523

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

Komachi, K. and Johnson, A. D. (1997). Residues in the WD repeats of Tup1 required for interaction with alpha2. Mol. Cell. Biol. 17(10): 6023-6028. PubMed Citation: 9315661

Korf, I., Fan, Y. and Strome, S. (1998). The Polycomb group in Caenorhabditis elegans and maternal control of germline development. Development 125: 2469-2478. PubMed Citation: 17210650

Kurzhals, R. L., Tie, F., Stratton, C. A., Harte, P. J. (2008). Drosophila ESC-like can substitute for ESC and becomes required for Polycomb silencing if ESC is absent. Dev. Biol. 313(1): 293-306. PubMed Citation: 18048023

Langlais, K. K., Brown, J. L. and Kassis, J. A. (2012). Polycomb group proteins bind an engrailed PRE in both the 'ON' and 'OFF' transcriptional states of engrailed. PLoS One 7: e48765. PubMed ID: 23139817

Lee, T. I. (2006). Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125(2): 301-13. Medline abstract: 16630818

Leeb, M., et al. (2010). Polycomb complexes act redundantly to repress genomic repeats and genes. Genes Dev. 24(3): 265-76. PubMed Citation: 20123906

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

Li, G., et al. (2010). Jarid2 and PRC2, partners in regulating gene expression. Genes Dev. 24(4): 368-80. PubMed Citation: 20123894

Mak, W., et al. (2002). Mitotically stable association of polycomb group proteins Eed and Enx1 with the inactive X Chromosome in trophoblast stem cells. Curr. Biol. 12: 1016-1020. 12123576

Margueron, R., et al. (2009). Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 461(7265): 762-7. PubMed Citation: 19767730

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

Müller, J., et al. (2000). Histone methyltransferase activity of a Drosophila polycomb group repressor complex. Cell 111: 197-208. 12408864

Nekrasov, M., Wild, B. and Müller, J. (2005). Nucleosome binding and histone methyltransferase activity of Drosophila PRC2. EMBO Rep. 6(4): 348-53. PubMed Citation: 15776017

Ng, J., et al. (1997). Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein. Mol. Cell. Biol. 17(11): 6663-6672. PubMed Citation: 9343430

Ng, J., et al. (2000). A Drosophila ESC-E(Z) protein complex is distinct from other polycomb group complexes and contains covalently modified ESC. Mol. Cell. Biol. 20(9): 3069-78. PubMed Citation: 10757791

Ohno, K., et al. (2008). ESC, ESCL and their roles in Polycomb Group mechanisms. Mech. Dev. 125: 527-541. PubMed Citation: 18276122

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: 479-490. PubMed Citation: 9267028

Parrish, J. Z., Kim, M. D., Jan, L. Y. and Jan, Y. N. (2006). Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites. Genes Dev. 20(7): 820-35. Medline abstract: 16547170

Parrish, J. Z., et al. (2007). Polycomb genes interact with the tumor suppressor genes hippo and warts in the maintenance of Drosophila sensory neuron dendrites. Genes Dev. 21: 956-972. Medline abstract: 17437999

Peytavi, R., et al. (1999). HEED, the product of the human homolog of the murine eed gene, binds to the matrix protein of HIV-1. J. Biol. Chem. 274(3): 1635-45. 9880543

Plath, K., et al. (2003). Role of histone H3 lysine 27 methylation in X inactivation, Science 300: 131-135. 12649488

Rinn, J. L., et al. (2007). Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129(7): 1311-23. Medline abstract: 17604720

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

Sathe, S. S. and Harte, P. J. (1995a). The Drosophila extra sex combs protein contains WD motifs essential for its function as a repressor of homeotic genes. Mech. Dev. 52: 77-87. PubMed Citation: 7577677

Sathe, S. S. and & Harte, P. J. (1995b). The extra sex combs protein is highly conserved between Drosophila virilis and Drosophila melanogaster. Mech. Dev. 52: 225-232. PubMed Citation: 8541211

Satijn, D. P., et al. (2001). The Polycomb group protein EED interacts with YY1, and both proteins induce neural tissue in Xenopus embryos. Mol. Cell Biol. 21(4): 1360-1369. 11158321

Schmitges, F. W., et al. (2011). Histone methylation by PRC2 is inhibited by active chromatin marks. Mol. Cell 42(3): 330-41. PubMed Citation: 21549310

Schumacher, A., Faust, C. and Magnuson, T. (1996). Positional cloning of a global regulator of anterior-posterior patterning in mice. Nature 383: 250-253. PubMed Citation: 8805699

Schumacher, A., et al. (1998). The murine Polycomb-group gene eed and its human orthologue: functional implications of evolutionary conservation. Genomics 54(1): 79-88. 9806832

Sewalt, R. G., et al. (1998). Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes. Mol. Cell. Biol. 18(6): 3586-3595. PubMed Citation: 9584199

Shindo, N., Sakai, A., Arai, D., Matsuoka, O., Yamasaki, Y., and Higashinakagawa, T. (2005). The ESC-E(Z) complex participates in the hedgehog signaling pathway. Biochem. Biophys. Res. Commun. 327: 1179-1187. Medline abstract: 15652519

Siebold, A. P., et al. (2009). Polycomb repressive complex 2 and Trithorax modulate Drosophila longevity and stress resistance. Proc. Natl. Acad. Sci. 107(1): 169-74. PubMed Citation: 20018689

Siepel, A. et al. (2005). Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15: 1034-1050. Medline abstract: 16024819

Silva, J., et al. (2003). Establishment of Histone H3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes. Developmental Cell 4: 481-495. 12689588

Simon, J., et al. (1995). The extra sex combs product contains WD40 repeats and its time of action implies a role distinct from other Polycomb group products. Mech. Dev. 53: 197-208. PubMed Citation: 8562422

Struhl, G. and Brower, D. (1982). Early role of the esc+ gene product in the determination of segments in Drosophila. Cell 31: 285-92. PubMed Citation: 7159925

Struhl, G. (1983). Role of the esc+ gene product in ensuring the selective expression of segment-specific homeotic genes in Drosophila. J. Embryol. Exp. Morphol. 76: 297-331. PubMed Citation: 6631324

Tee, W. W., Shen, S. S., Oksuz, O., Narendra, V. and Reinberg, D. (2014). Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs. Cell 156: 678-690. PubMed ID: 24529373

Tie, F., Furuyama, T. and Harte, P. J. (1998). The Drosophila Polycomb Group proteins ESC and E(Z) bind directly to each other and co-localize at multiple chromosomal sites. Development 125(17): 3483-3496. PubMed Citation: 9693151

Tie, F., et al. (2001). The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development 128: 275-286. 11124122

Tie, F., Prasad-Sinha, J., Birve, A., Rasmuson-Lestander, A. and Harte, P. J. (2003). A 1-megadalton ESC/E(Z) complex from Drosophila that contains polycomblike and RPD3. Mol. Cell. Biol. 23(9): 3352-62. 12697833

Tie, F., Stratton, C. A., Kurzhals, R. L. and Harte, P. J. (2007). The N terminus of Drosophila ESC binds directly to histone H3 and is required for E(Z)-dependent trimethylation of H3 lysine 27. Mol. Cell. Biol. 27: 2014-2026. PubMed Citation: 17210640

van der Vlag, J. and Otte, A. P. (1999). Transcriptional repression mediated by the human polycomb-group protein EED involves histone deacetylation. Nat. Genet. 23(4): 474-8. PubMed Citation: 10581039

van Lohuizen, M., et al. (1998). Interaction of mouse polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate Pc-G complexes. Mol. Cell. Biol. 18(6): 3572-3579. PubMed Citation: 9584197

Wang, J., et al. (2000). Mouse homolog of the Drosophila Pc-G gene esc exerts a dominant negative effect in Drosophila. Genesis 26(1): 67-76. 10660674

Wang, L., et al. (2006). Alternative ESC and ESC-like subunits of a Polycomb group histone methyltransferase complex are differentially deployed during Drosophila development. Mol. Cell. Bio. 26: 2637-2647. 16537908

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

Xu, L., Fong, Y. and Strome, S. (2001). The Caenorhabditis elegans maternal-effect sterile proteins, MES-2, MES-3, and MES-6, are associated in a complex in embryos. Proc. Natl. Acad. Sci. 98: 5061-5066. 11320248

Yung, P. Y., Stuetzer, A., Fischle, W., Martinez, A. M. and Cavalli, G. (2015). Histone H3 Serine 28 is essential for efficient Polycomb-mediated gene repression in Drosophila. Cell Rep 11(9):1437-45. PubMed ID: 26004180

date revised: 23 July 2015

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

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