BIOLOGICAL OVERVIEW

Polycomb Group proteins function as a molecular machine in gene silencing. Extra sex combs is unique among PcG proteins in that it is only expressed transiently and required only during the time when transition from transient to stable repression is occuring (Struhl, 1982). Other PcG proteins are available for a longer period during embryonic development. It is argued that PcG proteins cooperate in gene silencing. If so, what is there about the function of ESC that either allows or requires its shorter duration, compared with other PcG proteins?

Like other PcG proteins, ESC has no DNA binding motif. Instead, it holds five consensus WD motifs distributed throughout the protein. WD repeats function in protein interaction, and the presence of this motif implys a function for ESC in protein interaction and the assembly of a multi-protein complex involved in gene silencing. The yeast protein Tup1 carries seven copies of the WD repeat, and serves as a model for ESC function. Alpha 2 is a DNA-binding yeast protein that regulates specific genes of the cell mating type. Evidence suggests that Alpha 2 recruits the Tup 1 repressor via WD repeats. After recruitment, Tup 1 may act to provide at least partial repression of transcription (Komachi, 1994).

PcG proteins are required for stable long term transcriptional silencing of the homeotic genes. Among PcG genes, esc is unique in being critically required for establishment of PcG-mediated silencing during early embryogenesis, but not for its subsequent maintenance throughout development. Esc has been shown to be physically associated with the PcG protein Enhancer of Zeste [E(z)]. Esc, together with E(z), is present in a 600 kDa complex that is distinct from complexes containing other PcG proteins. This Esc complex has been purified and it also contains the histone deacetylase Rpd3 and the histone-binding protein p55 (Chromatin assembly factor 1 subunit), which is also a component of the chromatin remodeling complex NURF and the chromatin assembly complex CAF-1. The association of Esc and E(z) with p55 and Rpd3 is conserved in mammals. Rpd3 is required for silencing mediated by a Polycomb response element (PRE) in vivo and E(z) and Rpd3 are bound to the Ubx PRE in vivo, suggesting that they act directly at the PRE. It is proposed that histone deacetylation by this complex is a prerequisite for establishment of stable long-term silencing by other continuously required PcG complexes (Tie, 2001).

To test whether the association of Esc and E(z) with p55/Caf1 and Rpd3 has been conserved in mammals, the human complex containing the Esc homolog (EED) was examined for the presence of Rpd3 and p55 homologs. Database searches reveal that Drosophila Rpd3 is most closely related to two human histone deacetylases, HDAC1 and HDAC2 (77% and 75% identical to Rpd3). Similarly, there are two closely related p55 homologs in mammals, RbAp48 and RbAp46 (91% and 86% identical to p55). RbAp48 and RbAp46 have also been found together in the SIN3 and Mi-2 deacetylase complexes, as have HDAC1 and HDAC2. A test was performed to see whether all four proteins are associated with the human EED complex. A GST-ESC fusion protein encoding full-length Esc can pull down full-length in vitro translated Esc and a GST-ESC1-60 fusion protein encoding just the N-terminal 60 residues of Esc is sufficient to pull down full-length in vitro translated Esc. Similarly, GST-EED1-81, which contains the corresponding N-terminal region of EED, binds directly to in vitro translated EED. In addition to FLAG-Esc, GST-ESC1-60 also pulls down p55 and Rpd3 from Drosophila embryo nuclear extract. This strongly suggests that GST-ESC1- 60 specifically pulls down the Esc complex. GST-EED1-81 pulls down HDAC1, HDAC2 and RbAp48 from HeLa cell nuclear extract. RbAp46 has also been detected. Thus, the association of ESC with p55 and Rpd3 is mirrored in the conserved association of mammalian EED with RbAp48, RbAp46 and HDAC1 and HDAC2. These results confirm the previously reported association of EED with HDAC1 and HDAC2 (Tie, 2001).

The presence of p55 in the ESC complex provides a direct molecular link to chromatin. The highly conserved mammalian p55 homologs, RbAp48 and RbAp46, have been shown to bind directly to histone H4 and possibly H2A, but not H2B or H3. The N- and C-terminal regions of RbAp48 that mediate binding to histone H4 are virtually identical to the corresponding regions of Drosophila p55, strongly suggesting that p55 has the same histone-binding specificity (Tie, 2001).

What, then, is the role of p55 in the Esc complex? It is unlikely that p55 is responsible for the selective recruitment or targeting of Esc and E(Z) to the ~100 specific chromosomal sites at which they co-localize. The histone-binding activity of p55 does not, by itself, suggest a mechanism for such specificity and p55 binds to many more sites on the polytene chromosomes than Esc and E(Z), presumably reflecting its distribution in other complexes, such as CAF1 and NURF. It seems more likely that p55 acts after the Esc complex is recruited and serves to direct the deacetylase activity of Rpd3 to local histone substrates. This is analogous to the role proposed for RbAp46 in the heterodimeric HAT1 complex. RbAp46 greatly stimulates the acetyltransferase activity of the non-histone-binding HAT1 catalytic subunit, presumably by tethering it to its substrate via its histone-binding activity. Similarly, although recombinant Rpd3 can deacetylate histone H4 in vitro, free Rpd3 does not bind to H4 when the two are co-expressed in vivo and is unlikely to be able to deacetylate nucleosomal histones. This suggests that p55 may play a similar essential role in the Esc complex by targeting Rpd3 to histone substrates for deacetylation (Tie, 2001).

The presence of Rpd3 in the Esc complex suggests that histone deacetylation is an intrinsic activity of the Esc complex and that Rpd3 is required for PRE-mediated silencing. The related mammalian EED complex has been shown to contain the Rpd3 homologs HDAC1 and HDAC2, and immunoprecipitates containing this complex can deacetylate a histone H4 tail-peptide in vitro. In yeast, Rpd3-dependent repression in vivo has been shown to be associated with deacetylation of histones H4 and H3. Which nucleosomes would be deacetylated by the Esc complex? Histone deacetylation by yeast Rpd3 appears to be highly localized, extending only one or two nucleosomes from a site to which it is recruited. Since components of the Esc complex are physically associated with the Ubx PRE in vivo, Esc-mediated deacetylation may be restricted to nucleosomes comprising and immediately adjacent to PREs. Nucleosomes outside the PRE might also be targeted if the PRE has long-range interactions with the promoter or if the Esc complex itself also binds to the promoter or other regions outside the Ubx PRE, a possibility that the data presented here do not rule out. Although an effect is observed of several Rpd3 mutations on silencing of a PRE-mini-white reporter, which is an extremely sensitive assay, PcG phenotypes have not been reported for Rpd3 mutants. A hypomorphic Rpd3 allele associated with the insertion of a P-element transposon in the noncoding 5' untranslated region has been analyzed in the most detail. Homozygous mutant embryos derived from germline clones of this allele do not exhibit PcG phenotypes, but have a pair-rule phenotype similar to that of ftz mutants. Abundant ubiquitously distributed Rpd3 RNA and protein of maternal origin are detectable in early (0-2 hour) wild-type embryos, but are reduced no more than fivefold in these Rpd3 mutant embryos derived from germline clones. By stage 9-10, the level of maternally derived Rpd3 RNA and protein is greatly diminished. Localized zygotic expression of Rpd3 becomes detectable in the brain and ventral nervous system of wild-type embryos, but is not detectable in these mutant embryos, suggesting that this Rpd3 allele may have a stronger effect on zygotic expression than maternal expression. If Rpd3 protein derived from maternally synthesized RNA is sufficient to promote development of a normal cuticular phenotype, then it remains possible these mutant embryos may contain sufficient maternally derived protein to do so and that germline clones of a true null Rpd3 allele would display PcG phenotypes. Alternatively, it is possible that the function of Rpd3 in the Esc complex is not absolutely essential for Esc-dependent silencing or is redundant, i.e. when eliminated, it can be compensated by another histone deacetylase, either one normally associated with the Esc complex or a related one that can associate with the complex in the absence of Rpd3. A number of other histone deacetylases have been identified in Drosophila and at least two are reported to be ubiquitously distributed in the early embryo (Tie, 2001).

However, unlike mammals, which have two very closely related Rpd3 orthologs (HDAC1 and HDAC2), both of which are associated with mouse EED, the Drosophila genome contains no equally closely related homolog of Rpd3. The next most closely related Drosophila HDAC is an unequivocal ortholog of mammalian HDAC3, which is a class I HDAC like Rpd3. Interestingly, mouse HDAC3 has been reported to interact with the mouse Esc homolog EED in a yeast two-hybrid assay, consistent with the possibility that Rpd3 function in the Esc complex might be at least partially redundant. Further genetic analysis of Rpd3 should help to clarify its role in the Esc complex (Tie, 2001).

The 600 kDa Esc complex is distinct from complexes containing PC and other PcG proteins. This suggests that the Esc complex and other PcG complexes are likely to have separate functions. Furthermore, in embryos lacking any functional Esc protein, some weak residual Pc-dependent silencing activity is still detected, also supporting separate, if interdependent, functions. Similar conclusions have been drawn for the homologous mammalian PcG complexes, which have been reported to be expressed in temporally distinct stages of B cell differentiation, further suggesting they have distinct functions. In Drosophila, derepression of homeotic genes is detected slightly earlier in Esc mutants than in other PcG mutants, raising the possibility that Esc complex function might be required earlier than other PcG complexes. However, unlike the apparent temporal separation of the homologous complexes during mammalian B cell development, both Esc- and PC-containing complexes are present together throughout most of embryogenesis, before Esc disappears, and E(z), like other PcG proteins, is required continuously throughout development. The phenotypic similarities between Esc, E(z) and other PcG mutants, the genetic interactions among them and their common association with PREs, suggests that their functions, however distinct at the biochemical level, are interdependent (Tie, 2001).

What role might Esc-mediated histone deacetylation play in PcG silencing? Given the critical early requirement for Esc, Esc-mediated deacetylation of PRE-associated nucleosomes might be an essential prerequisite for the initial binding of one or more components of PRC1 or other PcG complexes to PREs. A schematic model is presented for such a function of the Esc complex in which Esc complex-mediated deacetylation of PRE associated histones is a critical step in establishing stable long-term PcG silencing. Alternatively, the Esc complex may be required for events subsequent to the initial binding of other PcG proteins to a PRE, perhaps for their assembly into active silencing complexes or for interaction of PRE-bound PcG complexes with the promoter. Indeed, repression of a reporter gene by a tethered GAL4-Pc fusion protein remains dependent on endogenous Esc and E(z) as well as other PcG proteins. This indicates that, at least for PC, constitutive binding to DNA does not bypass the requirement for Esc and E(z). This also suggests that while the biochemical evidence reveals no stable direct association of the Esc complex with other PcG complexes, it remains possible that there is a transient or less stable association in vivo that is essential for establishing PcG silencing (Tie, 2001).

The association of mammalian EED with the two closely related HDACs and two histone-binding proteins could reflect the existence of two separate EED complexes or some different functionality of the EED complex compared with the Esc complex. Consistent with this latter possibility, EED has recently been shown to be required after embryogenesis for aspects of adult hematopoietic development. Interestingly, analysis of the complete Drosophila genome sequence using the BLASTP and TBLASTN algorithms reveals that p55 has no other closely related Drosophila homologs, strongly suggesting that it is the functional counterpart of both RbAp48 and RbAp46 in Drosophila. Likewise, Rpd3 is the only Drosophila counterpart of mammalian HDAC1 and HDAC2. Given the remarkably high degree of similarity between RbAp48 and RbAp46 and HDAC1 and HDAC2, it is not yet clear whether each of these proteins has a distinct or redundant role in the EED complex. Perhaps this situation reflects a greater degree of functional specialization or versatility within the mammalian EED complexes. Since HDAC1 and HDAC2 have also been found together with RbAp48 and RbAp46 in other co-repressor complexes, it is also possible that the EED and Esc complexes represent specialized relatives of these complexes, perhaps more dedicated to a specific subset of genes (Tie, 2001).

GENE STRUCTURE

Exons - four

Bases in 3' UTR - 281

PROTEIN STRUCTURE

Amino Acids - 425

Structural Domains

The predicted ESC protein contains five copies of the WD motif and three other WD related sequences. The WD motif is found in G-protein beta subunits as well as non-G proteins involved in diverse cellular functions, including transcriptional repression. The sequence alterations of a number of esc mutations cause amino acid substitutions within the WD repeats, identifying them as essential for the function of the ESC protein as a repressor of homeotic gene expression. The WD motif is implicated in protein-protein interaction (Frei, 1985a and b, and Sathe, 1995a).

The Drosophila Extra sex combs (Esc) protein, a member of the Polycomb group (PcG), is a transcriptional repressor of homeotic genes. Genetic studies have shown that Esc protein is required in early embryos at about the time that other PcG proteins become engaged in homeotic gene repression. The Esc protein consists primarily of multiple copies of the WD repeat, a motif that has been implicated in protein-protein interaction. To further investigate the domain organization of Esc protein, esc homologs have been isolated and characterized from divergent insect species. Esc protein is highly conserved in housefly (72% identical to Drosophila Esc), butterfly (55% identical), and grasshopper (56% identical). The butterfly homolog provides Esc function in Drosophila, indicating that the sequence similarities reflect functional conservation. Homology modeling using the crystal structure of another WD repeat protein, the G-protein beta-subunit, predicts that Esc protein adopts a beta-propeller structure. The sequence comparisons and modeling suggest that there are seven WD repeats in Esc protein which together form a seven-bladed beta-propeller. Conserved regions in Esc protein have been located with respect to this predicted structure. Site-directed mutagenesis of specific loops, predicted to extend from the propeller surface, identifies conserved parts of Esc protein required for function in vivo. It is suggested that these regions might mediate physical interaction with Esc partner proteins (Ng, 1997).

date revised: 17 January 2001

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