Molecular Genetic Analysis of Suppressor 2 of zeste Identifies Key Functional Domains

ArticleinGenetics 182(4):999-1013 · July 2009with6 Reads
DOI: 10.1534/genetics.108.097360 · Source: PubMed
The Su(z)2 complex contains Posterior sex combs (Psc) and Suppressor 2 of zeste [Su(z)2], two paralogous genes that likely arose by gene duplication. Psc encodes a Polycomb group protein that functions as a central component of the PRC1 complex, which maintains transcriptional repression of a wide array of genes. Although much is known about Psc, very little is known about Su(z)2, the analysis of which has been hampered by a dearth of alleles. We have generated new alleles of Su(z)2 and analyzed them at the genetic and molecular levels. Some of these alleles display negative complementation in that they cause lethality when heterozygous with the gain-of-function Su(z)2(1) allele but are hemizygous and, in some cases, homozygous viable. Interestingly, alleles of this class identify protein domains within Su(z)2 that are highly conserved in Psc and the mammalian Bmi-1 and Mel-18 proteins. We also find several domains of intrinsic disorder in the C-terminal regions of both Psc and Su(z)2 and suggest that these domains may contribute to the essential functions of both proteins.
    • "Together, these genetic analyses suggest that Su(z)2 is a strong suppressor of dE2f1-dsRNA phenotypes. We then sought to extend this observation by examining additional Su(z)2 alleles described recently (Emmons et al. 2009). We examined the capacity of Su(z)2 point mutant alleles (Su(z)2 s15 , Su(z)2 s20 , Su(z)2 s21 , Su(z)2 s36 , Su(z)2 s84 , Su(z)2 s95 , and Su(z)2 sM ) to suppress the dE2f1-dsRNA phenotypes. "
    [Show abstract] [Hide abstract] ABSTRACT: The E2F transcription factors are important regulators of the cell cycle whose function is commonly misregulated in cancer. To identify novel regulators of E2F1 activity in vivo, we used Drosophila to conduct genetic screens. For this, we generated transgenic lines that allow the tissue-specific depletion of dE2F1 by RNAi. Expression of these transgenes using Gal4 drivers in the eyes and wings generated reliable and modifiable phenotypes. We then conducted genetic screens testing the capacity of Exelixis deficiencies to modify these E2F1-RNAi phenotypes. From these screens, we identified mutant alleles of Suppressor of zeste 2 [Su(z)2] and multiple Polycomb group genes as strong suppressors of the E2F1-RNA interference phenotypes. In validation of our genetic data, we find that depleting Su(z)2 in cultured Drosophila cells restores the cell-proliferation defects caused by reduction of dE2F1 by elevating the level of dE2f1. Furthermore, analyses of methylation status of histone H3 lysine 27 (H3K27me) from the published modENCODE data sets suggest that the genomic regions harboring dE2f1 gene and certain dE2f1 target genes display H3K27me during development and in several Drosophila cell lines. These in vivo observations suggest that the Polycomb group may regulate cell proliferation by repressing the transcription of dE2f1 and certain dE2F1 target genes. This mechanism may play an important role in coordinating cellular differentiation and proliferation during Drosophila development.
    Full-text · Article · Dec 2012
    • "Some had complex structures but many are simple oblongs of the approximate size expected for a globular protein of 169 kDa (8–10 nm). We note that more than half of the sequence in PSC is predicted to be intrinsically disordered [46,47], so that it is interesting that the protein has a compact rather than extended conformation.Figure 1. PSC compacts and oligomerizes 4-nucleosome arrays. (a) Representative glycerol gradient purification of nucleosomal templates with and without PSC for EM and STEM. "
    [Show abstract] [Hide abstract] ABSTRACT: Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity of mechanisms used to modify chromatin architecture.
    Full-text · Article · Oct 2012
    • "and Dunker 2010). Natively unfolded regions occur in other chromatin architectural proteins, including the PcG proteins RYBP and GAGA factor, and have been proposed to play a role in the function of PSC and Su(z)2 (Agianian et al. 1999; Emmons et al. 2009; Lo et al. 2009; Neira et al. 2009). The linker histone regions required for chromatin compaction are intrinsically disordered in solution. "
    [Show abstract] [Hide abstract] ABSTRACT: Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.
    Article · Oct 2011
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