Article

Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions

Department of Biology, Emory University, Atlanta, Georgia 30322, USA.
Genes & development (Impact Factor: 10.8). 06/2009; 23(11):1338-50. DOI: 10.1101/gad.1798209
Source: PubMed

ABSTRACT

Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila melanogaster genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). We performed chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP-chip) experiments with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines and found evidence that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression.

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    • "Recent genome-wide ChIP-chip studies provide evidence for an extensive overlap of the CP190 distribution pattern with dCTCF, BEAF-32 and Su(Hw) insulator proteins and the promoters of active genes (Bartkuhn et al. 2009; Bushey et al. 2009; Negre et al. 2010; Negre Cold Spring Harbor Laboratory Press on October 31, 2014 -Published by genome.cshlp.org Downloaded from et al. 2011; Schwartz et al. 2012; Soshnev et al. 2012). "
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    ABSTRACT: Insulators are multi-protein - DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.
    Full-text · Article · Oct 2014 · Genome Research
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    • "The generated heat maps highlighted a good correlation between DE genes upon Beaf32-KD and the presence of NFRs or of high nucleosome positioning signals in promoters or in gene bodies, respectively (Fig 1B, NFR/ " +1 " ), providing Beaf32 was bound to promoters (Supplementary Fig S2A). In agreement, high nucleosome positioning has been observed for highly active, housekeeping genes such as those regulated by Beaf32/DREF or dCTCF (Emberly et al, 2008; Bushey et al, 2009; Gilchrist et al, 2010). Beaf32-KD significantly affected nucleosome positioning in approximately 2,000 genes (Fig 1C, see " +1 " for " Beaf32KD " ), as evidenced by changes in MNase-Seq reads along their bodies upon Beaf32-KD compared to control cells (see Materials and Methods). "
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    ABSTRACT: Chromosomal domains in Drosophila are marked by the insulator-binding proteins (IBPs) dCTCF/Beaf32 and cofactors that participate in regulating long-range interactions. Chromosomal borders are further enriched in specific histone modifications, yet the role of histone modifiers and nucleosome dynamics in this context remains largely unknown. Here, we show that IBP depletion impairs nucleosome dynamics specifically at the promoters and coding sequence of genes flanked by IBP binding sites. Biochemical purification identifies the H3K36 histone methyltransferase NSD/dMes-4 as a novel IBP cofactor, which specifically co-regulates the chromatin accessibility of hundreds of genes flanked by dCTCF/Beaf32. NSD/dMes-4 presets chromatin before the recruitment of transcriptional activators including DREF that triggers Set2/Hypb-dependent H3K36 trimethylation, nucleosome positioning, and RNA splicing. Our results unveil a model for how IBPs regulate nucleosome dynamics and gene expression through NSD/dMes-4, which may regulate H3K27me3 spreading. Our data uncover how IBPs dynamically regulate chromatin organization depending on distinct cofactors.
    Preview · Article · Jun 2014 · The EMBO Journal
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    • "Insulators have been identified as another type of regulatory element that affects PRE activity, potentially by blocking the spreading of PcG proteins or H3K27me3 marks [102–104]. Genome-wide analysis shows that diverse insulator proteins such as Su(Hw), CP190 and dCTCF are broadly distributed throughout the genome [105,106]. Interestingly, PREs at many sites, including the Hox gene region, are flanked by insulator elements, suggesting that the flanking insulators protect neighbouring genes from inappropriate silencing by PREs as well as inappropriate activation by enhancers. The removal of insulator binding sites or the depletion of insulator proteins can result in lower H3K27me3 within these domains, but appears to have little effect on spreading beyond the borders, which might be expected if insulators were the sole causative agent [107]. "
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    ABSTRACT: Chromatin-binding proteins must navigate the complex nuclear milieu to find their sites of action, and a constellation of protein factors and other properties are likely to influence targeting specificity. Despite considerable progress, the precise rules by which binding specificity is achieved have remained elusive. Here, we consider early targeting events for two groups of chromatin-binding complexes in Drosophila: the Male-Specific Lethal (MSL) and the Polycomb group (PcG) complexes. These two serve as models for understanding targeting, because they have been extensively studied and play vital roles in Drosophila, and their targets have been documented at high resolution. Furthermore, the proteins and biochemical properties of both complexes are largely conserved in multicellular organisms, including humans. While the MSL complex increases gene expression and PcG members repress genes, the two groups share many similarities such as the ability to modify their chromatin environment to create active or repressive domains, respectively. With legacies of in-depth genetic, biochemical and now genomic approaches, the MSL and PcG complexes will continue to provide tractable systems for understanding the recruitment of multiprotein chromatin complexes to their target loci.
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