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


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 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.
    Genome Research 10/2014; 25(1). DOI:10.1101/gr.174169.114 · 14.63 Impact Factor
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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.
    Open Biology 03/2014; 4(3):140006. DOI:10.1098/rsob.140006 · 5.78 Impact Factor
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    • "Previous studies have shown a negative correlation between insulators and nucleosome occupancy19234647 and it has been suggested that insulator factors such as CTCF may possess chromatin remodelling features1920. In Drosophila, most of the CTCF target sites (as well as target sites of other insulator binding factors) are co-occupied by CP1902329. Sites that are co-bound by dCTCF/CP190 show low nucleosome occupancy, whereas sites bound by dCTCF alone show a regular nucleosomal occupancy22. Similarly, depletion of CP190 restores the dip in nucleosome occupancy at these regions suggesting that CP190 is the key player in disrupting the nucleosome organization22. "
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    ABSTRACT: Insulator mediated alteration in higher-order chromatin and/or nucleosome organization is an important aspect of epigenetic gene regulation. Recent studies have suggested a key role for CP190 in such processes. In this study, we analysed the effects of ectopically tethered insulator factors on chromatin structure and found that CP190 induces large-scale decondensation when targeted to a condensed lacO array in mammalian and Drosophila cells. In contrast, dCTCF alone, is unable to cause such a decondensation, however, when CP190 is present, dCTCF recruits it to the lacO array and mediates chromatin unfolding. The CP190 induced opening of chromatin may not be correlated with transcriptional activation, as binding of CP190 does not enhance luciferase activity in reporter assays. We propose that CP190 may mediate histone modification and chromatin remodelling activity to induce an open chromatin state by its direct recruitment or targeting by a DNA binding factor such as dCTCF.
    Scientific Reports 01/2014; 4:3917. DOI:10.1038/srep03917 · 5.58 Impact Factor
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