Genome wide ChIP-chip analyses reveal important roles for CTCF in Drosophila genome organization

The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.
Developmental Biology (Impact Factor: 3.55). 02/2009; 328(2):518-28. DOI: 10.1016/j.ydbio.2008.12.039
Source: PubMed


Insulators or chromatin boundary elements are defined by their ability to block transcriptional activation by an enhancer and to prevent the spread of active or silenced chromatin. Recent studies have increasingly suggested that insulator proteins play a role in large-scale genome organization. To better understand insulator function on the global scale, we conducted a genome-wide analysis of the binding sites for the insulator protein CTCF in Drosophila by Chromatin Immunoprecipitation (ChIP) followed by a tiling-array analysis. The analysis revealed CTCF binding to many known domain boundaries within the Abd-B gene of the BX-C including previously characterized Fab-8 and MCP insulators, and the Fab-6 region. Based on this finding, we characterized the Fab-6 insulator element. In genome-wide analysis, we found that dCTCF-binding sites are often situated between closely positioned gene promoters, consistent with the role of CTCF as an insulator protein. Importantly, CTCF tends to bind gene promoters just upstream of transcription start sites, in contrast to the predicted binding sites of the insulator protein Su(Hw). These findings suggest that CTCF plays more active roles in regulating gene activity and it functions differently from other insulator proteins in organizing the Drosophila genome.

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Available from: Victor V Lobanenkov
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    • "Su(Hw) belongs to Class II insulator proteins and, unlike Class I insulator proteins, is not associated with Pol II or promoters of active genes (48,50). Moreover, Su(Hw) tends to localize in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin according to color-coded classification (59). "
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    ABSTRACT: Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein.
    Full-text · Article · Apr 2013 · Nucleic Acids Research
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    • "Like insulator proteins, over half of AGO2-binding sites are located at promoters. Extensive promoter association has been reported for the insulator proteins CP190, CTCF, Mod(mdg4)2.2, and BEAF-32, but not Su(Hw) (Bushey et al. 2009; Jiang et al. 2009; Smith et al. 2009), with a preference for active promoters (Negre et al. 2010). Genome-wide, 61% of AGO2 sites in S2 cells are found within 250 base pairs (bp) upstream of a transcription start site (TSS), with a slight bias upstream of the TSS (Fig. 2C). "
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    ABSTRACT: A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
    Preview · Article · Aug 2011 · Genes & development
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    • "One of the ways to identify new boundaries may be via genome-wide binding studies of boundary interacting proteins. Such studies have been carried out recently (27–30), but all the binding sites identified may not reflect boundaries as these proteins are also known to have additional functions. In the current study we present a boundary search approach based on the logic that closely spaced genes that are differentially expressed must be insulated from the regulatory elements of one another and one of the mechanisms to achieve this may be the presence of a boundary element separating the domains of the two neighboring genes. "
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    ABSTRACT: Precise transcriptional control is dependent on specific interactions of a number of regulatory elements such as promoters, enhancers and silencers. Several studies indicate that the genome in higher eukaryotes is divided into chromatin domains with functional autonomy. Chromatin domain boundaries are a class of regulatory elements that restrict enhancers to interact with appropriate promoters and prevent misregulation of genes. While several boundary elements have been identified, a rational approach to search for such elements is lacking. With a view to identifying new chromatin domain boundary elements we analyzed genomic regions between closely spaced but differentially expressed genes of Drosophila melanogaster. We have identified a new boundary element between myoglianin and eyeless, ME boundary, that separates these two differentially expressed genes. ME boundary maps to a DNaseI hypersensitive site and acts as an enhancer blocker both in embryonic and adult stages in transgenic context. We also report that BEAF and GAF are the two major proteins responsible for the ME boundary function. Our studies demonstrate a rational approach to search for potential boundaries in genomic regions that are well annotated.
    Full-text · Article · May 2011 · Nucleic Acids Research
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