Abstract
Insulator or enhancer-blocking elements are proposed to play an important role in the regulation of transcription by preventing inappropriate enhancer/promoter interaction. The zinc-finger protein CTCF is well studied in vertebrates as an enhancer blocking factor, but Drosophila CTCF has only been characterised recently. To date only one endogenous binding location for CTCF has been identified in the Drosophila genome, the Fab-8 insulator in the Abdominal-B locus in the Bithorax complex (BX-C). We carried out chromatin immunopurification coupled with genomic microarray analysis to identify CTCF binding sites within representative regions of the Drosophila genome, including the 3-Mb Adh region, the BX-C, and the Antennapedia complex. Location of in vivo CTCF binding within these regions enabled us to construct a robust CTCF binding-site consensus sequence. CTCF binding sites identified in the BX-C map precisely to the known insulator elements Mcp, Fab-6, and Fab-8. Other CTCF binding sites correlate with boundaries of regulatory domains allowing us to locate three additional presumptive insulator elements; “Fab-2,” “Fab-3,” and “Fab-4.” With the exception of Fab-7, our data indicate that CTCF is directly associated with all known or predicted insulators in the BX-C, suggesting that the functioning of these insulators involves a common CTCF-dependent mechanism. Comparison of the locations of the CTCF sites with characterised Polycomb target sites and histone modification provides support for the domain model of BX-C regulation.
Highlights
Insulator elements are DNA sequences that regulate interactions between promoters and enhancers
Previous investigations have identified independent regulatory domains that control the expression of Bithorax complex genes in different segments of the fly, the molecular nature of the domain boundaries is unclear
Our major result is that we find CTCF binding sites precisely located at the boundaries of these regulatory domains, giving a common molecular basis for these boundaries
Summary
Insulator elements are DNA sequences that regulate interactions between promoters and enhancers. By preventing inappropriate enhancer/promoter communication, insulators are believed to play a key role in the genomic organisation of transcriptional regulation. Their mode of action is still unclear but may involve the formation of chromatin loops that partition the genome into separate regulatory domains [1,2,3,4,5]. Almost all characterised insulator elements are associated with the binding of CTCF, a DNA-binding protein that contains multiple zinc fingers. CTCF was initially identified as both a transcriptional activator and repressor [6,7,8], it was subsequently recognised as being essential for the enhancer blocking activity of several vertebrate insulators [9]. Drosophila CTCF has been identified [13], joining other known Drosophila enhancer blocking proteins such as Su(Hw) [14], Zw5, and BEAF32 [15,16]
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