CTCF Genomic Binding Sites in Drosophila and the Organisation of the Bithorax Complex

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.

The chromatin insulator CTCF and the emergence of metazoan diversity

“…In D. melanogaster, CTCF confers insulator activity to the Fab-6 and Fab-8 chromatin boundaries within the Bithorax Hox complex (BX-C) (10,41). With the exception of Fab-7, all known and postulated boundaries within BX-C bind CTCF in vivo (28 transformations (31,42). Together, these findings illustrate the importance of CTCF for ordered Hox gene expression in D. melanogaster.…”

“…First, CTCF-binding sites should exist between individual genes of a Hox cluster. This property is well documented in both vertebrates and Drosophila (10,28,40,43,55,56), suggesting that it existed in the ancestor of protostomes and deuterostomes. Second, owing to the stability of the proposed kernel, one should find signs of conservation at least at some CTCF-binding sites.…”

“…One of these sites (no. 10) was positioned within the Fab-7 boundary, in accordance with previous publications (28,44). Next, we computed the conservation score of the remaining 10 sites, on the basis of genomic alignments of 12 Drosophila species with a sequenced genome (45).…”

“…Studies in vertebrates and Drosophila have implicated CTCF in the coordination of chromatin organization via thousands of binding sites at a genome-wide scale (15)(16)(17)27). To explore whether the presence of CTCF is associated with a similar enrichment of binding sites in other Bilateria, we scanned a broad range of bilaterian and control genomes (11 Bilateria; four early branching metazoans; one protist, fungus, and plant each) with known binding motifs for CTCF (16,28) and slightly modified, but corrupt versions thereof (SI Appendix, Fig. S5).…”

“…Two arguments justify this approach: a strong conservation in all bilaterian CTCFs of the amino acids responsible for target site recognition (SI Appendix, Fig. S3) (29) and the similarity of CTCF-binding motifs derived from Drosophila and diverse mammals (16,28,30) despite more than 500 My of divergence.…”