Genome-Wide Mapping of Boundary Element-Associated Factor (BEAF) Binding Sites in Drosophila melanogaster Links BEAF to Transcription

Abstract

Insulator elements play a role in gene regulation that is potentially linked to nuclear organization. Boundary element-associated factors (BEAFs) 32A and 32B associate with hundreds of sites on Drosophila polytene chromosomes. We hybridized DNA isolated by chromatin immunoprecipitation to genome tiling microarrays to construct a genome-wide map of BEAF binding locations. A distinct difference in the association of 32A and 32B with chromatin was noted. We identified 1,820 BEAF peaks and found that more than 85% were less than 300 bp from transcription start sites. Half are between head-to-head gene pairs. BEAF-associated genes are transcriptionally active as judged by the presence of RNA polymerase II, dimethylated histone H3 K4, and the alternative histone H3.3. Forty percent of these genes are also associated with the polymerase negative elongation factor NELF. Like NELF-associated genes, most BEAF-associated genes are highly expressed. Using quantitative reverse transcription-PCR, we found that the expression levels of most BEAF-associated genes decrease in embryos and cultured cells lacking BEAF. These results provide an unexpected link between BEAF and transcription, suggesting that BEAF plays a role in maintaining most associated promoter regions in an environment that facilitates high transcription levels.Insulator elements participate in gene regulation by limiting potential interactions between promoters and regulatory elements. In transgene assays, an insulator can block enhancer-promoter interactions, but only when located between the enhancer and the promoter (21, 35). Similarly, insulators can block repression mediated by Polycomb group proteins (41). They can also protect bracketed transgenes from chromosomal position effects (36, 52). Because of these properties, insulators are thought to participate in genome organization and gene regulation by defining the boundaries of discrete regulatory domains (9,38,61,63). The mode of action of insulators is unclear but might involve acting as promoter decoys (20) or the formation of chromatin loops through interactions between insulators and perhaps also other nuclear substructures that remain to be biochemically defined (7,10,17,67).The scs and scsЈ elements from the 87A hsp70 heat shock locus were two of the first DNA sequences shown to have insulator activity (35,36,60). Two boundary element-associated factors (BEAFs), 32A and 32B, were identified based on their interaction with the scsЈ insulator element (but not the scs element) (29, 69). 32A and 32B are derived from the same gene and differ only by about 80 amino acids located at their amino termini. These unique regions harbor different atypical C 2 H 2 zinc finger DNA binding domains, termed BED fingers (3). BEAF binding sites are essential for scsЈ insulator activity (16), as is functional BEAF protein (22, 53). BEAF immunolocalizes to hundreds of sites on polytene chromosomes (69). Several of these genomic binding sites have been shown to have insulator activity (16), indicating that BEAF-dependen...

The BEAF-32 insulator coordinates genome organization and function during the evolution of Drosophila species

“…1B). This is consistent with previous reports suggesting that ;50% of BEAF-32-associated genes are arranged in a head-to-head orientation ( Jiang et al 2009). Based on the genomic distribution of BEAF-32 relative to genes, 50% is significantly greater than expected (P < 1 3 10 À4 )…”

“…BEAF-32 is the DNA-binding protein for one of these insulators with a role in the recruitment of other components to specific sites in the genome. In D. melanogaster, BEAF-32 associates preferentially with actively transcribed genes, although the specific mechanism by which it affects gene expression is not known (Bushey et al 2009;Jiang et al 2009). Here we identify the BEAF-32 insulator as a cis element located between head-to-head genes to attain differential regulation of transcription.…”

Dynamic changes in the genomic localization of DNA replication-related element binding factor during the cell cycle

“…[32][33][34][35] Analysis of the genome-wide distribution of these proteins has given some insights into the issue of whether the different Drosophila insulators play distinct or overlapping roles in genome organization and function. [36][37][38] For example, BEAF-32 is found more often close to gene promoters, where it appears to confer independent regulation to closely adjacent, divergently transcribed genes. 39 On the other hand, Su(Hw) tends to be present in intergenic regions next to genes transcribed at low levels.…”

Insulators form gene loops by interacting with promoters in Drosophila

“…The stalled promoters of the bithorax complex display insulator activity in embryos (Chopra et al, 2009). Many insulator proteins, such as CTCF, CP190, Mod(mdg4)-67.2 [Mod(mdg4) -FlyBase] and BEAF (BEAF-32), are frequently found bound to the promoters (Smith et al, 2009;Bartkuhn et al, 2009;Jiang et al, 2009;Bushey et al, 2009;Nègre et al, 2010).…”