The scs′ Boundary Element: Characterization of Boundary Element-Associated Factors

Abstract

Boundary elements are thought to define the peripheries of chromatin domains and to restrict enhancerpromoter interactions to their target genes within their domains. We previously characterized a cDNA encoding the BEAF-32A protein (32A), which binds with high affinity to the scs boundary element from the Drosophila melanogaster 87A7 hsp70 locus. Here, we report a second protein, BEAF-32B, that differs from 32A only in its amino terminus. Unlike 32A, it has the same DNA binding specificity as the complete BEAF activity affinity purified from Drosophila. We characterize three domains in these proteins. Heterocomplex formation is mediated by their identical carboxy-terminal domains, and DNA binding is mediated by their unique amino-terminal domains. The identical middle domains of 32A and 32B are dispensable for the functions described here, although they may be important for boundary element function. 32A and 32B apparently form trimers, and the ratio of 32A to 32B varies at different loci on polytene chromosomes as judged by immunofluorescence. The scs element contains a high-and low-affinity binding site for BEAF. We observed that interaction with the low-affinity site is facilitated by binding to the high-affinity site some 200 bp distant.Much evidence suggests that chromatin is organized into independent functional units called domains (reviewed in reference 37). Functional independence requires restricting the activity of DNA elements such as enhancers to the domain in which they reside. For instance, proper temporal, developmental, and tissue-specific gene expression requires that enhancers activate only their target promoters, which might be tens of kilobases away, despite their ability to activate diverse promoters (22). Domains are presumably separated by special nucleoprotein structures, or boundary elements, that restrict interactions between DNA elements located in different domains (reviewed in references 7 and 38). Boundary elements, also called insulators, are likely to accomplish this through effects on higher-order chromatin structure, nuclear organization, or both. Boundary elements should be identifiable by two in vivo assays. First, when bracketing a transgene, they should shield the transgene from position effects caused by integration into different chromosomal environments (position-independent expression); and second, activation of a promoter by an enhancer should be blocked by an intervening boundary element since the enhancer and promoter should then be in separate domains (enhancer blocking). In addition, they should not act as enhancers and they should localize to domain edges.The best-characterized putative domain boundary elements are from Drosophila melanogaster. These are the scs and scsЈ special chromatin structures found at the proximal and distal boundaries of the 87A7 hsp70 puff of polytene chromosomes (8, 35) and a 340-bp fragment from the gypsy retrotransposon (12). Bracketing a gene by scs and scsЈ leads to position-independent expression of that gene, while enhancer blockin...

Parental Allele-Specific Chromatin Configuration in a Boundary–Imprinting-Control Element Upstream of the Mouse H19 Gene

“…For example, the "specialized chromatin structures" at the boundaries of the Drosophila hsp70 genes, which are associated with multiple DNase I HS sites, can insulate the white gene from both positive and negative chromosomal position effects and can insulate the hsp70 promoter from activation by enhancers (29,55). Specific peptides (BEAF-32A and BEAF-32B) are constitutively bound to these elements, and immunostaining localizes these factors to interband regions and puffs of polytene chromosomes, suggesting a role in the organization of chromosomal domains (25,60). Another well-characterized insulator is the Drosophila retrotransposon element gypsy, which inhibits the interaction of distal enhancers with the transcription complex but does not affect the action of enhancers positioned proximally (19).…”

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

“…The recognition motif for both proteins shares the sequence 5'-CGATA-3' but the reported DREF binding site contains three additional nucleotides to form the motif 5'-TATCGATA-3'. 8,43 Although DREF and BEAF-32 contain a BED Zn finger DNA binding domain expression, 19 whereas in other cases they may facilitate silencing. 21 Recent results using 5C and Hi-C suggest that chromosomes are organized into topologically associating domains (TADs) during interphase, and that insulator proteins are enriched at the boundaries between these domains.…”

The Drosophila Boundary Element-Associated Factors BEAF-32A and BEAF-32B Affect Chromatin Structure

“…The boundary element-associated factors BEAF-32A and BEAF-32B bind to the scs9 insulator as well as to hundreds of other sites on polytene chromosomes (Zhao et al 1995;Hart et al 1997). The BEAF-binding sites in scs9 are essential for its insulator activity, and other genomic BEAF-binding sites that have been tested also function as insulators (Cuvier et al 1998).…”

“…Thus BEAF-utilizing insulators are common in Drosophila. BEAF-32A and -32B are 32-kDa proteins derived from the same gene (Hart et al 1997). They differ at their amino termini, which have different BED finger DNAbinding domains (Aravind 2000).…”