Identification of a Class of Chromatin Boundary Elements

Abstract

Boundary elements are thought to define the ends of functionally independent domains of genetic activity. An assay for boundary activity based on this concept measures the ability to insulate a bracketed, chromosomally integrated reporter gene from position effects. Despite their presumed importance, the few examples identified to date apparently do not share sequence motifs or DNA binding proteins. The Drosophila protein BEAF binds the scs boundary element of the 87A7 hsp70 locus and roughly half of polytene chromosome interband loci. To see if these sites represent a class of boundary elements that have BEAF in common, we have isolated and studied several genomic BEAF binding sites as candidate boundary elements (cBEs). BEAF binds with high affinity to clustered, variably arranged CGATA motifs present in these cBEs. No other sequence homologies were found. Two cBEs were tested and found to confer position-independent expression on a miniwhite reporter gene in transgenic flies. Furthermore, point mutations in CGATA motifs that eliminate binding by BEAF also eliminate the ability to confer position-independent expression. Taken together, these findings suggest that clustered CGATA motifs are a hallmark of a BEAF-utilizing class of boundary elements found at many loci. This is the first example of a class of boundary elements that share a sequence motif and a binding protein.Chromatin appears to be partitioned into chromosomal domains that are operationally defined by bracketing DNA regions called boundary elements or insulators (10; see reference 34 for a review). Boundary elements are presumably necessary to curtail the potentially promiscuous behavior of enhancers, limiting their action to the domain in which they reside. The biological activity of a boundary element is experimentally measured by either position-independent expression or enhancerblocking assays. If this view of chromosomal organization is correct, boundary elements play a very important functional role. Yet only a few examples have been identified, and each is so far a unique case, as they do not appear to have notable sequence homologies or to have binding activities in common.The best-characterized boundary elements are the scs and scsЈ regions found to bracket the 87A7 hsp70 heat shock puff of Drosophila melanogaster polytene chromosomes (33) and a 340-bp fragment from the gypsy retrotransposon (11). The scs/scsЈ and the gypsy-derived elements have a boundary function in both of the assays mentioned above. They confer position-independent expression on a bracketed reporter gene by insulating the transgene from both activating and repressive effects at the site of chromosomal integration, and they block communication between a specific enhancer and promoter when interposed (20,21,31). It is important to note that boundary elements do not inactivate promoters or enhancers; they only block communication when interposed (2, 3, 21, 32). For instance, if an enhancer and boundary element are located between two divergently transcribed promoters, t...

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

“…We used two assays, a position-independent expression assay and an enhancer-blocking assay. For the position-independent expression assay, we generated transgenic fly lines in which the mini-white gene was bracketed by the M2 derivative of scs9 (on the 59 side) and scs (on the 39 side) (Cuvier et al 1998). The M2 insulator has two copies of the high-affinity BEAF-binding site present in scs9.…”

“…The bracketed mini-white gene is insulated from most chromosomal position effects; $90% of fly lines have yellow or light-orange eyes. In the absence of the 59 insulator, ,50% of fly lines have such light eye pigmentation (Cuvier et al 1998). Therefore if BID interferes with BEAF function, about half of the M2 lines will have darker eye pigmentation in the presence of BID expression.…”

“…BEAF binds to several hundred sites in euchromatin (Zhao et al 1995). Despite their largely distinct chromosomal distributions, BEAF and D1 likely interact at the bases of the X, 2L, and 2R chromosome arms, where several hundred dispersed copies of a sequence (BE28) that has both BEAF-and D1-binding sites are found (Cuvier et al 1998. The w m4h gene is located near the base of the X chromosome, and the 1.688 g/cm 3 satellite is a component of the pericentromeric heterochromatin in this region.…”

“…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.…”

Genomic Organization of gypsy Chromatin Insulators in Drosophila melanogaster

“…To investigate the possible role of Su(Hw) in chromatin organization, genomic Su(Hw)-binding sites were isolated following a modification of the procedure of Cuvier et al (1998). A c-myc-tagged Su(Hw) protein was translated in vitro using a rabbit reticulocyte lysate.…”