IDENTIFICATION OF A CLASS OF CHROMATIN BOUNDARY ELEMENTS

Boundary elements are thought to define the ends of functionally indep endent domains of genetic activity. An assay for boundary activity bas ed on this concept measures the ability to insulate a bracketed, chrom o somally integrated reporter gene from position effects. Despite thei r presumed importance, the few examples identified to date apparently do not share sequence motifs or DNA binding proteins. The Drosophila p rotein BEAF binds the scs' boundary element of the 87A7 hsp70 locus an d 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 ca ndidate boundary elements (cBEs). BEAF binds with high affinity to clu stered, variably arranged CGATA motifs present in these cBEs. No other sequence homologies were found. Two cBEs were tested and found to con fer position-independent expression on a mini-white reporter gene in t ransgenic flies, Furthermore, point mutations in CGATA motifs that eli minate binding by BEAF also eliminate the ability to confer position-i ndependent expression. Taken together, these findings suggest that clu stered CGATA motifs are a hallmark of a BEAF-utilizing class of bounda ry elements found at many loci. This is the first example of a class o f boundary elements that share a sequence motif and a binding protein.