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 mini-white 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.

FIG. 1

Isolation of new genomic BEAF binding sites. (A) DNA fractions enriched in BEAF binding sites were prepared from fragmented genomic DNA by an enrichment procedure as outlined schematically in this panel. oligos, oligonucleotides. (B) The relative enrichment for BEAF binding fragments was assayed by competition gel shift analysis. The samples contained a fixed amount of BEAF to titrate about half of the radiolabeled scs′ D subfragment and two levels of competitor DNA (500 and 50 ng) obtained after each enrichment cycle, as indicated. IP, immunoprecipitation. (C) Some features of the scs′ boundary element are indicated. The arrowheads refer to the CGATA motifs (∗ for motifs mutated to CTCGA), the black bar indicates the nuclease-resitant core, and the open bars represent the nuclease-hypersensitive regions. The positions of the D and M subfragments are indicated. The dimerized MM boundary construct and its mutant derivative M*M* are also schematically represented.

FIG. 2

The selected cBEs share CGATA motifs and contain high-affinity sites for BEAF. (A) The position and arrangement of the CGATA motifs (arrowheads) are shown for the scs′ fragment and the new cBEs cBE76, -28, and -51. The shaded boxes highlight the sequences shown in panel B. (B) The highest sequence homologies found among the various cBEs and scs′ correspond to CGATA clusters. Only the bases shared by at least half of the sequences are shaded. The CGATA motifs are indicated by arrows above the sequences. The positions of the point mutations introduced into the scs′ D site are marked with asterisks. (C) The isolated cBEs contain high-affinity binding sites for BEAF. Affinity-purified BEAF was added in steps increased by a factor of 3. From the relative intensities of the shifted probes, we estimated that BEAF binds the cBEs as well as or better than the high-affinity D site of scs′.

FIG. 3

Footprint analysis of cBEs: protected regions and hypersensitive sites. Footprint analyses of cBE76 (A), cBE28 (B), and cBE51 (C) were done by using increasing amounts of either affinity-purified BEAF or bacterially expressed 32B protein, as indicated. Induced hypersensitive sites (hs) are marked. Boxed regions indicate the DNase I footprints, and the arrowheads indicate the CGATA motifs.

FIG. 4

cBEs do not activate transcription, and cBE76 does not block upstream enhancers in transient expression assays. Different CAT reporter constructs were assayed by transient transfection into Drosophila D1 cells. (A) Upstream activating sequence assay. Relative CAT activities obtained when the indicated sequences were inserted upstream of the minimal 227-bp hsp27 CAT reporter gene. Ecdysone or heat shock induction of a 1.2-kb hsp27 promoter (B), which contains the ERE and HSE, served as the positive control. (B) The enhancer-blocking capacities of cBE76 (both orientations), scs′, and a 200-bp fragment containing a Gal4 binding site were tested. These fragments were inserted into the 1,200-bp hsp27 promoter between the promoter and the upstream HSE and ERE as shown. Relative CAT activity obtained following treatment with ecdysone (ecd) or heat shock (h.s.) or without treatment (−) is shown.

FIG. 5

Two hypersensitive sites are present in the 5′ region of the IMPdH raspberry gene, one of which overlaps cBE76. Nuclei isolated from Kc cells were treated with DNase I for different lengths of time. The isolated DNA was subjected to indirect end labelling with a PvuII-XhoI fragment from the IMPdH gene as a probe (shown in the map on the right). A major hypersensitive site (hatched box) localizes to the cBE76 fragment (filled box). A second hypersensitive site is located 200 bp closer to the IMPDH gene. Lane EcoRI contained genomic DNA cut with PvuII and EcoRI (which cuts within cBE76) as a size standard.

FIG. 6

cBEs confer position-independent expression on a mini-white reporter gene, and point mutations in a BEAF binding site abolish this activity. Young (∼48-h-old), heterozygous females are shown, each representing one independent transgenic fly line obtained by P-element-mediated transformation with the indicated mini-white constructs. (A) Mini-white gene without bracketing elements. (B) The 990-bp scs PvuII fragment was inserted 3′ of the mini-white gene. (C through G) Derived from B by inserting the following DNA sequences 5′ of the mini-white gene: C, 515-bp scs′ fragment; D, cBE76; E, cBE28; F, scs′-derivative MM dimerized fragment; G, scs′-derivative M*M* dimerized fragment. MM consists of a 227-bp fragment containing the scs′ D (high-affinity) site as a dimer such that the spacing between BEAF binding sites is the same as that found in scs′ for the B (low-affinity) and D sites, and the M*M* sequence differs only in having point mutations in all CGATA motifs to eliminate binding by BEAF.