Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix

Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the boundary element-associated factor (BEAF), which binds to the scs' boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. In this report we show that BEAF is associated with the nuclear matrix and map the domain required for matrix association to the middle region of the protein. This region contains a predicted coiled-coil domain with several potential sites for posttranslational modification. We demonstrate that the DNA sequences that bind to BEAF in vivo are also associated with the nuclear matrix and colocalize with BEAF. These results suggest that boundary elements may function by tethering chromatin to nuclear architectural components and thereby provide a structural basis for compartmentalization of the genome into functionally independent domains.     

Identification of a multicopy chromatin boundary element at the borders of silenced chromosomal domains

The insulating properties required to delimit higher-order chromosomal domains have been shown to be shared by a variety of chromatin boundary elements (BEs). Boundary elements have been described in several species, from yeast to human, and we have previously reported the existence of a class of chromatin BEs in Drosophila melanogaster whose insulating activity requires the DNA-binding protein BEAF (boundary element-associated factor). Here we focus on the characterization of a moderately repeated 1.2 kb DNA sequence that encompasses boundary element 28 (BE28). We show that it directionally blocks enhancer/promoter communication in transgenic flies. This sequence contains a BEAF-binding sequence juxtaposed to an AT-rich sequence that harbors a strong nuclease-hypersensitive site. Using a combination of DNA-protein and protein blotting techniques, we found that this region is recognized by the A+T-binding D1 non-histone chromosomal protein of D. melanogaster, and we provide evidence that D1 and BEAF physically interact. In addition, the multicopy BE28 element maps to pericentric regions of the D. melanogaster 2L, 2R and X chromosome arms to which D1 has been shown to localize. In yeast, BEs that mark the periphery of silenced chromosomal domains have recently been shown to block the spreading of heterochromatin assembly. We propose that the BE28 repeat clusters could fulfill a similar function, acting as a local boundary between hetero- and euchromatin in a process involving interactions between the BEAF and D1 proteins.     

Identification of a Class of Chromatin Boundary Elements

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.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 enhancer-blocking 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 scs′ 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/scs′ 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, the enhancer cannot activate the promoter with the intervening boundary element but can activate the other promoter. Thus, the positional functioning of boundary elements is distinct from the bidirectional repressive effect of silencer elements.The boundary activity of the gypsy-derived element is known to be mediated by the binding of the zinc finger protein su(Hw) to its reiterated binding sites (31). The su(Hw) protein has been studied in some detail, and regions involved in DNA binding, enhancer blocking, and interactions with mod(mdg4) have been identified (8, 13, 22). Interactions between the mod(mdg4) gene product and the su(Hw) protein are necessary for boundary function (9). In addition to loss of enhancer blocking, it has been suggested that some mod(mdg4) mutations lead to an unmasked activity that represses certain promoters (3).To address the boundary activity of scs′ at a biochemical level, we previously characterized two cDNAs encoding the related scs′ boundary element-associated factors BEAF-32A and -32B (14, 38). The BEAF activity in Drosophila nuclear extracts appears to be composed predominantly of trimers of one 32A and two 32B subunits. Interactions between BEAF subunits results in cooperative binding to the three CGATA motifs of the high-affinity binding site in scs′ which, in turn, facilitates binding to the lower-affinity binding site located some 200 bp away (14).Evidence of a role for BEAF in boundary activity derives from an enhancer-blocking assay in Drosophila D1 cells: seven tandem copies of a 48-bp oligonucleotide containing the scs′ high-affinity binding site had enhancer-blocking activity (although less than that obtained by using scs′), while point mutations that eliminated BEAF binding further reduced this activity (38). We immunolocalized BEAF to numerous interbands and puff boundaries on polytene chromosomes, suggesting the existence of a common class of boundary elements in Drosophila and that the band-interband structure of polytene chromosomes could be related to the localization of boundary elements.In this study, we isolated some of these genomic BEAF binding sites and used transgenic flies to demonstrate that the newly isolated sequences tested represent boundary elements. The only homology found between these candidate boundary elements (cBEs) and scs′ are clusters of CGATA motifs. Despite the varied spacing and orientations of the motifs in the different clusters, BEAF interacts with all of the clusters. We also used transgenic flies to directly establish the functional importance of BEAF binding sites by mutagenesis of CGATA motifs. This strongly indicates that the hundreds of BEAF binding sites in the Drosophila genome represent an abundant class of boundary elements, providing the first example of a class of binding elements that share a sequence motif and a binding protein.