Nucleosome exclusion from the interspecies-conserved central AT-rich region of the Ars insulator

The Ars insulator is a boundary element identified in the upstream region of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, and possesses the ability to both block enhancer-promoter communications and protect transgenes from silent chromatin. To understand the molecular mechanism of the Ars insulator, we investigated the correlation between chromatin structure, DNA structure and insulator activity. Nuclease digestion of nuclei isolated from sea urchin embryos revealed the presence of a nuclease-hypersensitive site within the Ars insulator. Analysis of micrococcal nuclease-sensitive sites in the Ars insulator, reconstituted with nucleosomes, showed the exclusion of nucleosomes from the central AT-rich region. Furthermore, the central AT-rich region in naked DNA was sensitive to nucleotide base modification by diethylpyrocarbonate (DEPC). These observations suggest that non-B-DNA structures in the central AT-rich region may inhibit nucleosomal formation, which leads to nuclease hypersensitivity. Furthermore, comparison of nucleotide sequences between the HpArs gene and its ortholog in Strongylocentrotus purpuratus revealed that the central AT-rich region of the Ars insulator is conserved, and this conserved region showed significant enhancer blocking activity. These results suggest that the central AT-rich nucleosome-free region plays an important role in the function of the Ars insulator.     

CTCF-dependent enhancer blockers at the upstream region of the chicken alpha-globin gene domain

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5′ flanking region of the chicken α-globin domain, 11–16 kb upstream of the embryonic α-type π gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two α-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific α-globin gene domain located in a permanently open chromatin area.     

The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.     

The 87A7 chromomere. Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains

The chromatin fiber of eukaryotic chromosomes is thought to be organized into a series of discrete domains or loops. To learn more about these large-scale structures, we have examined the sequence and chromatin organization of the DNA segments surrounding the two hsp 70 genes at the Drosophila melanogaster cytogenetic locus 87A7. These studies indicate that this heat shock locus is flanked on both the proximal and distal sides by novel chromatin structures, which we have called, respectively, scs and scs' (specialized chromatin structures). Each structure is defined by two sets of closely spaced nuclease-hypersensitive sites arranged around a central nuclease-resistant segment. Our findings suggest that these two structures define the proximal and distal boundaries of the 87A7 chromomere and, hence, may be one of the first examples of anchor points for the organization of eukaryotic chromosomes into a series of discrete higher order domains. Moreover, these structures may provide focal points both for the decondensation of the chromomere when the hsp 70 genes are induced by heat shock and for the subsequent rewinding and condensation of the chromomere during recovery from heat shock.     

SARs stimulate but do not confer position independent gene expression.

Two minimal scaffold-associated regions (SARs) from Drosophila were tested in stably transformed cells for their effects on the expression of reporter genes. The expression of genes bounded by two SARs is consistently stimulated by about 20- to 40-fold, if the average of a pool of cell transformants is analyzed. However, analysis of individual, stable cell transformants demonstrates that flanking SAR elements do not confer position-independent expression on the reporter gene and that the extent of position-dependent variegation is similarly large with or without the flanking SAR elements. The SAR stimulation of expression is observed in stable but not in transiently transfected cell lines. The Drosophila scs and scs' boundary elements, which do not bind to the nuclear matrix in vitro, are only about one-tenth as active as SARs in stimulating expression in stable transformants. Interestingly, the SAR stimulatory effect can be blocked by a fragment containing CpG islands (approximately 70% GC), if positioned between the SAR and the enhancer. In contrast, when inserted in the same position, control fragments, such as the scs/scs' elements, do not interfere with SAR function.     

A position-effect assay for boundaries of higher order chromosomal domains.

Eukaryotic chromosomes are thought to be organized into a series of discrete higher order chromatin domains. This organization is believed to be important not only in the compaction of the chromatin fiber, but also in the utilization of genetic information. Each domain would define an independent unit of gene activity, insulated from the regulatory influences of adjacent domains. Critical to this model of chromosome organization and function are the domain boundaries: the special nucleoprotein structures that delimit each higher order domain and segregate the chromosome into units of independent gene activity. In the work reported here we have tested whether two putative domain boundaries, scs and scs', from the Drosophila 87A7 heat shock locus can establish a domain of independent gene activity in vivo and insulate against chromosomal position effects.     

Properties of intracellular bovine papillomavirus chromatin.

Episomal nucleoprotein complexes of bovine papillomavirus type 1 (BPV-1) in transformed cells were exposed to DNase I treatment to localize hypersensitive regions. Such regions, which are indicative for gene expression, were found within the noncoding part of the genome, coinciding with the origin of replication and the 5' ends of most of the early mRNAs. However, there were also regions of hypersensitivity within the structural genes. These intragenic perturbations of the chromatin structure coincide with regulatory sequences at the DNA level. One of these regions maps in close proximity to a Z-DNA antibody-binding site which is located near the putative BPV-1 enhancer sequence.     

Characterization of a topoisomerase-like activity at specific hypersensitive sites in the Drosophila histone gene cluster

It is well known that treatment of DNA-topoisomerase complexes with SDS induces cleavage of the DNA by trapping a reactive intermediate in which the topoisomerase is covalently linked to the terminal phosphates of the cut DNA. I have used this technique to examine potential topoisomerase binding sites in the histone gene chromatin of Drosophila Kc cells. Treatment of Kc nuclei with SDS induces Mg++-dependent DNA cleavage near the borders of two nuclease-hypersensitive sites located 5' and 3' of histone H4. It is likely that the SDS-induced cleavage at these hypersensitive sites is due to a topoisomerase because protein becomes tightly bound to the ends of the cleaved DNA fragments. Preliminary experiments suggest that a type II topoisomerase may be responsible for the cleavage.