Restriction enzymes permit quantitative determination of defined chromatin structures within the chromosome

Abstract

Open chromatin structures, operationally defined as nuclease-hypersensitive sites, are frequently found spanning the controlling regions of genes and they may ensure that trans-acting factors have ready access to their genomic substrates. The rapidity and extent of induction of a gene may be dependent on the probability that its promoter is folded into an open structure. We show that restriction enzymes can be used to estimate the probability that a given promoter region is contained within a defined structure in the chromosome. In the case of the Drasophila major heat-shock-protein gene, we show that an individual promoter element is folded in an accessible form in at least 75% of embryonic chromosomes. This efficient maintenance of the hypersensitive region may be a necessary precondition for a rapid heat-shock response.

The role of a positioned nucleosome at the Drosophila melanogaster hsp26 promoter.

“…The region between the two DH sites remains protected, indicating the presence of a nucleosome. XbaI restriction sites are located within the HSEs of these DH sites; consequently, the accessibility of these elements within nuclei can be assessed by the effectiveness of restriction enzyme cleavage at these sites (Jack et al, 1991;Lu et al, 1993a). The accessibility of the XbaI sites for transgene CarXRV is 101% (proximal XbaI site) and 99% (distal XbaI site) compared with CarX; the accessibility of the XbaI sites for CarXAV(Ri) is 86% (proximal XbaI site) and 81% (distal XbaI site) of CarX (Figures 2 and 4).…”

Position effect variegation in Drosophila is associated with an altered chromatin structure.

“…A nucleosome positioned specifically between the two DH sites (both before and after heat shock) may facilitate interaction between the two HSFs and possibly the GAGA factors, which are bound at either side of the nucleosome (Thomas and Elgin 1988). The use of restriction enzymes is an effective means of quantitatively measuring the accessibility of a given site on a chromosome within the nucleus (Fascher et al 1990;Jack and Eggert 1990;Archer et al 1991;Jack et al 1991;Lu et al 1993a;Verdin et al 1993;Schlossherr et al 1994). Nuclei were isolated from non-heat-shocked third-instar larvae and incubated with an excess amount of XbaI, which cleaves within the HSEs (Fig.…”

Nucleosome positioning and gene regulation

“…When DNA is associated with a histone octamer, a 10-1 1-bp periodic cleavage pattern, spanning approximately 140 bp, can be detected by high resolution analysis "011, 1977; for an example, see Thomas and Elgin, 19881. However, the production of such a cleavage pattern is insufficient by itself to infer the presence of a nucleosome, since the same cleavage pattern can also be generated by subnucleosomal components, even individual histones [Kerrigan and Kadonaga, 19921. Restriction enzymes cleave DNA in a sequencespecific fashion, and can be used to quantitate the accessibility of a particular site within isolated nuclei [Jack et al, 1991;Lu et al, 1992Lu et al, , 1993a. The degree of accessibility reflects features of the chromatin structure.…”

“…The degree of accessibility reflects features of the chromatin structure. Since the accessibility for restriction enzymes is high (50-80%) when the recognition sites are located in DH sites (nucleosome-free regions), and low ( 6 4 % ) when the recognition sites are associated with histone octamers, restriction enzymes are often used to map the borders of hypersensitive regions and the positions of nucleosomes [Jack et al, 1991;Straka and Horz, 1991;Reik et al, 19911. Restriction enzymes can also be used t o compare the accessibility of the same site in wild-type and mutant transgenes [e.g., see Straka and Horz, 1991, and Lu et al, 19921, or of a site in a given gene in different transcriptional states (repressed/induced) [e.g., see Jack et al, 19911.…”