The effect of salt extraction on the structure of transcriptionally active genes; evidence for a DNAseI-sensitive structure which could be dependent on chromatin structure at levels higher than the 30 nm fibre

Abstract

The procedure developed by Lawson and Cole (Biochemistry, 1979, 18 2161-2166) for removing lysine-rich histones from nuclei at low pH also quantitatively extracts proteins HMG14 and 17. The effect of this low pH extraction on the DNAseI-sensitive structures of active genes in avian red blood cells has been investigated. No major perturbation of a developmentally regulated DNAseI hypersensitive site in the beta-globin domain and at the 5' end of the alpha D gene was seen. The overall DNAseI-sensitive conformation of the beta A-globin gene (relative to the ovalbumin gene) is minimally affected by pH3 salt extraction, but there is some loss of sensitivity of the alpha D gene. Removal of HMG proteins at neutral pH had no effect on the sensitivity of active genes in erythroid or fibroblast nuclei. These results, together with those carried out on DNAseI sensitivity and HMG binding to monomer nucleosomes, indicate that there is a major structural feature of active genes responsible for DNAseI-sensitivity which is independent of HMG proteins or nucleosome core particle structure but may be dependent on higher order chromatin structures.

A Complex Chromatin Landscape Revealed by Patterns of Nuclease Sensitivity and Histone Modification within the Mouse β-Globin Locus

“…A long-standing assumption has been that nuclease sensitivity is the result of a more open or decondensed chromatin structure, but to date no direct molecular evidence supports this. Studies using limited trypsin digestion (38) or extraction under high salt conditions (21) suggested that chromatin higher-order structure, or at least higher-order structure dependent upon linker histones or high-mobility-group proteins, was not related to nuclease sensitivity. While our results show that histone acetylation and H3 K4 dimethylation may also be ruled out, other histone modifications that we have not tested might better correlate with the nuclease-sensitive structure; the list of known covalent histone modifications is already very long and continues to grow.…”

Locating mammalian transcription factor binding sites: A survey of computational and experimental techniques

“…Generalized nuclease sensitivity is a property inherent in all actively expressed genes (Gazit and Cedar 1980), correlating closely with the presence of acetylated histones. Whether a cause or an effect, the presence of acetylated histones accompanies the appearance of open chromatin extending over 10s to 100s of kilobases and is documented in the chicken lysozyme, chicken ovalbumin, human apolipoprotein B, c-fos, and chicken ␤-globin loci (Lawson et al 1982;Goodwin et al 1985;Jantzen et al 1986;Feng and Villeponteau 1992;Hebbes et al 1994). In contrast, hypersensitivity refers to regions showing extreme sensitivity to the cleavage effects of the enzyme that are localized to short stretches of DNA ranging from 100 to 400 bp in length (Gross and Garrard 1988).…”

Transcription-induced nucleosome ‘splitting’: an underlying structure for DNase I sensitive chromatin.

“…Neither over-expressing topoisomerase I nor pre-nicking DNA with MNase prior to DNase I digestion of nuclei reverses the split nucleosomal structures (data not shown), indicating that maintenance of the structures does not require torsional stress. Nevertheless, DNase I sensitivity and half-nucleosomal cutting patterns have proven difficult to preserve upon mononucleosome isolation (Lohr and Van Holde, 1979;Goodwin et al, 1985). The insoluble chromatin associated with active genes may be composed of a split nucleosome structure (see Xu et al, 1986).…”