Factors affecting nucleosome structure in transcriptionally active chromatin

作者：Lidia C. Boffa, Janis WALKER, Thelma A. Chen, Richard Sterner, Maria Rita Mariani, Vincent G. Allfrey · 发表于：European Journal of Biochemistry · 年份：1990 · DOI：10.1111/j.1432-1033.1990.tb19474.x · 被引用次数：52 · 研究领域：Genomics and Chromatin Dynamics、RNA and protein synthesis mechanisms、RNA Interference and Gene Delivery

The nucleosomes of transcriptionally active genes can be separated from those of inactive genes by affinity chromatography on organomercury‐agarose (Hg‐agarose) columns. The basis for this separation is the difference in accessibility of the sulfhydryl groups of histone H3 and certain non‐histone proteins in active and inactive chromatin. A new procedure distinguishing between different modes of binding of transcriptionally active nucleosomes to the Hg‐agarose column has been applied to study several factors which might influence the binding reaction. Nucleosomes that bind to the column because of salt‐labile associations with SH‐reactive non‐histone proteins, such as the high‐mobility‐group proteins, HMG‐1 and HMG‐2, were released by adding 0.5 M NaCl to the eluting buffer. The remaining nucleosomes, in which reactive histone H3 thiol groups can bind covalently to the organomercury, were then displaced from the column by 10 mM dithiothreitol. Both Hg‐agarose‐bound fractions contain the transcriptionally active DNA sequences of the cell, but inactive nucleosomes, such as those containing α‐globin DNA, pass through the column. The histones of both Hg‐agarose‐bound fractions have significantly higher levels of acetylation than do histones of the unbound fraction, but the content of tri‐ and tetra‐acetylated H3 and H4 is significantly higher in the nucleosomes with reactive H3 thiols. The rate of turnover of histone N‐acetyl groups is also far greater in the Hg‐agarose‐bound nuc...