Factors affecting nucleosome structure in transcriptionally active chromatin
Authors: BOFFA Lidia C. , WALKER Janis , CHEN Thelma A. , STERNER Richard , MARIANI Maria R. , ALLFREY Vincent G. 
Published: March 2005

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 nucleosomes than in the unbound nucleosomes. Although the overall levels of histone acetylation can be increased significantly by incubating HeLa cells in the presence of the deacetylase inhibitor, 5 mM sodium butyrate, this treatment has little if any effect on the total number of nucleosomes retained on the Hg‐agarose column. However, the ability of Hg‐agarose chromatography to detect localized changes in chromatin structure is evidenced by an 11‐fold increase in the Hg‐agarose binding of nucleosomes containing the DNA of the butyrate‐inducible alkaline phosphatase gene, compared to the Hg‐agarose‐bound nucleosomes of control cells. Although nascent RNA chains are present in the Hg‐agarose‐bound nucleosomes released by dithiothreitol, binding of the SH‐reactive nucleosomes to the Hg‐agarose column is not dependent on the presence of proteins associated with nascent RNA chains, since binding does not decrease following removal of the nascent transcripts by ribonuclease treatment.The inhibition of RNA polymerase II by α‐amanitin results in a loss of the affinity for Hg of nucleosomes containing c‐myc and histone H4 gene sequences, but there is no corresponding loss of Hg affinity with nucleosomes containing 28S ribosomal DNA sequences, which are transcribed by amanitin‐resistant RNA polymerase I.When RNA synthesis is inhibited by actinomycin D, which we find to intercalate preferentially into the DNA of transcriptionally active DNA sequences, there is no loss of Hg affinity of nucleosomes containing the histone H4 or c‐myc DNA sequences. The results indicate that the intercalation of actinomycin D into the DNA of transcribing chromatin can lock the nucleosomal transcription complex in the SH‐reactive configuration that is characteristic of the template‐active state.