Rapid and reversible changes in nucleosome structure accompany the activation, repression, and superinduction of murine fibroblast protooncogenes c-fos and c-myc [ Communicated by Igor Tamm, April 13, 1987 (received for review February 10, 1987 ABSTRACT A procedure for the isolation of transcriptionally active nucleosomes was used to monitor changes in chromatin structure during the activation, repression, and superinduction of the protooncogenes c-fos and c-myc. Nuclei were isolated from murine fibroblasts at successive times after stimulation of quiescent cell cultures with serum or plateletderived growth factor. The nucleosomes released by a brief micrococcal nuclease digestion were fractionated by Hg1I-

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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 a-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 Hgagarose 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 Hgagarose 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 I1 by a-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 result...

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Unfolding of the nucleosomes in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3 and permits the selective recovery of the unfolded nucleosomes by mercury-affinity chromatography. This new technique has been used to compare the nucleosomal proteins and their postsynthetic modifications in the unfolded and the compactly beaded nucleosomes of HeLa cells in logarithmic growth, and at different stages of the growth cycle. The Hg-bound nucleosomes are shown to be deficient in replicating DNA sequences, but to remain associated with fragments of nascent RNA chains (or RNP particles) during gradient centrifugations. Both nucleosome fractions contain a full complement of "core" histones but differ with respect to postsynthetic modifications. The Hg-bound nucleosomes contain high levels of the tri- and tetra-acetylated forms of histones H3 and H4. The unbound nucleosomes are deficient in acetylated histones but enriched in phosphorylated H2A. In synchronized HeLa cells, histone H2A and H4 gene sequences occur in the Hg-bound nucleosomes during the S-phase when their transcription takes place, but not in the G2-phase when the genes are repressed.