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Studies on nucleosomal histone acetylation and nonhistone chromosomal proteins in relation to chromatin structure

University of British Columbia

PART A - CHROMATIN SYNTHESIS Experiments are described that suggest micrococcal nuclease selectively excises from trout testis chromatin a population of mononucleo-somes containing increased levels of newly-synthesized histones and, possibly, newly-synthesized DNA. The results suggest that newly-synthesized chromatin has an increased sensitivity to micrococcal nuclease digestion. PART B - STRUCTURE OF TRANSCRIPTIONALLY ACTIVE CHROMATIN Different nucleases were used to probe the structure of transcriptionally active chromatin. Micrococcal nuclease and deoxyribonuclease II, enzymes which preferentially digest internucleosomal linker DNA, have both been used successfully to fractionate chromatin into transcriptionally active and inactive regions. Deoxyribonuclease I, an enzyme which digests both intra- and internucleosomal DNA, selectively destroys transcriptionally competent genes. After trout testis nuclei or chromatin was digested with one of the above nucleases, nucleosomes associated with the nuclease sensitive regions (transcriptionally active regions) were selectively eluted from the digested nuclei by the addition of low salt (0.1 M or 0.2 M NaCl); alternatively, nucleosomes were isolated from the digest products by virtue of their solubility in 0.1 M NaCl or 2 mM MgCl₂. The accumulated results suggest that nucleosomes containing highly acetylated histone H4 and normal levels of the acetylated.species of histones H3, H2A and H2B are associated with transcriptionally competent chromatin regions. Furthermore, the internucleosomal linker DNA associated with these regions contains low levels of histone Hi, and high levels of HMG-T1, HMG-T2 and HMG-T3, and lower levels of other nonhistone chromosomal proteins. The bulk of chromatin, which is transcriptionally inactive, is associated with nucleosomes containing low levels of acetylated H4 (i.e. unacetylated and monoacetylated H4 are the major species), and the internucleosomal linker DNA is associated mainly with histone H1. The role of H4 acetylation in transcriptionally competent chromatin may be to reduce interactions between core particles and thus render the extended chromatin region accessible to RNA polymerases. Conversely, unacetylated H4 may be involved in maintaining higher levels of compaction of the chromatin. PART C - EFFECT OF SODIUM N-BUTYRATE ON HISTONE ACETYLATION The biochemical mechanisms underlying the increased acetylation of histones found in butyrate - treated tissue culture cells were investigated. The results clearly indicate that the increased acetylation of histone in vivo is most probably due to an inhibition of deacetylase enzyme activity caused by butyrate. This inhibition is entirely reversible and appears to be a general phenomenon, since butyrate increases levels of acetylated H3 and H4 in all tissue culture cell types (Xenopus laevis embryonic cells (X58), rat ascites cells (IRC8), mouse fibroblasts (3T3), baby hamster kidney cells (BHK) and Friend erythroleukemic cells) studied.

Text

2010-03-15

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http://hdl.handle.net/2429/21885

Biochemistry and Molecular Biology

University of British Columbia

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