Affinity chromatographic purification of nucleosomes containing transcriptionally active DNA sequences.
The unfolding of nucleosome cores in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3, making them accessible to SH-reagents. This has suggested that nucleosomes from active genes could be retained selectively on organomercurial/agarose columns. When nucleosomes released from rat liver nuclei by limited digestion with micrococcal nuclease were passed through an Hg affinity column, a run-off fraction of compact, beaded nucleosomes was separated from a retained nucleosome fraction. Although both contained monomer-length DNA and a full complement of core histones, histones in the retained fraction were hyperacetylated. Dot blot hybridizations showed the Hg-bound nucleosome fraction to be enriched in DNA sequences transcribed by hepatocytes (serum albumin and transferrin genes), while a brain-specific gene (preproenkephalin) was not retained, but appeared in the nucleosomes of the run-off fraction. The results are discussed in light of other evidence linking hyperacetylation of histones H3 and H4 to conformational changes at the middle of the nucleosome core.
Allegra
P
P
Laboratory of Cell Biology, Rockefeller University, New York, N.Y. 10021.
Sterner
R
R
Clayton
D F
DF
Allfrey
V G
VG
D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D004247:DNA / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000032:analysis; D009693:Nucleic Acid Hybridization; D009707:Nucleosomes / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic
D006657:Histones; D009707:Nucleosomes; D004247:DNA