Isolation and characterization of acetylated histones H3 and H4 and their assembly into nucleosomes.
Nucleosome and chromatin structure/function relationships of histone acetylations are not understood. To address these questions we have developed chromatographic procedures that separate subtypes of H3 and the acetylated states of histone H3 and H4 in exceptionally pure forms. The sites of acetylation of the intermediately acetylated states of H3 have been determined and show a specific pattern of acetylation. An unexpected finding was the identification of a fifth site of acetylation in H3 at lysine 27. Nucleosome particles with fully acetylated H3 and H4 have been assembled on the Lytechinus variegatus 5 S rRNA DNA phasing sequence and characterized. These defined acetylated H3 and H4 particles migrate more slowly in polyacrylamide nucleoprotein particle gels than the control particles indicating a subtle effect of acetylation in nucleosome structure. However, DNA footprinting of these particles using DNase I show only small changes when compared to control particles over the core particle DNA length. It is shown further that H3 cysteines in the particle containing fully acetylated H3 and H4 were not accessible to iodoacetamide indicating that protein factors additional to H3 and H4 acetylation are required to make H3 cysteines accessible to the label. These findings are consistent with the proposal that histones H3, H4 acetylations exert their major effects outside of the core particle 146-base pair DNA, either on the DNA segment entering and leaving the nucleosome or possibly on the internucleosome interactions that involve the amino-terminal domains of the core histones in organization and stability of higher order chromatin structures.
Marvin
K W
KW
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Yau
P
P
Bradbury
E M
EM
D000107:Acetylation; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006657:Histones / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism*
D006657:Histones; D009707:Nucleosomes; D004247:DNA; D003850:Deoxyribonuclease I; D008239:Lysine; D003545:Cysteine; D007460:Iodoacetamide