Analysis of nucleosome assembly and histone exchange using antibodies specific for acetylated H4.
Using antibodies that specifically recognize the acetylated forms of histone H4, we show that it is possible to immunoprecipitate newly assembled (acetylated) nucleosomes. Newly replicated HeLa cell chromatin was labeled for 5-30 min with [3H]thymidine in the presence of sodium butyrate (thus inhibiting the deacetylation of newly deposited H4); bulk chromatin DNA was labeled for 24 h with [14C]thymidine. When soluble nucleosomes were incubated with immobilized antibodies, a comparison of the bound and unbound fractions showed up to a 65-fold enrichment for new chromatin DNA in the immunoprecipitate (bound), relative to the supernatant (unbound). No enrichment for new DNA was observed when preimmune control serum was used in a similar fashion. The enrichment for new DNA in the immunopellet was paralleled by a similar enrichment for all four newly synthesized histones. Acetylation was required for antibody recognition: When chromatin was replicated in the absence of butyrate (permitting histone deacetylation and chromatin maturation), equally low levels of new and old chromatin were immunoprecipitated, and no enrichment for new DNA was observed. Competition experiments confirmed these results. Analyses of histone deposition during the inhibition of DNA replication established that acetylated chromatin is the preferential target for H2A/H2B exchange. These experiments provide evidence for the highly selective assembly of newly synthesized H3, H2A, and H2B with acetylated H4, and for the involvement of histone acetylation in dynamic chromatin remodeling. In addition, immunoprecipitations of radiolabeled cytosolic extracts identified a possible somatic chromatin preassembly complex, containing newly synthesized H3 and new (acetylated) H4.
Perry
C A
CA
Department of Biology, Boston College, Chestnut Hill, Massachusetts 02167.
Dadd
C A
CA
Allis
C D
CD
Annunziato
A T
AT
D000107:Acetylation; D001667:Binding, Competitive; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D004996:Ethidium; D006367:HeLa Cells; D006657:Histones / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D009707:Nucleosomes / Q000378:metabolism*; D013194:Staining and Labeling
D002087:Butyrates; D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D020148:Butyric Acid; D004247:DNA; D004996:Ethidium