PARENTAL NUCLEOSOMES SEGREGATED TO NEWLY REPLICATED CHROMATIN ARE UNDERACETYLATED RELATIVE TO THOSE ASSEMBLED DE-NOVO

Antibodies specific for acetylated histone H4 were used to examine the acetylation state of parental histones that segregate to newly replicated DNA. To generate newly replicated chromatin containing only segregated parental nucleosomes, isolated nuclei were labeled with [H-3]TTP in vitro; alternatively, whole cells were labeled with [H-3]thymidine in the presence of cycloheximide. Soluble chromatin was prepared by micrococcal nuclease digestion, and subjected to immunoprecipitation with ''penta'' antibodies (Lin et al., 1989). In sharp contrast to nucleosomes containing newly synthesized, diacetylated H4 (Perry et al., 1993), chromatin replicated in vitro was only marginally susceptible to immunoprecipitation. Control experiments established that bona fide acetylated chromatin was selectively immunoprecipitated by the same techniques and that segregated nucleosomes were not disassembled during treatment with ''penta'' antibodies. When replication was coupled to an in vitro histone acetylation system, the enrichment for segregated nucleosomes in the immunopellet increased approximately 3-fold, demonstrating that changes in the acetylation state of segregated histones can be detected immunologically and that parental histones on new DNA are accessible to acetyltransferases during, or immediately after, DNA replication. In vivo pulse-chase experiments, performed in the presence of cycloheximide, confirmed these results. Uptake experiments further established that concurrent histone acetylation did not alter the rate of DNA synthesis in vitro. Our results provide evidence that replication-competent chromatin is not obligatorily acetylated, and indicate that the acetylation status of segregated histones may be maintained during chromatin replication. The possible significance of this, with respect to the regulation of chromatin higher order structures during DNA replication, and the propagation of transcriptionally active vs inactive chromatin structures, is discussed.