Newly synthesized histone H4 is deposited in a diacetylated isoform in a wide variety of organisms. In Tetrahymena a specific pair of residues, lysines 4 and 11, have been shown to undergo this modification in vivo. In this report, we demonstrate that the analogous residues, lysines 5 and 12, are acetylated in Drosophila and HeLa 114. These data strongly suggest that deposition-related acetylation sites in H4 have been highly, perhaps absolutely, conserved. In Tetrahymena and Drosophila newly synthesized histone H3 is also deposited in several modified forms. Using pulse-labeled H3 we have determined that, like H4, a specific, but distinct, subset of lysines is acetylated in these organisms. In Tetrahymena, lysines 9 and 14 are highly preferred sites of acetylation in new H3 while in Drosophila, lysines 14 and 23 are strongly preferred. No evidence has been obtained for acetylation of newly synthesized H3 in HeLa cells. Thus, although the pattern and sites of deposition-related acetylation appear to be highly conserved in H4, the same does not appear to be the case for histone H3.Modification of histones by acetylation of the E-amino group of specific lysine residues in the N-terminal domain of all four core histones is an active metabolic process whose exact function remains controversial. The primary focus of much current research is on how histone acetylation relates to the regulation of gene expression (1, 2). Less attention is being placed on understanding the biological function of depositionrelated acetylation, a reaction first described to affect specific histones during synthesis and deposition onto replicating chromatin (3,4).Biochemical analyses of deposition-related acetylation is hampered by the fact that only a fraction of the total histone is affected and once newly synthesized histone is deposited into nuclei, the pattern of acetylation is remodeled to fulfill transcription-related functions. In most systems, depositionrelated acetylation is witnessed only by administering a short pulse of label to preferentially label newly synthesized histones. Using this approach, numerous studies have reported that newly synthesized H4 is deposited as a modified isoform (3-9). This modification, although poorly understood, occurs in organisms ranging from protozoa to humans and thus appears to be highly conserved.In the ciliated protozoan Tetrahymena, deposition-related acetylation is particularly clear because of the separation between germ-line and somatic nuclei. Each vegetative cell contains a transcriptionally active, somatic macronucleus that governs the phenotype of the cell and a transcriptionally inert, germinal micronucleus that is responsible for genetic continuity. During conjugation, the sexual stage of the life cycle, essentially all of the newly synthesized H3 and H4 is selectively deposited into micronuclei; little if any new H3 or H4 is deposited into macronuclei (4). By stain and by label, histone extracted from these micronuclei is greatly enriched in diacetylated H4 and mono-and di...
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.
In a previous report [Annunziato, A.T. and Seale, R.L. (1983) J. Biol. Chem. 258:12675] a novel intermediate in chromatin assembly was described (detected by labeling new DNA in the presence of the deacetylase inhibitor sodium butyrate), which retained approximately 50% of the heightened sensitivity of newly replicated chromatin to DNaseI. It is now reported that nucleosomes replicated in butyrate are considerably more soluble in the presence of magnesium, relative to chromatin replicated under control conditions, and that this heightened magnesium-solubility is reflected in a concomitant increase in the preferential solubility of nucleosomes containing newly synthesized core histones. This differential solubility was accompanied by a 5- to 6-fold depletion of histone H1, and was completely abolished by the selective removal of H1 from isolated nuclei. The removal of H1 also markedly reduced the preferential DNaseI sensitivity of chromatin replicated in butyrate. Further, when mononucleosomes of control and (acetylated) nascent chromatin were compared, no differences in DNaseI sensitivity were detected. These results provide evidence that the interactions between newly assembled nucleosomes and histone H1 are altered when histone deacetylation is inhibited during chromatin replication, and suggest a mechanism for the control of H1 deposition during nucleosome assembly in vivo.
In the following report the relationship between histone methylation and histone acetylation has been examined in HeLa cells to better define the distribution of these two modifications. By labeling methylated histones in the presence or absence of sodium butyrate, we have found that the methylation of H3 is much more targeted to rapidly acetylated chromatin than is the methylation of H4, which largely involves the unacetylated subtype even in the presence of butyrate. Newly methylated H3 is highly likely to be complexed in nucleosomes that contain acetylated H4, as determined by immunoprecipitating radiolabeled chromatin with antibodies specific for acetylated H4 isoforms. In contrast, dynamically methylated H4 is underrepresented in acetylated chromatin, relative to newly methylated H3. The preferential methylation of acetylated H3 continues after pretreatment of cells with cycloheximide, indicating that not all acetylation-related methylation is associated with histone synthesis. This was confirmed by analyzing histone methylation in cells arrested at the G1/S boundary, in which histone synthesis was sharply lowered (relative to randomly cycling cells): under these conditions H3 methylation declined only approximately 4-fold, although ongoing methylation of H4 decreased approximately 20-fold. The continuing methylation of H3 in arrested cells included all H3 sequence variants, was selective for acetylated H3, and coincided with methyl group turnover that could not be ascribed to histone replacement synthesis. Most newly methylated H3 in arrested cells was complexed with acetylated H4 in chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.