An Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A

A yeast gene has been identified that encodes a novel, evolutionarily conserved Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.     

Differential underacetylation of histones H2A,H3 and H4 on the inactive X chromosome in human female cells

It has previously been shown that the acetylated forms of histone H4 are depleted or absent in both constitutive, centric heterochromatin and in the facultative heterochromatin of the inactive X chromosome (Xi) in female cells. By immunostaining of metaphase chromosomes from human lymphocytes with antibodies to the acetylated isoforms of histones H2A and H3, we now show that these histones too are underacetylated in both Xi and centric heterochromatin. Xi shows two prominent regions of residual H3 acetylation, one encompassing the pseudoautosomal region at the end of the short arm and one at about Xg22. Both these regions have been shown previously to be sites of residual H4 acetylation. H2A acetylation on Xi is higher overall than that of H3 or H4 and is particularly high around the pseudoautosomal region, but not at Xg22. The results suggest that the acetylated isoforms of H3 and H4 have at least some effects on chromosomal structure and function that are not shared by acetylated H2A.     

Immunofluorescent study of the basic chromosomal protein set of green algae and Euglena]

Using antisera to fractions H1, H2a, H3 and H4 of the calf thymus histones, a comparative immunofluorescent investigation of these proteins in the nuclei of Chlamydomonas reinhardii, Haematococcus pluvialis, Dunaliella salina and Euglena gracilis was carried out. It has been shown that according to the immunofluorescent test, the nuclei of these algae contain proteins close to fractions H2a, H3 and H4 of the calf thymus histones. H1 fraction in these algae is either absent or can be considered as a protein immunochemically non-related to H1 fraction of the calf thymus histone. For quantitative evaluation (in units of the immunological distance) of the difference between histones of the algae and of the calf thymus in situ by indirect immunofluorescence, it was suggested to use the ultimate dilutions of antisera to histones. It was shown that the ultimate dilutions were correlated with titres of antisera in the reaction of microcomplement fixation. Such an approach and the data obtained are of interest for studying into the evolution of nucleosome histones in unicellular and multicellular eukaryotes.     

Acetylated histone H3 and H4 mark the upregulated LMP2A promoter of Epstein-Barr virus in lymphoid cells

We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap.     

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.     

Biochemical Characterization of Hpa2 and Hpa3, Two Small Closely Related Acetyltransferases from Saccharomyces cerevisiae

Based on their sequences, the Saccharomyces cerevisiae Hpa2 and Hpa3 proteins are annotated as two closely related members of the Gcn5 acetyltransferase family. Here, we describe the biochemical characterization of Hpa2 and Hpa3 as bona fide acetyltransferases with different substrate specificities. Mutational and MALDI-TOF analyses showed that Hpa3 translation initiates primarily from Met-19 rather than the annotated start site, Met-1, with a minor product starting at Met-27. When expressed in Escherichia coli and assayed in vitro, Hpa2 and Hpa3 (from Met-19) acetylated histones and polyamines. Whereas Hpa2 acetylated histones H3 and H4 (at H3 Lys-14, H4 Lys-5, and H4 Lys-12), Hpa3 acetylated only histone H4 (at Lys-8). Additionally, Hpa2, but not Hpa3, acetylated certain small basic proteins. Hpa3, but not Hpa2, has been reported to acetylate d-amino acids, and we present results consistent with that. Overexpression of Hpa2 or Hpa3 is toxic to yeast cells. However, their deletions do not show any standard phenotypic defects. These results suggest that Hpa2 and Hpa3 are similar but distinct acetyltransferases that might have overlapping roles with other known acetyltransferases in vivo in acetylating histones and other small proteins.     

Characterization of pea histone deacetylases

The present paper is the first report on histone deacetylases from plants. Three enzyme fractions with histone deacetylase activity (HD0, HD1 and HD2) have been partially purified from pea (Pisum sativum) embryonic axes. They deacetylate biologically acetylated chicken histones and, to a lesser extent, chemically acetylated histones, this being a criterion of their true histone deacetylase nature. The three enzymes are able to accept nucleosomes as substrates. HD1 is not inhibited by n-butyrate up to 50 mM, whereas HD0 and HD2 are only slightly inhibited, thereby establishing a clear difference to animal histone deacetylases. The three activities are inhibited by acetate, Cu²⁺ and Zn²⁺ ions and mercurials, but are only scarcely affected by polyamines, in strong contrast with yeast histone deacetylase. Several criteria have been used to obtain cumulative evidence that HD0, HD1 and HD2 actually are three distinct enzymes. In vitro experiments with free histones show that HD0 deacetylates all four core histones, whereas HD1 and HD2 show a clear preference for H2A and H2B, the arginine-rich histones being deacetylated more slowly.     

Nucleosome-histone acetyltransferase from rat liver chromatin

Histone acetyltransferase capable of acetylating histones in nucleosome was extracted from rat liver chromatin. The enzyme was partially purified by hydroxyapatite chromatography. The enzyme preparation did not contain histones and required acceptors for enzyme reaction. The enzyme catalyzed acetylation of nucleosome-histones and free histones but did not catalyzed acetylation of DNA-histone mixture. Autoradiographic analysis revealed that the H4 was dominantly acetylated and other nucleosomal histones were also acetylated.