Specific antibodies reveal ordered and cell-cycle-related use of histone-H4 acetylation sites in mammalian cells

Antibodies specific for the acetylated forms of histone H4 (H4) were produced in rabbits with a synthetic peptide corresponding to the 18 N-terminal residues of tetra-acetylated H4 (i.e. with acetyllysine at positions 5, 8, 12 and 16). Specificity was determined by inhibition assays using four additional peptides, each acetylated at only a single site. Using an antiserum (R6) specific for the acetylation site at Lys-5 we have estimated the proportion of Lys-5 sites acetylated in the mono-, di- and tri-acetylated forms of H4 from randomly growing human HL-60 cells. The values obtained (7%, 29% and 61% respectively) differ from those expected if acetylation were random (i.e. 25%, 50% and 75%) or if site usage followed a set order for all H4 molecules (i.e. a jump from 0% to 100%). Antibodies from a second animal (R5) bound preferentially to peptides acetylated at Lys-12 and also bound to mono-acetylated H4 relatively weakly in several cell types. In contrast, mono-acetylated H4 from metaphase HeLa cells labelled more strongly with both antisera, indicating significant acetylation at Lys-5 and Lys-12. We conclude that (1) the sites at Lys-5 and Lys-12 are under-used in mono-acetylated H4 from a variety of mammalian cell types and Lys-8 and/or Lys-16 are therefore the first to be acetylated, (2) more than one order of site usage is possible and (3) there is a metaphase-specific shift in site usage. These results suggest that H4 acetylation plays a role in the modulation of chromatin structure in mammalian cells.     

Histone H4 acetylation analyses in patients with polysomy X: implications for the mechanism of X inactivation

In humans, it is thought that the X-inactivation phenomenon occurs no matter how many X chromosomes are present, and that only one of them remains active. Nevertheless, individuals who have an abnormal number of X chromosomes show a wide spectrum of abnormalities, which increase with the number of X chromosomes present in a given individual. It has been shown that the inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, and that this could be used as an accessible marker for distinguishing between Xi and Xa in spreads of metaphase chromosomes. We studied three X-polysomic patients for the presence of active chromatin by analysis of histone H4 acetylation on unfixed metaphase spreads. Using antisera to H4 acetylated at lysines 16, 8 and 5, respectively, we observed frequencies different from those expected from cells with only one underacetylated X chromosome. In particular, when antiserum to H4 acetylated at lysine 16 was used about 90% of the cells showed acetylation of all X chromosomes. This suggests a possible disturbance in the deacetylation process, probably due to the presence of multiple Xs. Received: 25 April 1997 / Accepted: 15 March 1998     

Dynamic histone acetylation in alfalfa cells. Butyrate interference with acetate labeling

Dynamic histone acetylation of alfalfa (Medicago sativa) was studied in suspension cultures by short-term labeling with radioactive acetate. The relative labeling rates for the acetylated histones were in order of decreasing incorporation; H3.2 greater than H3.1 greater than H4 greater than H2B.1 greater than H2A.3. Histone H3 showed at least seven sites of acetylation, histone H2B.1 had six sites and histone H4 had five sites. Low numbers of acetylation sites were observed for histone H2B.2 and all histone H2A variants. The mass ratio, steady state acetylation and dynamic acetylation between major variant H3.1 and minor variant H3.2 were approx. 2:1, 1:2 and 2:5, respectively. Treatment of alfalfa cells with 50 mM n-butyrate did not lead to histone hyperacetylation, but instead interfered with histone acetylation labeling by acetate. The extent of apparent inhibition increased with time and concentration of butyrate. It is likely that the conversion of butyrate to acetylCoA results in dilution of the specific radioactivity of [3H]acetate in the acetylCoA pool thereby inhibiting the labeling reaction. This interpretation is supported by 14C-labeling of alfalfa acetylated histones by [1-14C]butyrate.     

Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry

The acetylation isoforms of histone H4 from butyrate-treated HeLa cells were separated by C(4) reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a "zip" model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is di-methylated in all modified isoforms, as well as the non-acetylated isoform of H4.     

Euchromatic domains in plant chromosomes as revealed by H4 histone acetylation and early DNA replication.

Using specific polyclonal antisera raised against acetylated isoforms of histone H4, we have analyzed their distribution in the dioecious plant Silene latifolia (syn. Melandrium album) possessing heteromorphic sex chromosomes. Our previous studies on this species have shown that one of the two X chromosomes in homogametic female cells is heavily methylated and late replicating, as a possible consequence of dosage compensation. Here we report that there are no detectable differences in intensity and distribution of H4 acetylation between these two X chromosomes. In S. latifolia only distal-subtelomeric chromosome regions, on both the sex chromosomes and autosomes, display strong signals of H4 acetylation at N-terminal lysines 5, 8, and 12. These acetylated domains correspond to the very early replicating distal chromosome regions as revealed by 5-bromodeoxyuridine pulses followed by the indirect immunofluorescence microscopy. The distribution of H4 acetylated at lysine 16 was uniform along the chromosomes. The unique distal-subtelomeric H4 acetylation signals were also observed in three other Silene species (S. vulgaris, S. pendula, and S. chalcedonica), but not in two non-related plant species tested (Allium cepa and Nicotiana tobacum). The presented data as well as our recent studies on the structure of S. latifolia chromosome ends indicate that Silene species possess the specific distal-subtelomeric location of euchromatin, gene-rich regions on chromosomes.     

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.     

Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes

Unfixed metaphase chromosome preparations from human lymphocyte cultures were immunofluorescently labelled using antibodies to defined histone epitopes. Both mouse monoclonal antibody HBC-7, raised against the N-terminal region of H2B, and rabbit serum R5/12, which recognizes H4 acetylated at Lys-12, gave non-uniform labelling patterns, whereas control antibodies against total histone fractions H4 and H1 produced homogeneous fluorescence. HBC-7 bound approximately uniformly to the bulk of the chromosomes, but the major heterochromatic domains of chromosomes 1, 9, 15, 16 and the Y showed significantly brighter fluorescence. Serum R5/12 indicated an overall reduction in acetylation of H4 in metaphase chromosomes compared with interphase nuclei, although some specific chromosomal locations had considerably elevated acetylation levels. Acetylation levels in the major heterochromatic domains appeared extremely low. To investigate further the differences noted in heterochromatin labelling, metaphases from cultures grown in the presence of various agents known to induce undercondensation of the major heterochromatic domains were similarly immunolabelled. Decondensed heterochromatin no longer exhibited higher than normal immunofluorescence levels with HBC-7. The higher resolution afforded by stretching the centromeric heterochromatin of chromosomes 1, 9 and 16 confirmed the low level of H4 acetylation in these domains. We consider the implications of these observations in relation to chromatin conformation and activity.by W.C. Earnshaw     

Histone H4 Acetylation and Replication Timing in Chinese Hamster Chromosomes

The distribution of acetylated isoforms of histone H4 along Chinese hamster chromosomes has been studied by immunostaining with antibodies recognizing H4 acetylated at defined lysines in its N-terminal domain. The heterochromatic long arm of the X chromosome in both female (CHO) and male (DON) cell lines is underacetylated at three out of four lysines (5, 8, and 12). In contrast, the level of acetylation at lysine 16, which is the first to be acetylated in mammals, was similar in X chromosomes and autosomes. Labeling of the cells with bromodeoxyuridine (BrdU) to mark late-replicating chromosome domains, followed by double immunostaining with antibodies to BrdU and acetylated H4, showed a close, though not perfect, correlation between late replication and low levels of H4 acetylation. The results show that levels of histone acetylation are associated with the replication timing of defined domains on both the X chromosome and autosomes, but the exceptions we observe suggest that this link is not absolute or essential.     

Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer

The mechanism of DNA hypermethylation-associated tumor suppressor gene silencing in cancer remains incompletely understood. Here, we show by chromatin immunoprecipitation that for three genes (P16, MLH1, and the O(6)-methylguanine-DNA methyltransferase gene, MGMT), histone H3 Lys-9 methylation directly correlates and histone H3 Lys-9 acetylation inversely correlates with DNA methylation in three neoplastic cell lines. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) resulted in moderately increased Lys-9 acetylation at silenced loci with no effect on Lys-9 methylation and minimal effects on gene expression. By contrast, treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza-dC) rapidly reduced Lys-9 methylation at silenced loci and resulted in reactivation for all three genes. Combined treatment with 5Aza-dC and TSA was synergistic in reactivating gene expression through simultaneous effects on Lys-9 methylation and acetylation, which resulted in a robust increase in the ratio of Lys-9 acetylated and methylated histones at loci showing dense DNA methylation. By contrast to Lys-9, histone H3 Lys-4 methylation inversely correlated with promoter DNA methylation, was not affected by TSA, and was increased moderately at silenced loci by 5Aza-dC. Our results suggest that reduced H3 Lys-4 methylation and increased H3 Lys-9 methylation play a critical role in the maintenance of promoter DNA methylation-associated gene silencing in colorectal cancer.     

Duplication and maintenance of heterochromatin domains

To investigate the mechanisms that assure the maintenance of heterochromatin regions, we took advantage of the fact that clusters of heterochromatin DNA replicate late in S phase and are processed in discrete foci with a characteristic nuclear distribution. At the light microscopy level, within these entities, we followed DNA synthesis, histone H4 acetylation, heterochromatin protein 1 (Hp1alpha and -beta), and chromatin assembly factor 1 (CAF-1). During replication, Hp1alpha and -beta domains of concentration are stably maintained, whereas heterochromatin regions are enriched in both CAF-1 and replication-specific acetylated isoforms of histone H4 (H4Ac 5 and 12). We defined a time window of 20 min for the maintenance of this state. Furthermore, treatment with Trichostatin A (TSA), during and after replication, sustains the H4Ac 5 and 12 state in heterochromatin excluding H4Ac 8 and 16. In comparison, early replication foci, at the same level, did not display any specific enrichment in H4Ac 5 and 12. These data emphasize the specific importance for heterochromatin of the replication-associated H4 isoforms. We propose that perpetuation of heterochromatin involves self-maintenance factors, including local concentration of Hp1alpha and -beta, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1.     

Properties of the type B histone acetyltransferase Hat1: H4 tail interaction, site preference, and involvement in DNA repair

The Hat1 histone acetyltransferase catalyzes the acetylation of H4 at lysines 5 and 12, the same sites that are acetylated in newly synthesized histone H4. By performing histone acetyltransferase (HAT) assays on various synthetic H4 N-terminal peptides, we have examined the interactions between Hat1 and the H4 tail domain. It was found that acetylation requires the presence of positively charged amino acids at positions 8 and 16 of H4, positions that are normally occupied by lysine; however, lysine per se is not essential and can be replaced by arginine. In contrast, replacing Lys-8 and -16 of H4 with glutamines reduces acetylation to background levels. Similarly, phosphorylation of Ser-1 of the H4 tail depresses acetylation by both yeast Hat1p and the human HAT-B complex. These results strongly support the model proposed by Ramakrishnan and colleagues for the interaction between Hat1 and the H4 tail (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427-438) and may have implications for the genetic analysis of histone acetylation. It was also found that Lys-12 of H4 is preferentially acetylated by human HAT-B, in further agreement with the proposed model of H4 tail binding. Finally, we have demonstrated that deletion of the hat1 gene from the fission yeast Schizosaccharomyces pombe causes increased sensitivity to the DNA-damaging agent methyl methanesulfonate in the absence of any additional mutations. This is in contrast to results obtained with a Saccharomyces cerevisiae hat1Delta strain, which must also carry mutations of the acetylatable lysines of H3 for heightened methyl methanesulfonate sensitivity to be observed. Thus, although the role of Hat1 in DNA damage repair is evolutionarily conserved, the ability of H3 acetylation to compensate for Hat1 deletion appears to be more variable.     

Differential acetylation of histone H4 lysine during development of in vitro fertilized,cloned and parthenogenetically activated bovine embryos

The oocyte is remarkable in its ability to remodel parental genomes following fertilization and to reprogram somatic nuclei after nuclear transfer (NT). To characterise the patterns of histone H4 acetylation and DNA methylation during development of bovine gametogenesis and embryogenesis, specific antibodies for histone H4 acetylated at lysine 5 (K5), K8, K12 and K16 residues and for methylated cytosine of CpG dinucleotides were used. Oocytes and sperm lacked the staining for histone acetylation, when DNA methylation staining was intense. In IVF zygotes, both pronuclei were transiently hyper-acetylated. However, the male pronucleus was faster in acquiring acetylated histones, and concurrently it was rapidly demethylated. Both pronuclei were equally acetylated during the S to G2-phase transition, while methylation staining was only still observed in the female pronucleus. In parthenogenetically activated oocytes, acetylation of the female pronucleus was enriched faster, while DNA remained methylated. A transient de-acetylation was observed in NT embryos reconstructed using a non-activated ooplast of a metaphase second arrested oocyte. Remarkably, the intensity of acetylation staining of most H4 lysine residues peaked at the 8-cell stage in IVF embryos, which coincided with zygotic genome activation and with lowest DNA methylation staining. At the blastocyst stage, trophectodermal cells of IVF and parthenogenetic embryos generally demonstrated more intense staining for most acetylated H4 lysine, whilst ICM cells stained very weakly. In contrast methylation of the DNA stained more intensely in ICM. NT blastocysts showed differential acetylation of blastomeres but not methylation. The inverse association of histone lysine acetylation and DNA methylation at different vital embryo stages suggests a mechanistically significant relationship. The complexities of these epigenetic interactions are discussed.     

Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3.

Heterochromatin at yeast telomeres and silent mating (HM) loci represses adjacent genes and is formed by the binding and spreading of silencing information regulators (SIR proteins) along histones. This involves the interaction between the C terminus of SIR3 and the N terminus of histone H4. Since H4 is hypoacetylated in heterochromatin we wished to determine whether acetylation is involved in regulating the contacts between SIR3 and H4. Binding of H4 peptide (residues 1-34) acetylated at lysines Lys-5, Lys-8, Lys-12, and Lys-16 to an immobilized SIR3 protein fragment (residues 510-970) was investigated using surface plasmon resonance. We find that acetylation of H4 lysines reduces binding (K(a)) of H4 to SIR3 in a cumulative manner so that the fully acetylated peptide binding is decreased approximately 50-fold relative to unacetylated peptide. Thus, by affecting SIR3-H4 binding, acetylation may regulate the formation of heterochromatin. These data help explain the hypoacetylated state of histone H4 in heterochromatin of eukaryotes.     

Butyrate-induced histone hyperacetylation in human and mouse cells: estimation of putative sites of histone acetylation in vivo

Human and mouse cells in culture were treated with various concentrations of sodium butyrate. Acid-extracted histones of control and butyrate-treated cells were analyzed by two-dimensional gel electrophoresis. All core histones of the control cells contained modified forms. All core histones of the butyrate-treated cells were hyperacetylated. Depending on the number of acetylation sites per molecule, each histone or histone variant exhibited a characteristic number of acetylated forms. This number was the same for each histone common in human and mouse cells treated with butyrate. Histones 2A.1, 2A.2, and 2A.X have two sites of inner acetylation; 2A.Z has 3; 2B's have 5; and each one of the H3 variants as well as H4 have 4.     

Histone acetylation in chicken erythrocytes. Rates of deacetylation in immature and mature red blood cells.

We analysed the rates of histone deacetylation in chicken mature and immature red blood cells. A multiplicity of deacetylation rates was observed for the histones and these rates may be subdivided into two major categories based on the extent of histone acetylation. In one set of experiments, cells were labelled with [3H]acetate in the presence of the deacetylase inhibitor n-butyrate, thereby accumulating radiolabel in the hyperacetylated forms of the histone. These hyperacetylated forms are deacetylated rapidly. [3H]Acetate-labelled tetra-acetylated H4 (H4Ac4) in mature cells was deacetylated with an initial half-life (t1/2) of approximately 5 min (time required for the removal of one-half of the labelled acetyl groups). In immature cells, all [3H]acetate-labelled H4Ac4 was deacetylated with a t1/2 of approximately 5 min. Erythrocytes were also labelled with [3H]acetate for extended periods in the absence of the deacetylase inhibitor. During this period, radiolabel accumulated predominantly in the mono- and di-acetylated forms of the histone. Using this protocol, the rate of deacetylation of H4Ac1 was observed to be approximately 145 min for mature cells, and approximately 90 min for immature cells, demonstrating that the less extensively acetylated histone is deacetylated slowly. These results are discussed in the context of the rates of histone acetylation in chicken red blood cells described in the companion paper [Zhang & Nelson (1988) Biochem. J. 250, 233-240].     

Reversible effects of Na-butyrate on histone acetylation

Histones were labeled by incubating HeLa cells in the presence of radioactive leucine for 20 hours. Following a 5 hour chase in non-radioactive medium the cells were exposed to 7 mM Na-butyrate to increase the level of histone acetylation. Histones were then extracted, fractionated by high-resolution electrophoresis in acetic acid-urea gels and the specific activity of the parental form of H4 histone and that of each acetylated form was calculated. No differences were found in the specific activities indicating that the major effect of butyrate on histone acetyl levels involves histones which were synthesized before the administration of butyrate. The effect is reversible and within 15 minutes after the removal of the drug most of the acetylated forms of H4 histone are converted to the unmodified form.