The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association

Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na(+), K(+), Mg(2+), Ca(2+), cobalt-hexammine(3+), spermidine(3+) and spermine(4+). Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K(+), (as well as Rb(+) and Cs(+)) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K(+) (and Rb(+)/Cs(+)) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome-nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.     

Comprehensive analysis of dynamics of histone H4 acetylation in mitotic barley cells

Nucleosomal histones are covalently modified at specific amino acid residues. In the case of histone H4, four lysines (K5, K8, K12, and K16) are acetylated. In the current studies, we examined the dynamics of histone H4 acetylation at K8 and K12 in mitotic barley cells using a three-dimensional immunofluorescent method. Based on the results and previous studies on the dynamics of K5 and K16 acetylation, we provide a comprehensive view of the dynamics of H4 acetylation. Interphase nuclei exhibit strong acetylation in the centromeric region at K5, K8 and K12. In the case of K12, strong acetylation at nucleolar organizing regions was observed from prophase to anaphase. The dynamics of K12 were closely related to those of K5. On the other hand, K8 exhibited a pattern of almost uniform acetylation from prophase to telophase and strong acetylation in distal regions of chromosomes at both metaphase and anaphase, which is very similar to the dynamics of K16 acetylation. Thus, it appears that there is pair-wise acetylation of K12 and K5 in the nucleolar organizing regions and of K8 and K16 in the gene-rich regions. Together, these results suggest that pair-wise dynamics of H4 acetylation regulate chromosomal structure and function during the cell cycle.     

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.     

Effects of acetylation of histone H4 at lysines 8 and 16 on activity of the Hat1 histone acetyltransferase

During nucleosome assembly in vivo, newly synthesized histone H4 is specifically diacetylated on lysines 5 and 12 within the H4 NH(2)-terminal tail domain. The highly conserved "K5/K12" deposition pattern of acetylation is thought to be generated by the Hat1 histone acetyltransferase, which in vivo is found in the HAT-B complex. In the following report, the activity and substrate specificity of the human HAT-B complex and of recombinant yeast Hat1p have been examined, using synthetic H4 NH(2)-terminal peptides as substrates. As expected, the unacetylated H4 peptide was a good substrate for acetylation by yeast Hat1p and human HAT-B, while the K5/K12-diacetylated peptide was not significantly acetylated. Notably, an H4 peptide previously diacetylated on lysines 8 and 16 was a very poor substrate for acetylation by either yeast Hat1p or human HAT-B. Treating the K8/K16-diacetylated peptide with histone deacetylase prior to the HAT-B reaction raised acetylation at K5/K12 to 70-80% of control levels. These results present strong support for the model of H4-Hat1p interaction proposed by Dutnall et al. (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427-438) and provide evidence for the first time that site-specific acetylation of histones can regulate the acetylation of other substrate sites.     

Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1.     

Identification of five sites of acetylation in alfalfa histone H4.

Radioactive acetylation in vivo of plant histone H4 of alfalfa, Arabidopsis, tobacco, and carrot revealed five distinct forms of radioactive, acetylated histone. In histone H4 of eukaryotes ranging from fungi to man, acetylation is restricted to four lysines (residues 5, 8, 12, and 16) possibly caused by a quantitative methylation of lysine-20. Chemical and proteolytic fragmentation of the amino terminally blocked alfalfa H4 protein, dynamically acetylated by radioactive acetate in vivo, allowed protein sequencing and identification of selected peptides. Peptide identification was facilitated by analyzing fully characterized calf histone H4 in parallel. Acetylation in vivo of alfalfa histone H4 was restricted to the lysines in the amino-terminal domain of the protein, residues 1-23. Lysine-20 was shown to be free of methylation, as in pea histone H4. This apparently makes lysine-20 accessible as a novel target for histone acetylation. The in vivo pattern of lysine acetylation (16 greater than 12 greater than 8 greater than or equal to 5 = 20) revealed a preference for lysines -16 and -12 without an apparent strict sequential specificity of acetylation.     

Histone H4 acetylation in human cells. Frequency of acetylation at different sites defined by immunolabeling with site-specific antibodies

Histone H4 can be reversibly acetylated at lysine residues 5, 8, 12 and 16. It is possible that acetylation of individual residues will exert specific effects on chromatin function, but this hypothesis is difficult to test with present techniques for analysis of acetylation. To address this problem, we have prepared antibodies which distinguish H4 molecules acetylated at each of the sites used in vivo. By electrophoresis and immunolabeling we have shown that, in H4 from human cells, the four lysine residues are acetylated in a preferred, but not exclusive order, namely lysine 16, followed by 12 and 8, followed by 5.     

PARP1 ADP-ribosylates lysine residues of the core histone tails

The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tails.     

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.     

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.     

Structural cooperativity in histone H3 tail modifications

Post-translational modifications of histone H3 tails have crucial roles in regulation of cellular processes. There is cross-regulation between the modifications of K4, K9, and K14 residues. The modifications on these residues drastically promote or inhibit each other. In this work, we studied the structural changes of the histone H3 tail originating from the three most important modifications; tri-methylation of K4 and K9, and acetylation of K14. We performed extensive molecular dynamics simulations of four types of H3 tails: (i) the unmodified H3 tail having no chemical modification on the residues, (ii) the tri-methylated lysine 4 and lysine 9 H3 tail (K4me3K9me3), (iii) the tri-methylated lysine 4 and acetylated lysine 14 H3 tail (K4me3K14ace), and (iv) tri-methylated lysine 9 and acetylated lysine 14 H3 tail (K9me3K14ace). Here, we report the effects of K4, K9, and K14 modifications on the backbone torsion angles and relate these changes to the recognition and binding of histone modifying enzymes. According to the Ramachandran plot analysis; (i) the dihedral angles of K4 residue are significantly affected by the addition of three methyl groups on this residue regardless of the second modification, (ii) the dihedral angle values of K9 residue are similarly altered majorly by the tri-methylation of K4 residue, (iii) different combinations of modifications (tri-methylation of K4 and K9, and acetylation of K14) have different influences on phi and psi values of K14 residue. Finally, we discuss the consequences of these results on the binding modes and specificity of the histone modifying enzymes such as DIM-5, GCN5, and JMJD2A.     

Histone H4 from cuttlefish testis is sequentially acetylated. Comparison with acetylation of calf thymus histone H4

The differently acetylated subfractions of histone H4 isolated from cuttlefish testis and from calf thymus were separated by ion exchange chromatography on sulfopropyl-Sephadex, using a shallow linear gradient of guanidine hydrochloride in the presence of 6 M urea at pH 3.0. The tetra-, tri-, di-, mono-, and nonacetylated forms of cuttlefish H4 represent 2, 6.4, 18, 32.2, and 41.4% of the whole histone, respectively. The di-, mono-, and nonacetylated forms of calf H4 represent 11.7, 41.3, and 44% of the whole histone, respectively. The acetylation sites were determined in each subfraction by identification of the acetylated peptides. In each acetylated H4 subfraction, the acetylated tryptic peptides were identified by peptide mapping and amino acid analysis with reference to the peptide map of nonacetylated H4. In cuttlefish testis H4, lysine 12 is the main site of acetylation in the monoacetylated subfraction; lysines 5 and 12 are found acetylated in diacetylated H4; lysines 5, 12, and 16 are found acetylated in triacetylated H4. From these results and the stoichiometry of the different H4 subfractions, it can be concluded that lysine 5 is acetylated after lysine 12. In calf thymus, lysine 16 is the only site of acetylation in the monoacetylated subfraction. All the diacetylated forms are acetylated in lysine 16, the second site of acetylation being, in decreasing order, lysine 12, lysine 5, or lysine 8. These observations suggest that acetylation occurs in a sequential manner. Moreover, the sites of acetylation depend upon the biological event in which acetylation is involved.     

Switching on Chromatin: Mechanistic Role of Histone H4-K16 Acetylation

DNA in eukaryotic organisms does not exist free in cells, but instead is present as chromatin, a complex assembly of DNA, histone proteins, and chromatin-associated proteins. Chromatin exhibits a complex hierarchy of structures, but in its simplest form it is composed of long linear arrays of nucleosomes. Nucleosomes contain 147 base pairs of DNA wrapped around a histone octamer, consisting of two copies each of histones H2A, H2B, H3 and H4, where 15-38 amino terminal residues of each histone protein extends past the DNA gyres to form histone “tails” 1. Chromatin provides a versatile regulatory platform for nearly all cellular processes that involve DNA, and improper chromatin regulation results in a wide range of diseases, including various cancers and congenital defects. One major way that chromatin regulates DNA utilization is through a wide range of post-translational modification of histones, including serine and threonine phosphorylation, lysine acetylation, methylation, ubiquitination, and sumoylation, and arginine methylation 2. Histone H4 K16 acetylation is a modification that occurs on the H4 histone tail and is one of the most frequent of the known histone modifications. We have demonstrated that this mark both disrupts formation of higher-order chromatin structure and changes the functional interaction of chromatin-associated proteins 3. Our results suggest a dual mechanism by which H4 K16 acetylation can ultimately facilitate genomic functions.     

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.     

Influence of histone tails and H4 tail acetylations on nucleosome-nucleosome interactions

Nucleosome–nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg²⁺, Ca²⁺, cobalt(III)hexammine³⁺, spermidine³⁺ and spermine⁴⁺] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome–nucleosome interaction, while the H4 K → Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome–nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome–nucleosome stacking and DNA–DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.