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BACKGROUND: Centromeric domains often consist of repetitive elements that are assembled in specialized chromatin, characterized by hypoacetylation of histones H3 and H4 and methylation of lysine 9 of histone H3 (K9-MeH3). Perturbation of this underacetylated state by transient treatment with histone deacetylase inhibitors leads to defective centromere function, correlating with delocalization of the heterochromatin protein Swi6/HP1. Likewise, deletion of the K9-MeH3 methyltransferase Clr4/Suvar39 causes defective chromosome segregation. Here, we create fission yeast strains retaining one histone H3 and H4 gene; the creation of these strains allows mutation of specific N-terminal tail residues and their role in centromeric silencing and chromosome stability to be investigated. RESULTS: Reduction of H3/H4 gene dosage to one-third does not affect cell viability or heterochromatin formation. Mutation of lysines 9 or 14 or serine 10 within the amino terminus of histone H3 impairs centromere function, leading to defective chromosome segregation and Swi6 delocalization. Surprisingly, silent centromeric chromatin does not require the conserved lysine 8 and 16 residues of histone H4. CONCLUSIONS: To date, mutation of conserved N-terminal residues in endogenous histone genes has only been performed in budding yeast, which lacks the Clr4/Suvar39 histone methyltransferase and Swi6/HP1. We demonstrate the importance of conserved residues within the histone H3 N terminus for the maintenance of centromeric heterochromatin in fission yeast. In sharp contrast, mutation of two conserved lysines within the histone H4 tail has no impact on the integrity of centromeric heterochromatin. Our data highlight the striking divergence between the histone tail requirements for the fission yeast and budding yeast silencing pathways.  相似文献   

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N-terminal modifications of nucleosomal core histones are involved in gene regulation, DNA repair and recombination as well as in chromatin modeling. The degree of individual histone modifications may vary between specific chromatin domains and throughout the cell cycle. We have studied the nuclear patterns of histone H3 and H4 acetylation and of H3 methylation in Arabidopsis. A replication-linked increase of acetylation only occurred at H4 lysine 16 (not for lysines 5 and 12) and at H3 lysine 18. The last was not observed in other plants. Strong methylation at H3 lysine 4 was restricted to euchromatin, while strong methylation at H3 lysine 9 occurred preferentially in heterochromatic chromocenters of Arabidopsis nuclei. Chromocenter appearance, DNA methylation and histone modification patterns were similar in nuclei of wild-type and kryptonite mutant (which lacks H3 lysine 9-specific histone methyltransferase), except that methylation at H3 lysine 9 in heterochromatic chromocenters was reduced to the same low level as in euchromatin. Thus, a high level of H3methylK9 is apparently not necessary to maintain chromocenter structure and does not prevent methylation of H3 lysine 4 within Arabidopsis chromocenters.  相似文献   

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Chen Y  Wan B  Wang KC  Cao F  Yang Y  Protacio A  Dou Y  Chang HY  Lei M 《EMBO reports》2011,12(8):797-803
Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2L(WH) (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.  相似文献   

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The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosylmethionine to specific epsilon-N-lysine residues in the N-terminal regions of histones H3 and H4. These enzymes are located exclusively within the nucleus and are firmly bound to chromatin. The chromosomal bound enzymes do not methylate free or nonspecifically associated histones, while histones H3 and H4 within newly synthesized chromatin are methylated. These enzymes can be solubilized by limited digestion (10-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease. Histone H3 lysine methyltransferase remained associated with a short DNA fragment throughout purification. Dissociation of the enzyme from the DNA fragment with DNAase digestion resulted in complete loss of enzyme activity; however, when this enzyme remained associated with DNA it was quite stable. Activity of the dissociated enzyme could not be restored upon the addition of sheared calf thymus or Escherichia coli DNA. Histone H3 lysine methyltransferase was found to methylate lysine residues in chromosomal bound or soluble histone H3, while H3 associated with mature nucleosomes was not methylated. The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest was extremely labile, losing all activity upon further purification. We isolated a methyltransferase by DEAE-cellulose chromatography, which would transfer methyl groups to arginine residues in soluble histone H4. However, this enzyme would not methylate nucleosomal or chromosomal bound histone H4, nor were methylated arginine nucleosomal or chromosomal bound histone H4, nor were methylated arginine residues detectable upon incubating intact nuclei or chromatin with S-adenosylmethionine.  相似文献   

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The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.  相似文献   

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Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.  相似文献   

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The coordinated process of DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.  相似文献   

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Heat shock or arsenite treatment alter the pattern of histone methylation in Drosophila cells. Both types of stress induce a rapid increase in the methylation level of histone H2B. The methylated amino acid residue of H2B has been identified by thin layer chromatography and electrophoresis as methylproline and is located at the N-terminal end of H2B. Heat shock also induces a decrease in the level of methylation of histone H3. Under normal growth temperature conditions, histone H3 is shown to be methylated on lysine residues. However under heat shock conditions, there is a decrease in the extent of methylation of lysine residues and the appearance of new methylation on arginine residues in H3. These new heat shock-induced methylated residues have been identified as the symmetrical and asymmetrical forms of dimethylarginine. The methylated amino acid residue of histone H4 is lysine with mono-, di-, and trimethyl forms found in both control and heat or chemically stressed cells. These stress-induced changes in the methylation level of the N-terminal proline residue of histone H2B and shift in the methylation sites of histone H3 may be involved in the restructuration of chromatin accompanying the inactivation of normal genes in response to stress. Moreover, we suggest that the hypermethylation of H2B may also be involved in its protection from increased ubiquitin-mediated proteolytic activity under these conditions of cellular stress.  相似文献   

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Ultraviolet B (UVB) induces phosphorylation of histone H3 at serine 10, and mitogen-activated protein kinases are involved in this signal transduction pathway. Here we provide evidence that Fyn kinase, a member of the Src kinase family, is involved in the UVB-induced phosphorylation of histone H3 at serine 10. UVB distinctly increased Fyn kinase activity and phosphorylation. Fyn kinase inhibitors 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-d)pyramide and leflunomide, an Src kinase inhibitor, suppressed both UVB-induced phosphorylation of histone H3 at serine 10 and Fyn kinase activity and phosphorylation. UVB-induced phosphorylation of histone H3 at serine 10 was blocked by either a dominant-negative mutant of Fyn (DNM-Fyn) kinase or small interfering RNA of Fyn kinase. UVB-induced phosphorylation and activities of ERKs and protein kinase B/Akt were markedly inhibited by DNM-Fyn kinase. However, DNM-Fyn kinase did not inhibit UVB-induced phosphorylation of p38 MAPK or c-Jun N-terminal kinases. Active Fyn kinase phosphorylated histone H3 at serine 10 in vitro, and the phosphorylated Fyn kinase could translocate into the nucleus of HaCaT cells. These results indicate that Fyn kinase plays a key role in the UVB-induced phosphorylation of histone H3 at serine 10.  相似文献   

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Poux AN  Marmorstein R 《Biochemistry》2003,42(49):14366-14374
Histone acetyltransferase (HAT) proteins often exhibit a high degree of specificity for lysine-bearing protein substrates. We have previously reported on the structure of the Tetrahymena Gcn5 HAT protein (tGcn5) bound to its preferred histone H3 substrate, revealing the mode of substrate binding by the Gcn5/PCAF family of HAT proteins. Interestingly, the Gcn5/PCAF HAT family has a remarkable ability to acetylate lysine residues within diverse cognate sites such as those found around lysines 14, 8, and 320 of histones H3, H4, and p53, respectively. To investigate the molecular basis for this, we now report on the crystal structures of tGcn5 bound to 19-residue histone H4 and p53 peptides. A comparison of these structures with tGcn5 bound to histone H3 reveals that the Gcn5/PCAF HATs can accommodate divergent substrates by utilizing analogous interactions with the lysine target and two C-terminal residues with a related chemical nature, suggesting that these interactions play a general role in Gcn5/PCAF substrate binding selectivity. In contrast, while the histone H3 complex shows extensive interactions with tGcn5 and peptide residues N-terminal to the target lysine, the corresponding residues in histone H4 and p53 are disordered, suggesting that the N-terminal substrate region plays an important role in the enhanced affinity of the Gcn5/PCAF HAT proteins for histone H3. Together, these studies provide a framework for understanding the substrate selectivity of HAT proteins.  相似文献   

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