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1.
Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes. The histone H3 kinase was not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity could be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity was regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibited kinase activity. The presence of PP1 on chromatin suggested that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generated an H3 kinase activity that was also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, we found that stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1.  相似文献   

2.
Yeast disruptor of telomeric silencing-1 (DOT1) is involved in gene silencing and in the pachytene checkpoint during meiotic cell cycle. Here we show that the Dot1 protein possesses intrinsic histone methyltransferase (HMT) activity. When compared with Rmt1, another putative yeast HMT, Dot1 shows very distinct substrate specificity. While Rmt1 methylates histone H4, Dot1 targets histone H3. In contrast to Rmt1, which can only modify free histones, Dot1 activity is specific to nucleosomal substrates. This was also confirmed using native chromatin purified from yeast cells. We also demonstrate that, like its mammalian homolog PRMT1, Rmt1 specifically dimethylates an arginine residue at position 3 of histone H4 N-terminal tail. In surprising contrast, methylation by Dot1 occurs in the globular domain of nucleosomal histone H3. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis suggests that H3 lysine 79 is trimethylated by Dot1. The intrinsic nucleosomal histone H3 methyltransferase activity of Dot1 is certainly a key aspect of its function in gene silencing at telomeres, most likely by directly modulating chromatin structure and Sir protein localization. In agreement with a role in regulating localization of histone deacetylase complexes like SIR, an increase of bulk histone acetylation is detected in dot1- cells.  相似文献   

3.
Using circular dichroism to probe the extent of DNA condensation in chromatin, we have demonstrated that a major nucleolar protein, nucleolin can decondense chromatin. By means of various binding assays we show that nucleolin has a strong affinity for histone H1 and that the phosphorylated N-terminal domain, rich in lengthy stretches of acidic amino acids, is responsible for this ionic interaction. Additional experiments clearly demonstrate that nucleolin is unable to act as a nucleosome core assembly or disassembly factor and hence has little affinity for the core histone octamer. We propose that this nucleolar protein induces chromatin decondensation by binding to histone H1, and that nucleolin can therefore be regarded as a protein of the high-mobility-group type.  相似文献   

4.
Eukaryotic chromatin is highly dynamic and turns over rapidly even in the absence of DNA replication. Here we show that the acidic histone chaperone nucleosome assembly protein 1 (NAP-1) from yeast reversibly removes and replaces histone protein dimer H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange. Transient removal of H2A-H2B dimers facilitates nucleosome sliding along the DNA to a thermodynamically favorable position. Histone exchange as well as nucleosome sliding is independent of ATP and relies on the presence of the C-terminal acidic domain of yeast NAP-1, even though this region is not required for histone binding and chromatin assembly. Our results suggest a novel role for NAP-1 (and perhaps other acidic histone chaperones) in mediating chromatin fluidity by incorporating histone variants and assisting nucleosome sliding. NAP-1 may function either untargeted (if acting alone) or may be targeted to specific regions within the genome through interactions with additional factors.  相似文献   

5.
Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.  相似文献   

6.
1. Histones H1 and H5 in chromatin and in free solution can be cross-linked to higher multimers. Is this due to a specific protein/protein interaction? If so, this interaction might be the structural basis of the condensation of the chromosomal nucleofilament, known to be mediated by histones H1 and H5. 2. Since only the central domain of H1 and H5 exhibits tertiary folding and globular structure, this is the most likely site of specific interaction. 3. Formaldehyde has been used to test whether the central domains of histone H1 from calf thymus or from sea urchin sperm or histone H5 from chicken erythrocytes self-interact. 4. The cross-linking shown by each globular peptide was compared with that of its parent histone. 5. In all three cases the peptide cross-linked to a much lower extent than its intact parent histone and the observed cross-linked rates were roughly in proportion to the relative number of lysine residues parent histone and peptide. 6. It is concluded that there is no specific self-interaction between the globular domains of either H1 or H5 molecules in free solution. 7. This result suggests that specific H1/H1 protein/protein interactions are not the basic cause of chromatin condensation.  相似文献   

7.
MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphorylates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phosphorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphorylation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.  相似文献   

8.
Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.  相似文献   

9.
10.
We have shown okadaic acid (OA) and calyculin-A (CLA) inhibition of mouse oocyte phosphoprotein phosphatase 1 (PPP1C) and/or phosphoprotein phosphatase 2A (PPP2CA) results in aberrant chromatin condensation, as evidenced by the inability to resolve bivalents. Phosphorylation of histone H3 at specific residues is thought to regulate chromatin condensation. Therefore, we examined changes in histone H3 phosphorylation during oocyte meiosis and the potential regulation by protein PPPs. Western blot and immunocytochemical analysis revealed histone H3 phosphorylation changed during mouse oocyte meiosis, with changes in chromatin condensation. Germinal vesicle-intact (GV-intact; 0 h) oocytes had no phospho-Ser10 but did have phospho-Ser28 histone H3. Oocytes that had undergone germinal vesicle breakdown (GVBD; 2 h) and progressed to metaphase I (MI; 7 h) and MII (16 h) had phosphorylated Ser10 and Ser28 histone H3 associated with condensed chromatin. To determine whether OA-induced aberrations in chromatin condensation were due to alterations in levels of histone H3 phosphorylation, we assessed phosphorylation of Ser10 and Ser28 residues following PPP inhibition. Oocytes treated with OA (1 microM) displayed increased phosphorylation of histone H3 at both Ser10 and Ser28 compared with controls. To begin to elucidate which OA-sensitive PPP is responsible for regulating chromatin condensation and histone H3 phosphorylation, we examined spatial and temporal localization of OA-sensitive PPPs, PPP1C, and PPP2CA. PPPC2A did not localize to condensed chromatin, whereas PPP1beta (PPP1CB) associated with condensing chromatin in GVBD, MI, and MII oocytes. Additionally, Western blot and immunocytochemistry confirmed presence of the PPP1C regulatory inhibitor subunit 2 (PPP1R2) in oocytes at condensed chromatin during meiosis and indicated a change in PPP1R2 phosphorylation. Inhibition of oocyte glycogen synthase kinase 3 (GSK3) appeared to regulate phosphorylation of PPP1R2. Furthermore, inhibition of GSK3 resulted in aberrant oocyte bivalent formation similar to that observed following PPP inhibition. These data suggest that PPP1CB is the OA/CLA-sensitive PPP that regulates oocyte chromatin condensation through regulation of histone H3 phosphorylation. Furthermore, GSK3 inhibition results in aberrant chromatin condensation and appears to regulate phosphorylation of PPP1R2.  相似文献   

11.
Loss of linker histone H1 in cellular senescence   总被引:9,自引:0,他引:9       下载免费PDF全文
  相似文献   

12.
Growth-associated H1 histone kinase, a homolog of the yeast cdc2+/CDC28 protein kinases that control entry into mitosis, is a chromatin-bound cyclic nucleotide-independent enzyme found only in growing cells. In a procedure involving salt extraction of chromatin, ammonium sulfate precipitation, and three chromatographic steps, the enzyme has been purified greater than 10,000-fold from Novikoff hepatoma cells. Enzyme purified by this procedure catalyzes the transfer to H1 histone of 2.7 mumol of phosphate/min/mg, a specific activity within the range of those reported for a number of homogeneous or nearly homogeneous protein kinases. Further purification to near homogeneity was achieved by an additional step of sucrose density gradient fractionation. Enzyme activity in the sucrose gradient is associated with two polypeptides of apparent Mr 60,000 and 33,000 on sodium dodecyl sulfate-gel electrophoresis. Substrate specificity studies show that in addition to H1, proteins with H1-like structure and function including histone H1(0), the erythrocyte-specific H5 histone, and the testis-specific H1t histone are phosphorylated. Nucleosome core histone H3, high mobility group proteins 1, 2, 14, and 17, protamine, casein, and ribosomal protein S6 are not substrates.  相似文献   

13.
Histone lysine methylation is an important chromatin modification that can be catalyzed to a mono-, di-, or tri-methyl state. An ongoing challenge is to decipher how these different methyllysine histone marks can mediate distinct aspects of chromatin function. The fission yeast checkpoint protein Crb2 is rapidly targeted to sites of DNA damage after genomic insult, and this recruitment requires methylation of histone H4 lysine 20 (H4K20). Here we show that the tandem tudor domains of Crb2 preferentially bind the di-methylated H4K20 residue. Loss of this interaction by disrupting either the tudor-binding motif or the H4K20 methylating enzyme Set9/Kmt5 ablates Crb2 localization to double-strand breaks and impairs checkpoint function. Further we show that dimethylation, but not tri-methylation, of H4K20 is required for Crb2 localization, checkpoint function, and cell survival after DNA damage. These results argue that the di-methyl H4K20 modification serves as a binding target that directs Crb2 to sites of genomic lesions and defines an important genome integrity pathway mediated by a specific methyl-lysine histone mark.  相似文献   

14.
During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone, which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone-dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domain-containing protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation.  相似文献   

15.
Edwards CR  Dang W  Berger SL 《Biochemistry》2011,50(48):10473-10483
Histones undergo post-translational modifications that are linked to important biological processes. Previous studies have indicated that lysine methylation correlating with closed or repressive chromatin is absent in the budding yeast Saccharomyces cerevisiae, including at H4 lysine 20 (K20). Here we provide functional evidence for H4 K20 monomethylation (K20me1) in budding yeast. H4 K20me1 is detectable on endogenous H4 by western analysis using methyl-specific antibodies, and the signal is abrogated by H4 K20 substitutions and by competition with H4 K20me1 peptides. Using chromatin immunoprecipitation, we show that H4 K20me1 levels are highest at heterochromatic locations, including subtelomeres, the silent mating type locus, and rDNA repeats, and lowest at centromeres within euchromatin. Further, an H4 K20A substitution strongly reduced heterochromatic reporter silencing at telomeres and the silent mating type locus and led to an increase in subtelomeric endogenous gene expression. The correlation between the location of H4 K20me1 and the effect of the H4 K20A substitution suggests that this modification plays a repressive function. Our findings reveal the first negative regulatory histone methylation in budding yeast and indicate that H4 K20me1 is evolutionarily conserved from simple to complex eukaryotes.  相似文献   

16.
Pre-messenger RNA splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Despite the striking evolutionary conservation of the spliceosomal components and their functions, controversy persists about the relative importance of splicing in Saccharomyces cerevisiae—particularly given the paucity of intron-containing genes in yeast. Here we show that splicing of one pre-messenger RNA, SUS1, a component of the histone H2B ubiquitin protease machinery, is essential for establishing the proper modification state of chromatin. One protein complex that is intimately involved in pre-mRNA splicing, the yeast cap-binding complex, appears to be particularly important, as evidenced by its extensive and unique genetic interactions with enzymes that catalyze histone H2B ubiquitination. Microarray studies show that cap binding complex (CBC) deletion has a global effect on gene expression, and for ∼20% of these genes, this effect is suppressed when ubiquitination of histone H2B is eliminated. Consistent with this finding of histone H2B dependent effects on gene expression, deletion of the yeast cap binding complex leads to overubiquitination of histone H2B. A key component of the ubiquitin-protease module of the SAGA complex, Sus1, is encoded by a gene that contains two introns and is misspliced when the CBC is deleted, leading to destabilization of the ubiquitin protease complex and defective modulation of cellular H2B levels. These data demonstrate that pre-mRNA splicing plays a critical role in histone H2B ubiquitination and that the CBC in particular helps to establish the proper state of chromatin and proper expression of genes that are regulated at the level of histone H2B ubiquitination.  相似文献   

17.
Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.  相似文献   

18.
19.
In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 microM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 microM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to > or = 22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.  相似文献   

20.
Reassociation of histone H1 with nucleosomes.   总被引:6,自引:0,他引:6  
The role of histone H1 in nucleosome heterogeneity and structure has been studied using a reconstitution procedure. Histone H1 and non-histone proteins are removed selectively from enzymatically fragmented chromatin by Dowex 50W-X2 treatment. The resulting "stripped" chromatin then is reassociated with purified histone H1 using step gradient dialysis. Material reconstituted in this manner was examined by gel electrophoresis, protein cross-linking, and chromatin fingerprinting. The results demonstrate that the histone H1 molecule efficiently binds to nucleosomes with fidelity in an apparent noncooperative manner. Polynucleosomes possess two specific binding sites for histone H1 per histone octamer; the first binding site is of higher affinity than the second. The 160-base pair nuclease digestion barrier and nucleosome electrophoretic class (MIII)n are established upon binding the 1st histone H1 molecule. Upon binding the 2nd histone H1 molecule, polynucleosomes assume a highly compact conformation. The experimental approach introduced here should permit determining whether nucleosomes possess independent specific binding sites for other chromosomal proteins, and should allow reconstitution of the other electrophoretic forms of nucleosomes which we have described previously.  相似文献   

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