Basal c-Jun N-terminal kinases promote mitotic progression through histone H3 phosphorylation

Phosphorylation of histone H3 at serine 10 (S10) is essential for the onset of mitosis. Here, we show that basal c-Jun N-terminal kinases (JNKs) are required for mitotic histone H3 S10 phosphorylation in human primary fibroblast IMR90 cells. Inhibition of JNKs by specific pharmacologic inhibitors, expression of dominant-negative JNK1 and 2 mutants, or RNAi of JNK1 and 2 prevented phosphorylation of histone H3 at S10 in vivo. The JNK-specific inhibitor SP600125 blocked mitotic entry, as shown by its ability to prevent CDK1 dephosphorylation and cyclin A degradation. Basal JNK phosphorylation increased at G2/M-phase, although total JNK protein levels remained unchanged. In addition, basal JNKs were localized in nuclei and centrosomes during this time, suggesting that the nuclear localization of JNKs during G2/M is tightly coupled with histone H3 phosphorylation. Basal JNKs were able to phosphorylate histone H3 in vitro and co-precipitation of histone H3 and JNKs was only detected at G2/M. Taken together, these data strongly suggest that basal JNKs play a key role in controlling histone H3 phosphorylation for mitotic entry at G2/M-phase.     

Site-specific loss of acetylation upon phosphorylation of histone H3

Post-translational modification of histones is a central aspect of gene regulation. Emerging data indicate that modification at one site can influence modification of a second site. As one example, histone H3 phosphorylation at serine 10 (Ser(10)) facilitates acetylation of lysine 14 (Lys(14)) by Gcn5 in vitro (, ). In vivo, phosphorylation of H3 precedes acetylation at certain promoters. Whether H3 phosphorylation globally affects acetylation, or whether it affects all acetylation sites in H3 equally, is not known. We have taken a genetic approach to this question by mutating Ser(10) in H3 to fix either a negative or a neutral charge at this position, followed by analysis of the acetylation states of the mutant histones using site-specific antibodies. Surprisingly, we find that conversion of Ser(10) to glutamate (S10E) or aspartate (S10D) causes almost complete loss of H3 acetylation at lysine 9 (Lys(9)) in vivo. Acetylation of Lys(9) is also significantly reduced in cells bearing mutations in the Glc7 phosphatase that increase H3 phosphorylation levels. Mutation of Ser(10) in H3 and the concomitant loss of Lys(9) acetylation has minimal effects on expression of a Gcn5-dependent reporter gene. However, synergistic growth defects are observed upon loss of GCN5 in cells bearing H3 Ser(10) mutations that are reminiscent of delays in G(2)/M progression caused by combined loss of GCN5 and acetylation site mutations. Together these results demonstrate that H3 phosphorylation directly causes site-specific and opposite changes in acetylation levels of two residues within this histone, Lys(9) and Lys(14), and they highlight the importance of these histone modifications to normal cell functions.     

Site specificity of yeast histone acetyltransferase B complex in vivo

Saccharomyces cerevisiae Hat1, together with Hat2 and Hif1, forms the histone acetyltransferase B (HAT-B) complex. Previous studies performed with synthetic N-terminal histone H4 peptides found that whereas the HAT-B complex acetylates only Lys12, recombinant Hat1 is able to modify Lys12 and Lys5. Here we demonstrate that both Lys12 and Lys5 of soluble, non-chromatin-bound histone H4 are in vivo targets of acetylation for the yeast HAT-B enzyme. Moreover, coimmunoprecipitation assays revealed that Lys12/Lys5-acetylated histone H4 is bound to the HAT-B complex in the soluble cell fraction. Both Hat1 and Hat2, but not Hif1, are required for the Lys12/Lys5-specific acetylation and for histone H4 binding. HAT-B-dependent acetylation of histone H4 was detected in the soluble fraction of cells at distinct cell cycle stages, and increased when cells accumulated excess histones. Strikingly, histone H3 was not found in any of the immunoprecipitates obtained with the different components of the HAT-B enzyme, indicating the possibility that histone H3 is not together with histone H4 in this complex. Finally, the exchange of Lys for Arg at position 12 of histone H4 did not interfere with histone H4 association with the complex, but prevented acetylation on Lys5 by the HAT-B enzyme, in vivo as well as in vitro.     

Copper induces histone hypoacetylation through directly inhibiting histone acetyltransferase activity

The abnormal accumulation of Cu2+ is closely correlated with the incidence of different diseases, such as Alzheimer's disease and Wilson disease. To study in vivo functions of Cu2+ will lead to a better understanding of the nature of these diseases. In the present study, effect of Cu2+ on histone acetylation was investigated in human hepatoma cells. Exposure of cells to Cu2+ resulted in a significant decrease of histone acetylation, as indicated by the decrease of the overall histone acetylation and the decrease of histone H3 and H4 acetylation. Since histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlled the state of histone acetylation in vivo, we tested their contribution to the inhibition of Cu2+ on histone acetylation. One hundred nanomolar trichostatin A, the specific inhibitor of HDAC, did not attenuate the inhibitory effect of Cu2+ on histone acetylation. Combined with that Cu2+ showed no effect on the in vitro activity of HDAC, these results led to the conclusion that it is HAT, but not HDAC that is involved in Cu2+ -induced histone hypoacetylation. This conclusion was confirmed by the facts that (1) Cu2+ significantly inhibited the in vitro activity of HAT, (2) Cu2+ -treated cells possessed a lower HAT activity than control cells, and (3) 50 or 100 microM bathocuproine disulfonate, a chelator of Cu2+, significantly attenuated the inhibition of Cu2+ on HAT activity and histone acetylation in the similar pattern. Combined with that Cu2+ showed no or obvious cytotoxicity at 100 or 200 microM in human hepatoma cells, and the previous study that Cu2+ inhibits the histone H4 acetylation of yeast cells at nontoxic or toxic levels, the data presented here suggest that inhibiting histone acetylation is probably one general in vivo function of Cu2+, where HAT is its molecular target.     

Chaperone control of the activity and specificity of the histone H3 acetyltransferase Rtt109

Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109Delta suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers diacetylated on both H4-K5/12 and H3-K9/56.     

Distinct methylation patterns in histone H3 at Lys-4 and Lys-9 correlate with up- & down-regulation of genes by ethanol in hepatocytes

Ethanol induced liver injury is associated with a global change in gene expression but its mechanisms are not known. We studied whether alcohol-induced gene expression is associated with post-translational methylations of histone H3. Primary culture of rat hepatocytes was treated with ethanol (50 or 100 mM) for 24 h and the status of methylation of H3 at lys 4 (H3dimeK4) or lys 9 (H3dimeK9) was monitored by Western blotting using antibodies to dimethylated histone H3 at lys 4 or lys 9. The cells exposed to ethanol showed strikingly opposing behaviors in methylation patterns; H3dimeK9 methylation was decreased whereas H3dimeK4 increased. Similar results were obtained in the interphase nuclei. Their binding on the metaphase chromosomes exhibits distinct site specific pattern of accumulation. Next, chromatin immunoprecipitation of the ethanol treated samples with antibodies for methylated lys 4 or lys 9 histone H3 followed by amplification of the immunoprecipitated DNA, was used to determine their association with the promoters of genes up- or downregulated by ethanol. Lys4 methylation was associated with ethanol upregulated genes (Adh, GST-yc2) whereas lys 9 methylation with downregulated genes (Lsdh, cytP4502c11) demonstrating a difference between these two methylations. These results suggest that exposure of hepatocytes to ethanol changes the expression of several susceptible genes which are associated with site specific modification of dimethylated forms of histone H3 amino termini at their regulatory regions.     

Radiosensitization of yeast cells by inhibition of histone h4 acetylation

Deletion of genes for proteins involved in histone H4 acetylation produces sensitivity to DNA-damaging agents in both Saccharomyces cerevisiae and mammalian cells. In the present studies, we show that treating wild-type yeast cells with histone acetyl transferase (HAT) inhibitors, which are chemicals that cause a global decrease in histone H4 acetylation, sensitizes the cells to ionizing radiation. Using HAT inhibitors, we have placed histone H4 acetylation into the RAD51-mediated homologous recombination repair pathway. We further show that yeast cells with functionally defective HAT proteins have normal phospho-H2A (gamma-H2A) induction after irradiation but a reduced rate of loss of gamma-H2A. This argues that HAT-defective cells are able to detect DNA double-strand breaks normally but have a defect in the repair of these lesions. We also show that cells treated with HAT inhibitors have intact G1 and G2 checkpoints after exposure to ionizing radiation, suggesting that G1 and G2 checkpoint activation is independent of histone H4 acetylation.     

Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.     

Role of Jade-1 in the histone acetyltransferase (HAT) HBO1 complex

Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.     

The Rtt109-Vps75 histone acetyltransferase complex acetylates non-nucleosomal histone H3

Acetylation of lysine 56 of histone H3 (H3-Lys-56) occurs in S phase and disappears during G(2)/M phase of the cell cycle. However, it is not clear how this modification is regulated during the progression of the cell cycle. We and others have shown that the histone acetyltransferase (HAT) Rtt109 is the primary HAT responsible for acetylating H3-Lys-56 in budding yeast. Here we show that Rtt109 forms a complex with Vps75 and that both recombinant Rtt109-Vps75 complexes and native complexes purified from yeast cells acetylate H3 present in H3/H4/H2A/H2B core histones but not other histones. In addition, both recombinant and native Rtt109-Vps75 HAT complexes exhibited no detectable activity toward nucleosomal H3, suggesting that H3-Lys-56 acetylation is at least in part regulated by the inability of Rtt109-Vps75 complexes to acetylate nucleosomal H3 during G(2)/M phase of the cell cycle. Further, Rtt109 bound mutant H3/H4 tetramers composed of histones lacking their N-terminal tail domains less efficiently than wild-type H3/H4 tetramers, and Rtt109-Vps75 complexes displayed reduced HAT activity toward these mutant H3/H4 tetramers. Thus, the N termini of H3/H4 tetramers are required for efficient acetylation of H3 by the Rtt109-Vps75 complex. Taken together, these studies provide insights into how H3-Lys-56 acetylation is regulated during the cell cycle.     

Inhibition of histone acetyltransferase by glycosaminoglycans

Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans (GAGs) are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans (HSPGs) being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin-derived oligosaccharides (>8 sugars) and N-desulfated or O-desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive-like inhibitor causing an approximately 50-fold increase in the apparent Km of pCAF for histone H4. HSPGs isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous GAGs might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an approximately 50% reduction of histone H3 acetylation after 6 h of treatment. In addition, Chinese hamster ovary cells deficient in GAG synthesis showed increased levels of acetylated histone H3 compared to wild-type parent cells. GAGs represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation.     

The genomic landscapes of histone H3-Lys9 modifications of gene promoter regions and expression profiles in human bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) of nonembryortic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem ceils, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide historic H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways,cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency.