Novel phosphorylation of histone H3 at threonine 11 that temporally correlates with condensation of mitotic and meiotic chromosomes in plant cells

A novel mitosis-specific phosphorylation site in histone H3 at threonine 11 has been described for mammalian cells. This modification is restricted to the centromeric region while phosphorylation at the classical H3 sites, Ser10 and Ser28 occurs along the entire chromosomal arms. Using phosphorylation state-specific antibodies we found that phosphorylation at threonine 11 occurs also in plant cells, during mitosis as well as meiosis. However, in contrast to animal cells, ph(Thr11)H3 was distributed along the entire length of condensed chromosomes, whereas H3 phosphorylated at Ser10 and Ser28 appeared to be restricted to centromeric/pericentromeric chromatin. Phosphorylation at Thr11 started in prophase and ended in telophase, it correlated with the condensation of mitotic and meiotic chromosomes and was independent of the distribution of late replicating heterochromatin and Giemsa-banding positive regions. Interestingly, treatment of cells with the phosphatase inhibitor cantharidin revealed a high level of Thr11 phosphorylation in interphase cells, in this case particularly in pericentromeric regions. These data show that histone modifications are highly dynamic. Moreover, animal and plant organisms may have evolved individual histone codes.     

Thr 3 phosphorylated histone H3 concentrates at centromeric chromatin at metaphase

Thr 3 was one of the newly characterized phosphorylation sites on histone H3. However, the functional significance of histone H3 Thr 3 phosphorylation during mitosis is unclear. In this study, SDS-PAGE and Western blotting analysis showed that histone H3 Thr 3 was phosphorylated specially during mitosis in MCF-10A and ECV-304 cells. Using indirect immunofluorescence labeling and laser confocal microscopy, we demonstrated that histone H3 Thr 3 phosphorylation occurred from prophase to anaphase and dephosphorylated completely in telophase. Remarkably, Thr 3 phosphorylated histone H3 mostly concentrated at centromeric chromatin at metaphase, which was distinct with Ser 10 phosphorylation aggregated at the telomere, but similar to that characteristic of Thr 11 phosphorylated H3 which is largely restricted to the centromeric chromatin. Using chromatin immunoprecipitation (ChIP) assay, we provided direct evidence that the Thr 3 phosphorylated H3 is associated with centromeric DNA at metaphase. These findings suggested that at metaphase Thr 3 phosphorylated histone H3 may also participate in kinetochore assembly to promote faithful chromosome segregation and serve as another recognition code for kinetochore proteins.     

Thr11磷酸化组蛋白H3在MCF-7细胞有丝分裂中的动态分布

组蛋白H3在氨基末端Ser10、Ser28、Thr11和Thr3等氨基酸残基的磷酸化修饰是一类在时间上和空间上与细胞有丝分裂相关的翻译后修饰事件。为了研究Thr11位点磷酸化作用的功能,利用SDS-PAGE、Western Blot、间接免疫荧光标记技术和激光共聚焦显微技术检测分析了人乳腺癌细胞(MCF-7)中Thr11磷酸化组蛋白H3在有丝分裂过程中的动态分布,以研究其在有丝分裂过程中的功能。结果显示:在MCF-7细胞中,组蛋白H3 Thr11的磷酸化发生在早前期细胞染色体的着丝粒处,成点状分布,继而在早中期达到最高水平,并以点状集中在赤道板上,在有丝分裂后期开始脱磷酸化,并于末期完成脱磷酸化。事实表明,H3 Thr11的磷酸化与细胞有丝分裂过程存在着时间和空间上的相关性。Thr11磷酸化H3只存在于着丝粒表明它可能参与有丝分裂期间功能性动原体的组成。这与Ser10磷酸化H3的分布及可能的功能截然相反。     

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.     

Mitosis-specific histone H3 phosphorylation in vitro in nucleosome structures

A mechanism of mitosis-specific enhancement of histone H3 phosphorylation was analyzed in vitro in terms of nucleosome structure. The incorporation of [32P]phosphate into DNA-bound H3 was approximately 5-7 times higher than in DNA-free H3 using the catalytic subunit of cAMP-dependent protein kinase. The two major N-terminal serine sites, including the mitosis-specific site (Ser10) and Ser28, were extensively phosphorylated in the DNA-bound forms. These phosphorylation patterns were identical to those of nucleosomal H3. In contrast, the H3 in DNA-free octamers was very slightly phosphorylated. The major site of H3 phosphorylation in DNA-free H3 was Thr118 in the C-terminus. Results indicate that DNA-binding is essential for the high level of mitosis-specific H3 phosphorylation, and that the nucleosome structure promotes H3 N-terminal phosphorylation in vitro. It also suggests the possibility that H1 prevents H3 phosphorylation during interphase of the cell cycle.     

Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.     

The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate

Yeast ESA1 is a member of the MYST subfamily of histone acetyltransferases (HATs), which use acetyl-coenzyme A (CoA) to acetylate specific Lys residues within histones to regulate gene expression. The structure of an ESA1-CoA complex reveals structural similarity to the catalytic core of the GCN5/PCAF subfamily of HAT proteins. Here we report additional structural and functional studies on ESA1 that demonstrate that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate. This Cys residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT subfamily. However, this mode of catalysis differs dramatically from the GCN5/PCAF subfamily, which mediate direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated substrate lysine of the histone. These results demonstrate that different HAT subfamilies can use distinct catalytic mechanisms, which have implications for their distinct biological roles and for the development of HAT-specific inhibitors.     

Modifications of human histone H3 variants during mitosis

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.     

Decylubiquinol impedes mitochondrial respiratory chain complex I activity

In this study, indirect immunofluorescence labeling was used to examine the cellular dynamic distribution of Thr11 phosphorylated H3 at mitosis in MCF-7 cells. The Thr11 phosphorylation was observed beginning at prophase at centromeres. Upon progression of mitosis, fluorescence signal was enhanced in the central region of the metaphase plate and maintained till anaphase at centromeres. During telophase, the fluorescent signal of Thr11 phosphorylated H3 disappears from centromeres, but the signal appears again at the midbody during cytokinesis, which suggests that the modified histones may take part in the formation of the midbody and play a crucial role in cytokinesis. Chromatin immunoprecipitation (ChIP) was used to confirm that Thr11 phosphorylated H3 is specifically associated with centromere DNA at prophase to metaphase, which is coincident with the results observed by immunofluorescence. In conclusion, there was a precise spatial and temporal correlation between H3 phosphorylation of Thr11 and stages of chromatin condensation. The timing of Thr11 phosphorylation and dephosphorylation in mitosis were similar to that reported for Ser10 phosphorylation of H3. The Thr11 phosphorylated H3 localized at centromeres during mitosis, which was different from the Ser10 phosphorylated H3 localized at telomere regions and Thr3 phosphorylated H3 localized along the chromosome arms. The results suggest that the Thr11 phosphorylation of histone H3 may play a specific role which was different from Ser10 and Thr3 phosphorylation in mitosis.     

Molecular basis for Gcn5/PCAF histone acetyltransferase selectivity for histone and nonhistone substrates

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.