Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae

Apoptosis is a highly coordinated cell suicide mechanism in vertebrates. Phosphorylation of serine 14 of histone H2B, catalyzed by Mst1 kinase, has been linked to chromatin compaction during apoptosis. We extend these results to unicellular eukaryotes by demonstrating that H2B is specifically phosphorylated at serine 10 (S10) in a hydrogen peroxide-induced cell death pathway in S. cerevisiae. H2B S10A mutants are resistant to cell death elicited by H(2)O(2) while H2B S10E phospho-site mimics promote cell death and induce the "constitutive" formation of condensed chromatin. Ste20 kinase, a yeast homolog of mammalian Mst1 kinase, translocates into the nucleus in a caspase-independent fashion and directly phosphorylates H2B at S10. Conservation of targeted H2B phosphorylation and the enzyme system responsible for the process point to an ancient mechanism of chromatin remodeling that likely plays an important role in governing cellular homeostasis in a wide range of organisms.     

Phosphorylation of histone H2B serine 32 is linked to cell transformation

Various types of post-translational modifications of the histone tails have been revealed, but a few modifications have been found within the histone core sequences. Histone core post-translational modifications have the potential to modulate nucleosome structure and DNA accessibility. Here, we studied the histone H2B core domain and found that phosphorylation of H2B serine 32 occurs in normal cycling and mitogen-stimulated cells. Notably, this phosphorylation is elevated in skin cancer cell lines and tissues compared with normal counterparts. The JB6 Cl41 mouse skin epidermal cell line is a well established model for tumor promoter-induced cell transformation and was used to study the function of H2B during EGF-induced carcinogenesis. Remarkably, cells overexpressing a nonphosphorylatable H2BS32A mutant exhibited suppressed growth and EGF-induced cell transformation, possibly because of decreased activation of activator protein-1, compared with control cells overexpressing wild type H2B. We identified ribosomal S6 kinase 2 (RSK2) as the kinase responsible for H2BS32 phosphorylation. Serum-starved JB6 cells contain very little endogenous H2BS32 phosphorylation, and EGF treatment induced this phosphorylation. The phosphorylation was attenuated in RSK2 knock-out MEFs and RSK2 knockdown JB6 cells. Taken together, our results demonstrate a novel role for H2B phosphorylation in cell transformation and show that H2BS32 phosphorylation is critical for controlling activator protein-1 activity, which is a major driver in cell transformation.     

H2B (Ser10) Phosphorylation is Induced during Apoptosis and Meiosis in S. cerevisiae

The nucleosome, composed of an octamer of highly conserved histone proteins and associated DNA, is the fundamental unit of eukaryotic chromatin. How arrays of nucleosomes are folded into higher-order structures, and how the dynamics of such compaction are regulated, are questions that remain largely unanswered. Our recent studies demonstrated that phosphorylation of histone H2B is necessary to induce cell death that exhibits phenotypic hallmarks of apoptosis including DNA fragmentation and chromatin condensation in yeast (serine 10)1 and in mammalian cells (serine 14).2 In this article, we extend these findings by uncovering a role for H2B phosphorylation at serine 10 (Ser10) in another biological event that is associated with dramatic alterations in higher-order chromatin structure, meiosis. Our data show strong staining, indicative of H2B (Ser10) phosphorylation, during the pachytene stage of yeast meiotic prophase. These data broaden the use of this phosphorylation mark in chromatin remodeling that closely correlates with chromatin compaction. How phosphorylation marks are translated into meaningful downstream events during processes as diverse as apoptosis and meiosis remains a challenge for future studies.     

Carbohydrates induce mono-ubiquitination of H2B in yeast

Histone modifications have emerged to be a major regulatory mechanism for gene expression (1-4). However, it is not clear how histone modifications are physiologically regulated. Here, we show that mono-ubiquitinated H2B at lysine 123 (uH2B) in the yeast (Saccharomyces cerevisiae) is present in exponential phase and absent in stationary phase. A wide array of carbohydrates or sugars, including glucose, fructose, mannose, and sucrose, are capable of inducing uH2B in stationary phase yeast. In contrast, non-metabolic glucose analogs are defective in inducing uH2B. Furthermore, uH2B induction is inhibited by iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase in glycolysis. Moreover, uH2B induction is markedly impaired in yeast mutants, in which glycolytic genes are deleted. These data indicate that glycolysis is required for the carbohydrate-induced mono-ubiquitination of H2B at lysine 123. Therefore, our study reveals a novel paradigm of metabolic regulation of histone modifications.     

PP1/Repo-man dephosphorylates mitotic histone H3 at T3 and regulates chromosomal aurora B targeting

The transient mitotic histone H3 phosphorylation by various protein kinases regulates chromosome condensation and segregation, but the counteracting phosphatases have been poorly characterized [1-8]. We show here that PP1γ is the major histone H3 phosphatase acting on the mitotically phosphorylated (ph) residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify Repo-Man, a chromosome-bound interactor of PP1γ [9], as a selective regulator of H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph dephosphorylation by an indirect mechanism but directly and specifically targets H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the end of mitosis. Consistent with these findings, Repo-Man modulates in a PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase B and its substrate MCAK. Our study defines a novel mechanism by which PP1 counteracts Aurora B.     

Histone H2A phosphorylation in DNA double-strand break repair

DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process. One of the best-characterized chromatin modification events in DNA-damage responses is the phosphorylation of the SQ motif found in histone H2A or the H2AX histone variant in higher eukaryotes. This modification is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved function. One function that histone modifications can have is to provide a unique binding site for interacting factors. Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implications of these interactions.     

Cellular aging is associated with increased ubiquitylation of histone H2B in yeast telomeric heterochromatin

Epigenetic changes in chromatin state are associated with aging. Notably, two histone modifications have recently been implicated in lifespan regulation, namely acetylation at H4 lysine 16 in yeast and methylation at H3 lysine 4 (H3K4) in nematodes. However, less is known about other histone modifications. Here, we report that cellular aging is associated with increased ubiquitylation of histone H2B in yeast telomeric heterochromatin. An increase in ubiquitylation at histone H2B lysine 123 and methylations at both H3K4 and H3 lysine 79 (H3K79) was observed at the telomere-proximal regions of replicatively aged cells, coincident with decreased Sir2 abundance. Moreover, deficiencies in the H2B ubiquitylase complex Rad6/Bre1 as well as the deubiquitylase Ubp10 reduced the lifespan by altering both H3K4 and H3K79 methylation and Sir2 recruitment. Thus, these results show that low levels of H2B ubiquitylation are a prerequisite for a normal lifespan and the trans-tail regulation of histone modifications regulates age-associated Sir2 recruitment through telomeric silencing.     

Regulation of ultraviolet B-induced phosphorylation of histone H3 at serine 10 by Fyn kinase

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.     

The apoptotic ring: A novel entity with phosphorylated histones H2AX and H2B,and activated DNA damage response kinases

We recently showed that histone H2AX phosphorylated on serine 139 (γ-H2AX), a hallmark of DNA damage response (DDR), also forms early during apoptosis induced by death receptor activation. Here, we extend and discuss our findings on apoptotic γ-H2AX, which differs from the well-established DDR with nuclear foci. During apoptosis induced by death receptors agonists (TRAIL and FasL) and staurosporine, γ-H2AX is initiated in the nuclear periphery immediately inside the nuclear envelope while total H2AX remains distributed throughout the nucleus. This process is readily detectable by immunofluorescence microscopy and we refer to it as the “γ-H2AX ring”. It is conserved both in cancer and normal cells. The γ-H2AX ring contains the activated checkpoints kinases, ATM, Chk2 and DNA-PK; the latter being the main effector for the apoptotic γ-H2AX phosphorylation. Notably, we show here that the γ-H2AX ring coincides with phosphorylated H2B on serine 14 (PS14-H2B), another histone modification associated with apoptosis. The coordinated phosphorylations of H2AX and H2B suggest a previously unrecognized histone phosphorylation signature for apoptosis consisting of γ-H2AX together with PS14-H2B and possibly PY142-H2AX. This signature (“phospho-histone 2 code”) together with the γ-H2AX ring provides a new feature to monitor and study apoptosis.     

ste20基因突变抑制葡萄糖诱导的酿酒酵母细胞凋亡

近年来,酿酒酵母的细胞调亡研究取得了很大进展。多种因素可以诱导其调亡,譬如过氧化氢（H2O2）、醋酸、高渗透压和高盐浓度等。葡萄糖是酿酒酵母生长所必须的重要营养物质之一。同时,在其他营养元素缺乏的条件下,只用葡萄糖培养将迅速的诱导酿酒酵母的细胞凋亡。Ste20是PAK（p21 activated kinase）家族的成员,它参与酿酒酵母的信息素应答、假菌丝生长和侵入生长等途径。有研究表明,ste20突变株能抵抗由信息素和过氧化物诱导的细胞调亡。我们发现STE20基因突变也能抑制葡萄糖诱导的凋亡,用葡萄糖处理时,与野生型相比,ste20突变株细胞能保持完整的细胞膜和细胞核结构。H2O2诱导酿酒酵母细胞凋亡时,需要Ste20激酶磷酸化组蛋白H2B第十号丝氨酸（S10）。因此,葡萄糖诱导的酿酒酵母细胞凋亡作用可能通过类似于过氧化氢诱导的酿酒酵母细胞凋亡的途径进行的。     

Discovering common combinatorial histone modification patterns in the human genome

Histone modifications play a crucial role in regulating gene expression and cell lineage determination and maintenance at the epigenetic level. To systematically investigate this phenomenon, this paper presented a statistical hybrid clustering algorithm to identify common combinatorial histone modification patterns. We applied the algorithm to 39 histone modification marks in human CD4 + T cells and detected 854 common combinatorial histone modification patterns. Our results could cover 211 (76.17%) patterns among 277 patterns identified by the tandem mass spectrometry experiments. Based on the frequency statistical analysis, it was found that the co-occurrence frequencies of 20 backbone modifications are greater than or close to 0.2 in the 854 patterns. we also found that 15 modifications (H2BK120ac, H4K91ac, H2BK20ac, etc.), three histone acetylations (H2AK9ac, H4K16ac, and H4K12ac) and five histone methylations (H3K79me1, H3K79me2, 3K79me3, H4K20me1, and H2BK5me1) were most likely prone to coexist respectively in these patterns. In addition, we found that DNA methylation tends to combine with histone acetylation rather than histone methylation.     

Phosphorylation of histone H2A is associated with centromere function and maintenance in meiosis

Histone phosphorylation is dynamically regulated during cell division, for example phosphorylation of histone H3 (H3)-Ser10, H3-Thr11 and H3-Ser28. Here we analyzed maize (Zea mays L) for Thr133-phosphorylated histone H2A, which is important for spindle checkpoint control and localization of the centromere cohesion protector Shugoshin in mammals and yeast. Immunostaining results indicate that phosphorylated H2A-Thr133 signals bridged those of the centromeric H3 histone variant CENH3 by using a plant displaying yellow fluorescent protein-CENH3 signals and H2A-Thr133 is phosphorylated in different cell types. During mitosis, H2A-Thr133 phosphorylation becomes strong in metaphase and is specific to centromere regions but drops during later anaphase and telophase. Immunostaining for several maize dicentric chromosomes revealed that the inactive centromeres have lost phosphorylation of H2A-Thr133. During meiosis in maize meiocytes, H2A phosphorylation becomes strong in the early pachytene stage and increases to a maximum at metaphase I. In the maize meiotic mutant afd1 (absence of first division), sister chromatids show equational separation at metaphase I, but there are no changes in H2A-Thr-133 phosphorylation during meiosis compared with the wild type. In sgo1 mutants, sister chromatids segregate randomly during meiosis II, and phosphorylation of H2A-Thr-133 is observed on the centromere regions during meiosis II. The availability of such mutants in maize that lack sister cohesion and Shugoshin indicate that the signals for phosphorylation are not dependent on cohesion but on centromere activity.