Striatal histone modifications in models of levodopa-induced dyskinesia

Despite recent advances in the treatment of Parkinson disease (PD), levodopa remains the most effective and widely used therapy. A major limitation to the use of levodopa is the development of abnormal involuntary movements, termed levodopa-induced dyskinesia (LDID), following chronic levodopa treatment. Since recent studies have suggested that modifications of chromatin structure may be responsible for many long-lasting changes in brain function, we have examined post-translational modifications of striatal histones in two models of LDID: an acute murine model and a chronic macaque monkey model, both exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In the primate model, which closely resembles human LDID, we observed that chronic levodopa and the appearance of LDID was associated with marked deacetylation of histone H4, hyperacetylation and dephosphorylation of histone H3, and enhancement of the phosphorylation of extracellular signal-regulated kinase (ERK). In the murine model of acutely rather than chronically induced LDID, dopamine depletion and levodopa treatment also induced deacetylation of histone H4 and phosphorylation of ERK, but histone H3 exhibited decreased trimethylation and reduced rather than enhanced acetylation. These data demonstrate striking changes in striatal histones associated with the induction of LDID in both animal models. The pattern of changes observed, as well as the behavioral features, differed in the two models. However, both models exhibit marked deacetylation of histone H4, suggesting that inhibitors of H4 deacetylation may be useful in preventing or reversing LDID.     

Histone deacetylation is required for progression through mitosis in tobacco cells

Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.     

β-N-Acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3

O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.     

Global regulation of post-translational modifications on core histones.

Full-length masses of histones were analyzed by mass spectrometry to characterize post-translational modifications of bulk histones and their changes induced by cell stimulation. By matching masses of unique peptides with full-length masses, H4 and the variants H2A.1, H2B.1, and H3.1 were identified as the main histone forms in K562 cells. Mass changes caused by covalent modifications were measured in a dose- and time-dependent manner following inhibition of phosphatases by okadaic acid. Histones H2A, H3, and H4 underwent changes in mass consistent with altered acetylation and phosphorylation, whereas H2B mass was largely unchanged. Unexpectedly, histone H4 became almost completely deacetylated in a dose-dependent manner that occurred independently of phosphorylation. Okadaic acid also partially blocked H4 hyperacetylation induced by trichostatin-A, suggesting that the mechanism of deacetylation involves inhibition of H4 acetyltransferase activity, following perturbation of cellular phosphatases. In addition, mass changes in H3 in response to okadaic acid were consistent with phosphorylation of methylated, acetylated, and phosphorylated forms. Finally, kinetic differences were observed with respect to the rate of phosphorylation of H2A versus H4, suggesting differential regulation of phosphorylation at sites on these proteins, which are highly related by sequence. These results provide novel evidence that global covalent modifications of chromatin-bound histones are regulated through phosphorylation-dependent mechanisms.     

Expression and secretion of human apolipoprotein A-I in the heart

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.     

光色素信号通路中磷酸化修饰研究进展

光是植物的唯一能量来源, 植物在进化过程中产生不同的光敏色素来感知光信号。光信号通路中元件通常被特异翻译后修饰调节。光敏色素是一种自磷酸化的丝氨酸/苏氨酸蛋白激酶, 可以被一些蛋白磷酸酶去磷酸化。通过对光敏色素A (phyA)和光敏色素B (phyB)的自磷酸化位点研究, 发现自磷酸化对光敏色素的功能及其介导的信号通路起着非常重要的作用。光激活的光敏色素诱导光敏色素作用因子(PIF)磷酸化, 这对于PIF的正常降解及光形态建成的起始是必需的。该文主要介绍了光敏色素信号通路磷酸化修饰的最新进展, 以期为深入研究光敏色素信号转导机制提供参考。     

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.     

Histone H2B deacetylation at lysine 11 is required for yeast apoptosis induced by phosphorylation of H2B at serine 10

Chromatin alterations, induced by covalent histone modifications, mediate a wide range of DNA-templated processes, including apoptosis. Apoptotic chromatin condensation has been causally linked to the phosphorylation of histone H2B (serine 14 in human; serine 10 in yeast, H2BS10ph) in human and yeast cells. Here, we extend these studies by demonstrating a unidirectional, crosstalk pathway between H2BS10 phosphorylation and lysine 11 acetylation (H2BK11ac) in yeast. We demonstrate that the H2BK11 acetyl mark, which exists in growing yeast, is removed upon H(2)O(2) treatment but before H2BS10ph occurs, in a unidirectional fashion. H2B K11Q mutants are resistant to cell death elicited by H(2)O(2), while H2B K11R mutants that mimic deacetylation promote cell death. Our results suggest that Hos3 HDAC deacetylates H2BK11ac, which in turn mediates H2BS10ph by Ste20 kinase. Together, these studies underscore a concerted series of enzyme reactions governing histone modifications that promote a switch from cell proliferation to cell death.     

p120 serine and threonine phosphorylation is controlled by multiple ligand-receptor pathways but not cadherin ligation

p120-catenin (p120) regulates cadherin turnover and is required for cadherin stability. Extensive and dynamic phosphorylation on tyrosine, serine and threonine residues in the N-terminal regulatory domain has been postulated to regulate p120 function, possibly through modulation of the efficiency of p120/cadherin interaction. Here we have utilized novel phospho-specific monoclonal antibodies to four major p120 serine and threonine phosphorylation sites to monitor individual phosphorylation events and their consequences. Surprisingly, membrane-localization and not cadherin interaction is the main determinant in p120 serine and threonine phosphorylation and dephosphorylation. Furthermore, the phospho-status of these four residues had no obvious effect on p120's role in cadherin complex stabilization or cell-cell adhesion. Interestingly, dephosphorylation was dramatically induced by PKC activation, but PKC-independent pathways were also evident. The data suggest that p120 dephosphorylation at these sites is modulated by multiple cell surface receptors primarily through PKC-dependent pathways, but these changes do not seem to reduce p120/cadherin affinity.     

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.     

Histone H3 lysine 4 monomethylation (H3K4me1) and H3 lysine 9 monomethylation (H3K9me1): Distribution and their association in regulating gene expression under hyperglycaemic/hyperinsulinemic conditions in 3T3 cells

Hyperglycemia/hyperinsulinemia are leading cause for the induction type 2 diabetes and the role of post-translational histone modifications in dysregulating the expression of genes has emerged as potential important contributor in the progression of disease. The paradoxical nature of histone H3-Lysine 4 and Lysine 9 mono-methylation (H3K4me1 and H3K9me1) in both gene activation and repression motivated us to elucidate the functional relationship of these histone modifications in regulating expression of genes under hyperglycaemic/hyperinsulinemic condition. Chromatin immunoprecipitation–microarray analysis (ChIP-chip) was performed with H3 acetylation, H3K4me1 and H3K9me1 antibody. CLUSTER analysis of ChIP-chip (Chromatin immunoprecipitation–microarray analysis) data showed that mRNA expression and H3 acetylation/H3K4me1 levels on genes were inversely correlated with H3K9me1 levels on the transcribed regions, after 30 min of insulin stimulation under hyperglycaemic condition. Interestingly, we provide first evidence regarding regulation of histone de/acetylases and de/methylases; Myst4, Jmjd2b, Aof1 and Set by H3Ac, H3K4me1 and H3K9me1 under hyperinsulinemic/hyperglycaemic condition. ChIP–qPCR analysis shows association of increased H3Ac/H3K4me1 and decreased levels of H3K9me1 in up regulation of Myst4, Jmjd2, Set and Aof1 genes. We further analyse promoter occupancy of histone modifications by ChIP walking and observed increased occupancy of H3Ac/H3K4me1 on promoter region (−1000 to −1) of active genes and H3K9me1 on inactive genes under hyperglycemic/hyperinsulinemic condition. To best of our knowledge this is the first report that shows regulation of chromatin remodelling genes by alteration in the occupancy of histone H3Ac/H3K4/K9me on both promoter and transcribed regions.     

Increases in intracellular calcium dephosphorylate histone H3 at serine 10 in human hepatoma cells: potential role of protein phosphatase 2A-protein kinase CbetaII complex

We present evidence that increases in intracellular calcium, induced by treatment with calcium ionophore A23187 or the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, dephosphorylated histone H3 at serine10 (histone H3-Ser10) in a dose-dependent manner in human hepatoma HepG2 cells. Inhibition of p42/44MAPK, pp90RSK, or p38MAPK did not affect the ability of A23187 to dephosphorylate histone H3-Ser10. This response is significantly blocked by okadaic acid, indicating a requirement for protein phosphatase 2A (PP2A). A23187 increased the activity of PP2A towards phosphorylated histone H3-Ser10. Furthermore, pretreatment with calphostin C, a selective protein kinase C (PKC) inhibitor, blocked A23187-dependent dephosphorylation of histone H3-Ser10, and coimmunoprecipitation analysis showed PP2A association with the PKCbetaII isoform. Unlike untreated cells, coimmunoprecipitated complex from A23187-treated cells showed greater dephosphorylation of histone H3-Ser10 in a PP2A-dependent manner. Inhibition of PP2A increased phosphorylation at Ser660 that determines calcium sensitivity and activity of PKCbetaII isoform, thus supporting a role for intracomplex regulation. Finally, chromatin immunoprecipitation assays following exposure to A23187 and okadaic acid revealed regulatory role of histone H3-Ser10 phosphorylation in selective gene induction. Altogether, our findings suggest a novel role for calcium in modulating histone H3-Ser10 phosphorylation level and led us to propose a model emphasizing PP2A activation, occurring downstream following perturbations in calcium homeostasis, as key event in dephosphorylating histone H3-Ser10 in mammalian cells.     

A native peptide ligation strategy for deciphering nucleosomal histone modifications

Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins. A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications. Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications. As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities. Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent with peptide studies. However, in contrast to analyses using peptide substrates, we find that the histone acetyltransferase activity of the Gcn5-containing SAGA complex is not stimulated by H3 phosphorylation in the context of nucleosomal arrays. This difference between peptide and array substrates suggests that the ability to generate specifically modified nucleosomal arrays should provide a powerful tool for understanding the effects of post-translational histone modifications.     

Eleostearic acid induces RIP1-mediated atypical apoptosis in a kinase-independent manner via ERK phosphorylation,ROS generation and mitochondrial dysfunction

RIP1 is a serine/threonine kinase, which is involved in apoptosis and necroptosis. In apoptosis, caspase-8 and FADD have an important role. On the other hand, RIP3 is a key molecule in necroptosis. Recently, we reported that eleostearic acid (ESA) elicits caspase-3- and PARP-1-independent cell death, although ESA-treated cells mediate typical apoptotic morphology such as chromatin condensation, plasma membrane blebbing and apoptotic body formation. The activation of caspases, Bax and PARP-1, the cleavage of AIF and the phosphorylation of histone H2AX, all of which are characteristics of typical apoptosis, do not occur in ESA-treated cells. However, the underlying mechanism remains unclear. To clarify the signaling pathways in ESA-mediated apoptosis, we investigated the functions of RIP1, MEK, ERK, as well as AIF. Using an extensive study based on molecular biology, we identified the alternative role of RIP1 in ESA-mediated apoptosis. ESA mediates RIP1-dependent apoptosis in a kinase independent manner. ESA activates serine/threonine phosphatases such as calcineurin, which induces RIP1 dephosphorylation, thereby ERK pathway is activated. Consequently, localization of AIF and ERK in the nucleus, ROS generation and ATP reduction in mitochondria are induced to disrupt mitochondrial cristae, which leads to cell death. Necrostatin (Nec)-1 blocked MEK/ERK phosphorylation and ESA-mediated apoptosis. Nec-1 inactive form (Nec1i) also impaired ESA-mediated apoptosis. Nec1 blocked the interaction of MEK with ERK upon ESA stimulation. Together, these findings provide a new finding that ERK and kinase-independent RIP1 proteins are implicated in atypical ESA-mediated apoptosis.     

Evolution of histone H3: emergence of variants and conservation of post-translational modification sites

Histone H3 proteins are highly conserved across all eukaryotes and are dynamically modified by many post-translational modifications (PTMs). Here we describe a method that defines the evolution of the family of histone H3 proteins, including the emergence of functionally distinct variants. It combines information from histone H3 protein sequences in eukaryotic species with the evolution of these species as described by the tree of life (TOL) project. This so-called TOL analysis identified the time when the few observed protein sequence changes occurred and when distinct, co-existing H3 protein variants arose. Four distinct ancient duplication events were identified where replication-coupled (RC) H3 variants diverged from replication-independent (RI) forms, like histone H3.3 in animals. These independent events occurred in ancestral lineages leading to the clades of metazoa, viridiplantae, basidiomycota, and alveolata. The proto-H3 sequence in the last eukaryotic common ancestor (LECA) was expanded to at least 133 of its 135 residues. Extreme conservation of known acetylation and methylation sites of lysines and arginines predicts that these PTMs will exist across the eukaryotic crown phyla and in protists with canonical chromatin structures. Less complete conservation was found for most serine and threonine phosphorylation sites. This study demonstrates that TOL analysis can determine the evolution of slowly evolving proteins in sequence-saturated datasets.