Middle‐Down Characterization of the Cell Cycle Dependence of Histone H4 Posttranslational Modifications and Proteoforms

Post‐translational modifications (PTMs) of histones are important epigenetic regulatory mechanisms that are often dysregulated in cancer. We employ middle‐down proteomics to investigate the PTMs and proteoforms of histone H4 during cell cycle progression. We use pH gradient weak cation exchange‐hydrophilic interaction liquid chromatography (WCX‐HILIC) for on‐line liquid chromatography‐mass spectrometry analysis to separate and analyze the proteoforms of histone H4. This procedure provides enhanced separation of proteoforms, including positional isomers, and simplifies downstream data analysis. We use ultrahigh mass accuracy and resolution Fourier transform‐ion cyclotron resonance (FT‐ICR) mass spectrometer to unambiguously distinguish between acetylation and tri‐methylation (?m = 0.036 Da). In total, we identify and quantify 233 proteoforms of histone H4 in two breast cancer cell lines. We observe significant increases in S1 phosphorylation during mitosis, implicating an important role in mitotic chromatin condensation. A decrease of K20 unmodified proteoforms is observed as the cell cycle progresses, corresponding to an increase of K20 mono‐ and di‐methylation. Acetylation at K5, K8, K12, and K16 declines as cells traverse from S phase to mitosis, suggesting cell cycle–dependence and an important role during chromatin replication and condensation. These new insights into the epigenetics of the cell cycle may provide new diagnostic and prognostic biomarkers.     

Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1.     

The histone deacetylase inhibitor butyrate improves metabolism and reduces muscle atrophy during aging

Sarcopenia, the loss of skeletal muscle mass and function during aging, is a major contributor to disability and frailty in the elderly. Previous studies found a protective effect of reduced histone deacetylase activity in models of neurogenic muscle atrophy. Because loss of muscle mass during aging is associated with loss of motor neuron innervation, we investigated the potential for the histone deacetylase (HDAC) inhibitor butyrate to modulate age‐related muscle loss. Consistent with previous studies, we found significant loss of hindlimb muscle mass in 26‐month‐old C57Bl/6 female mice fed a control diet. Butyrate treatment starting at 16 months of age wholly or partially protected against muscle atrophy in hindlimb muscles. Butyrate increased muscle fiber cross‐sectional area and prevented intramuscular fat accumulation in the old mice. In addition to the protective effect on muscle mass, butyrate reduced fat mass and improved glucose metabolism in 26‐month‐old mice as determined by a glucose tolerance test. Furthermore, butyrate increased markers of mitochondrial biogenesis in skeletal muscle and whole‐body oxygen consumption without affecting activity. The increase in mass in butyrate‐treated mice was not due to reduced ubiquitin‐mediated proteasomal degradation. However, butyrate reduced markers of oxidative stress and apoptosis and altered antioxidant enzyme activity. Our data is the first to show a beneficial effect of butyrate on muscle mass during aging and suggests HDACs contribute to age‐related muscle atrophy and may be effective targets for intervention in sarcopenia and age‐related metabolic disease.     

“Direct” and “Indirect” Effects of Histone Modifications: Modulation of Sterical Bulk as a Novel Source of Functionality

The chromatin-regulatory principles of histone post-translational modifications (PTMs) are discussed with a focus on the potential alterations in chromatin functional state due to steric and mechanical constraints imposed by bulky histone modifications such as ubiquitin and SUMO. In the classical view, PTMs operate as recruitment platforms for histone “readers,” and as determinants of chromatin array compaction. Alterations of histone charges by “small” chemical modifications (e.g., acetylation, phosphorylation) could regulate nucleosome spontaneous dynamics without globally affecting nucleosome structure. These fluctuations in nucleosome wrapping can be exploited by chromatin-processing machinery. In contrast, ubiquitin and SUMO are comparable in size to histones, and it seems logical that these PTMs could conflict with canonical nucleosome organization. An experimentally testable hypothesis that by adding sterical bulk these PTMs can robustly alter nucleosome primary structure is proposed. The model presented here stresses the diversity of mechanisms by which histone PTMs regulate chromatin dynamics, primary structure and, hence, functionality.     

Estrogen,through estrogen receptor 1, regulates histone modifications and chromatin remodeling during spermatogenesis in adult rats

Estrogen receptors (ESR1 and ESR2) play crucial roles in various processes during spermatogenesis. To elucidate individual roles of ESRs in male fertility, we developed in vivo selective ESR agonist administration models. Adult male rats treated with ESR1 and ESR2 agonist for 60 days show spermatogenic defects leading to reduced sperm counts and fertility. While studying epigenetic changes in the male germ line that could have affected fertility, we earlier observed a decrease in DNA methylation and its machinery upon ESR2 agonist treatment. Here, we explored the effects on histone modifications, which could contribute to decreased male fertility upon ESR agonist administration. ESR1 agonist treatment affected testicular levels of histone modifications associated with active and repressed chromatin states, along with heterochromatin marks. This was concomitant with deregulation of corresponding histone modifying enzymes in the testis. In addition, there was increased retention of histones along with protamine deficiency in the caudal spermatozoa after ESR1 agonist treatment. This could be due to the observed decrease in several chromatin remodeling proteins implicated in mediating histone-to-protamine exchange during spermiogenesis. The activating and repressing histone marks in spermatozoa, which play a critical role in early embryo development, were deregulated after both the ESR agonist treatments. Together, these epigenetic defects in the male germ line could affect the spermatozoa quality and lead to the observed decrease in fertility. Our results thus highlight the importance of ESRs in regulating different epigenetic processes during spermatogenesis, which are crucial for male fertility.     

Posttranslational Modifications of the Histone 3 Tail and Their Impact on the Activity of Histone Lysine Demethylases In Vitro

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k _cat/K _m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K _m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.     

植物PHD结构域蛋白的结构与功能特性

植物同源结构域(plant homeodomain,PHD)是锌指结构域家族的一类转录调控因子,其最主要的功能是可以识别各种组蛋白修饰密码,包括组蛋白甲基化和乙酰化等;此外PHD结构域还可以与DNA结合.含有PHD结构域的蛋白,或者本身具有组蛋白修饰酶活性,或者可以与各类组蛋白修饰酶相互作用,还有部分与DNA甲基化相关...     

Enhanced top-down characterization of histone post-translational modifications

Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.     

ProSight Annotator: Complete control and customization of protein entries in UniProt XML files

The effectiveness of any proteomics database search depends on the theoretical candidate information contained in the protein database. Unfortunately, candidate entries from protein databases such as UniProt rarely contain all the post-translational modifications (PTMs), disulfide bonds, or endogenous cleavages of interest to researchers. These omissions can limit discovery of novel and biologically important proteoforms. Conversely, searching for a specific proteoform becomes a computationally difficult task for heavily modified proteins. Both situations require updates to the database through user-annotated entries. Unfortunately, manually creating properly formatted UniProt Extensible Markup Language (XML) files is tedious and prone to errors. ProSight Annotator solves these issues by providing a graphical interface for adding user-defined features to UniProt-formatted XML files for better informed proteoform searches. It can be downloaded from http://prosightannotator.northwestern.edu .     

组蛋白泛素化修饰及其在DNA损伤应答中的作用

泛素化修饰是真核生物细胞内重要的翻译后修饰类型,通过调节蛋白质活性、稳定性和亚细胞定位广泛参与细胞内各项信号传导与代谢过程,对维持正常生命活动具有重要意义。组蛋白作为染色质中主要的蛋白成分,与DNA复制转录、修复等行为密切相关,是研究翻译后修饰的热点。DNA损伤后,组蛋白泛素化修饰通过调节核小体结构、激活细胞周期检查点、影响修复因子的招募与装配等诸多途径参与损伤应答。同时,组蛋白泛素化修饰还能调节其他位点翻译后修饰,并通过这种串扰(crosstalk)作用调节DNA损伤应答。本文介绍了组蛋白泛素化修饰的主要位点和相关组分(包括E3连接酶、去泛素化酶与效应分子),以及这些修饰作用共同编译形成的信号网络在DNA损伤应答中的作用,最后总结了目前该领域研究所面临的一些问题,以期为科研人员进一步探索组蛋白密码在DNA损伤应答中的作用提供参考。