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Identification of Lysine 37 of Histone H2B as a Novel Site of Methylation  [PDF]
Kathryn E. Gardner,Li Zhou,Michael A. Parra,Xian Chen,Brian D. Strahl
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0016244
Abstract: Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the complex number of histone modifications that contribute to chromatin function.
Global turnover of histone post-translational modifications and variants in human cells
Barry M Zee, Rebecca S Levin, Peter A DiMaggio, Benjamin A Garcia
Epigenetics & Chromatin , 2010, DOI: 10.1186/1756-8935-3-22
Abstract: In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC) pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same peptide. Lastly, we observed little difference in the turnover between nearly all modified forms of the H3.1, H3.2 and H3.3 variants, with the notable exception that H3.2K36me2 has a faster turnover than this mark on the other H3 variants.Quantitative proteomics provides complementary insight to previous work aimed at quantitatively measuring histone turnover, and our results suggest that turnover rates are dependent upon site-specific post-translational modifications and sequence variants.In eukaryotes, stable genetic storage is accomplished through the local organization of DNA around histone proteins to form the chromatin fiber. Histones have been long recognized as the structural scaffolds of chromatin, but more recent research has suggested that they possess a broader role. The epigenetic influence of histones is mediated primarily by post-translational modifications (PTMs) and also by selective deposition of histone variants, which in combination influence gene transcription
Histone Variants and Their Post-Translational Modifications in Primary Human Fat Cells  [PDF]
?sa Jufvas,Peter Str?lfors,Alexander V. Vener
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015960
Abstract: Epigenetic changes related to human disease cannot be fully addressed by studies of cells from cultures or from other mammals. We isolated human fat cells from subcutaneous abdominal fat tissue of female subjects and extracted histones from either purified nuclei or intact cells. Direct acid extraction of whole adipocytes was more efficient, yielding about 100 μg of protein with histone content of 60% –70% from 10 mL of fat cells. Differential proteolysis of the protein extracts by trypsin or ArgC-protease followed by nanoLC/MS/MS with alternating CID/ETD peptide sequencing identified 19 histone variants. Four variants were found at the protein level for the first time; particularly HIST2H4B was identified besides the only H4 isoform earlier known to be expressed in humans. Three of the found H2A potentially organize small nucleosomes in transcriptionally active chromatin, while two H2AFY variants inactivate X chromosome in female cells. HIST1H2BA and three of the identified H1 variants had earlier been described only as oocyte or testis specific histones. H2AFX and H2AFY revealed differential and variable N-terminal processing. Out of 78 histone modifications by acetylation/trimethylation, methylation, dimethylation, phosphorylation and ubiquitination, identified from six subjects, 68 were found for the first time. Only 23 of these modifications were detected in two or more subjects, while all the others were individual specific. The direct acid extraction of adipocytes allows for personal epigenetic analyses of human fat tissue, for profiling of histone modifications related to obesity, diabetes and metabolic syndrome, as well as for selection of individual medical treatments.
Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications
Tao Wu, Tiezheng Yuan, Sau-Na Tsai, Chunmei Wang, Sai-Ming Sun, Hon-Ming Lam, Sai-Ming Ngai
BMC Plant Biology , 2009, DOI: 10.1186/1471-2229-9-98
Abstract: In soybean leaves, mono-, di- and tri-methylation at Lysine 4, Lysine 27 and Lysine 36, and acetylation at Lysine 14, 18 and 23 were detected in HISTONE H3. Lysine 27 was prone to being mono-methylated, while tri-methylation was predominant at Lysine 36. We also observed that Lysine 27 methylation and Lysine 36 methylation usually excluded each other in HISTONE H3. Although methylation at HISTONE H3 Lysine 79 was not reported in A. thaliana, mono- and di-methylated HISTONE H3 Lysine 79 were detected in soybean. Besides, acetylation at Lysine 8 and 12 of HISTONE H4 in soybean were identified. Using a combination of mass spectrometry and nano-liquid chromatography, two variants of HISTONE H3 were detected and their modifications were determined. They were different at positions of A31F41S87S90 (HISTONE variant H3.1) and T31Y41H87L90 (HISTONE variant H3.2), respectively. The methylation patterns in these two HISTONE H3 variants also exhibited differences. Lysine 4 and Lysine 36 methylation were only detected in HISTONE H3.2, suggesting that HISTONE variant H3.2 might be associated with actively transcribing genes. In addition, two variants of histone H4 (H4.1 and H4.2) were also detected, which were missing in other organisms. In the histone variant H4.1 and H4.2, the amino acid 60 was isoleucine and valine, respectively.This work revealed several distinct variants of soybean histone and their modifications that were different from A. thaliana, thus providing important biological information toward further understanding of the histone modifications and their functional significance in higher plants.Histone modifications and histone variants play critical roles in regulating gene expression, modulating the cell cycle, and are responsible for maintaining genome stability [1-3]. The fundamental structural unit of chromatin in eukaryotic cells is the nucleosome, that consists of 146 base pairs (bp) of DNA wrapped around a histone octamer, each of which is formed by two cop
Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies
Chih Liu, Stephanie Tangsombatvisit, Jacob M Rosenberg, Gil Mandelbaum, Emily C Gillespie, Or P Gozani, Ash A Alizadeh, Paul J Utz
Arthritis Research & Therapy , 2012, DOI: 10.1186/ar3707
Abstract: We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen.We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE.Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified.Neutrophil extracellular traps (NETs) were first described in 2004 as web-like structures that trap and neutralize microbes at sites of infection [1]. Neutrophils, a first line of defense against microorganisms during such encounters, produce these highly modified chromatin webs through a cellular suicide program distinct from apoptosis and necrosis, termed "NETosis" [2]. In addition to neutrophil antimicrobial proteins, NETs are comprised of chromatin components, including histones. Because NETs are extracellular and typically in an inflammatory environment, their proximity to components of the adaptive and innate immune systems might provide an immunogenic substrate for autoimmune responses during regular encounters with commensal and pathogenic microbes.Indeed, an emerging and growing body of literature supports a putative link bet
Post-translational modifications of histones H3 and H4 associated with the histone methyltransferases Suv39h1 and G9a
Philippe Robin, Lauriane Fritsch, Ophélie Philipot, Fedor Svinarchuk, Slimane Ait-Si-Ali
Genome Biology , 2007, DOI: 10.1186/gb-2007-8-12-r270
Abstract: The amino-terminal tails of nucleosomal histones protrude from the DNA and are subject to covalent modifications. These modifications include lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, ADP-ribosylation, and ubiquitination [1]. Histone lysine methylation can have different effects depending on the residue that is modified: methylation of histone H3 at Lys4 (H3K4) is associated with gene activation, whereas methylation of H3K9, H3K27, and H4K20 generally correlates with transcriptional repression [2-4]. The roles of H3K36 and H3K79 methylation remain elusive; indeed, these modifications are associated with both transcriptional activation and repression [5,6].Lysine residues can be mono-, di-, or trimethylated, inducing different biological responses [3,7,8]. Thus, for example, highly condensed heterochromatic regions show a high degree of trimethylated H3K9 (H3K9me3), whereas euchromatic regions are preferentially enriched in mono- and dimethylated H3K9 [2,3]. Histone lysine methylation is mediated by histone methyltransferases (HMTs), many of which contain a conserved SET [Su(var)3-9, Enhancer-of-zeste, Trithorax] domain, such as Suv39h1 (Suppressor of variegation 39h1) and G9a [1,2,9]. Suv39h1 belongs to a family of peri-centromeric proteins and is responsible for H3K9 trimethylation [10-13]. G9a (EuHMTase-2) is the major methylase responsible for mono- and dimethylation of H3K9 in euchromatic regions [14,15], but it may also be present in heterochomatic regions [16].Covalent modifications of histones can regulate gene expression directly or through recruitment of non-histone effector proteins [2,17]. These effector proteins bind modified chromatin using a variety of chromatin-binding domains. For example, bromodomains recognize acetylated lysines, whereas chromo, MBT, Tudor, W40 domains and PHD fingers, recognize methylated lysines [17,18]. Repressive methyl-lysine modifications are recognized by chromodomain-containi
In Vivo Chromatin Organization of Mouse Rod Photoreceptors Correlates with Histone Modifications  [PDF]
Caroline Kizilyaprak,Danièle Spehner,Didier Devys,Patrick Schultz
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0011039
Abstract: The folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo chromatin structure, histone modifications and transcriptional activity.
Mass Spectrometry-Based Proteomics for the Analysis of Chromatin Structure and Dynamics  [PDF]
Monica Soldi,Alessandro Cuomo,Michael Bremang,Tiziana Bonaldi
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms14035402
Abstract: Chromatin is a highly structured nucleoprotein complex made of histone proteins and DNA that controls nearly all DNA-dependent processes. Chromatin plasticity is regulated by different associated proteins, post-translational modifications on histones (hPTMs) and DNA methylation, which act in a concerted manner to enforce a specific “chromatin landscape”, with a regulatory effect on gene expression. Mass Spectrometry (MS) has emerged as a powerful analytical strategy to detect histone PTMs, revealing interplays between neighbouring PTMs and enabling screens for their readers in a comprehensive and quantitative fashion. Here we provide an overview of the recent achievements of state-of-the-art mass spectrometry-based proteomics for the detailed qualitative and quantitative characterization of histone post-translational modifications, histone variants, and global interactomes at specific chromatin regions. This synopsis emphasizes how the advances in high resolution MS, from “Bottom Up” to “Top Down” analysis, together with the uptake of quantitative proteomics methods by chromatin biologists, have made MS a well-established method in the epigenetics field, enabling the acquisition of original information, highly complementary to that offered by more conventional, antibody-based, assays.
A tale of histone modifications
Patrick A Grant
Genome Biology , 2001, DOI: 10.1186/gb-2001-2-4-reviews0003
Abstract: In eukaryotes, genomic DNA is packaged with histone proteins into chromatin, compacting DNA some 10,000-fold. Such condensation of DNA provides a considerable obstacle to the nuclear machinery that drives processes such as replication, transcription or DNA repair. Importantly, the structure of chromatin dynamically changes, permitting localized decondensation and remodeling that facilitates the progress of nuclear machinery. An emerging theme in the field of chromatin research has been the significant role that posttranslational modifications of histones play in regulating nuclear function. Over the past few years, considerable progress has been made into the identification of the enzymatic machines that modify these proteins, and this review is devoted to our current understanding of the array of core histone modifications and the factors that regulate them.The basic repeating unit of chromatin is the nucleosome, typically composed of an octamer of the four core histones H2A, H2B, H3 and H4 and 146 basepairs of DNA wrapped around the histones [1]. Each core histone is composed of a structured domain and an unstructured amino-terminal 'tail' of 25-40 residues. This unstructured tail extends through the DNA gyres and into the space surrounding the nucleosomes. Histone tails provide sites for a variety of posttranslational modifications, including acetylation, phosphorylation and methylation. It is becoming increasingly apparent that such modifications of histone tails determine the interactions of histones with other proteins, which may in turn also regulate chromatin structure [2]. Identifying the multitude of histone modifications, the enzymes that generate them and the nuclear response to any given pattern of alterations poses a fascinating challenge.The acetylation and deacetylation of the ε-amino groups of conserved lysine residues present in histone tails has long been linked to transcriptional activity [3] and has been the most intensively studied histone modi
Histone modifications: from genome-wide maps to functional insights
Fred van Leeuwen, Bas van Steensel
Genome Biology , 2005, DOI: 10.1186/gb-2005-6-6-113
Abstract: The DNA in the nucleus of a eukaryotic cell is packed into chromatin, the fundamental building block of which, the nucleosome, consists of an octamer of the four histones H2A, H2B, H3 and H4, around which the DNA is wrapped. The histones within chromatin are subject to extensive post-translational modification, including acetylation, methylation, phosphorylation, ubiquitination, and ribosylation. Many enzymes have been identified that are responsible for the addition or removal of modifications at one or a few specific histone amino-acid residues, and many histone modifications are believed to play important roles in the regulation of transcription. Although some histone modifications may cause alterations in the structure or overall charge of the nucleosome [1,2], it is likely that most act by controlling the docking of specific regulatory factors. For example, the chromodomain of heterochromatin protein 1 (HP1) binds to the tail of histone H3 only when lysine 9 of H3 (H3K9) is methylated, and this may contribute to repression of transcription [3]. Similarly, the bromodomains of various transcriptional activators and nucleosome-remodeling factors recognize specific acetylated lysine residues within histone H3 or H4 [4]. While some modifications attract specific regulatory factors, others appear to block protein binding. This is illustrated by the inhibitory effect of acetylation on the binding of the silencing protein Sir3 to histone H4 [5]. Undoubtedly, many more factors will be discovered that recognize particular modification states of histones.Given the large number of histone modifications that appear to be involved in the control of gene expression, the integration of their regulatory roles is an important issue. How do these modifications work together? For example, H3K4 methylation and H3K9 acetylation have both been implicated in gene activation; do these two modifications typically act together on a common set of genes, or are they part of separate signal
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