Histone Molecules Research Articles

Molecular Mechanism of Protein Arginine Deiminase 2: A Study Involving Multiple Microsecond Long Molecular Dynamics Simulations.

Peptidylarginine deiminase 2 (PAD2) is a Ca2+-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. This kind of structural modification in histone molecules may affect gene regulation, leading to effects that may trigger several diseases, including breast cancer, which makes PAD2 an attractive target for anticancer drug development. To design new effective inhibitors to control activation of PAD2, improving our understanding of the molecular mechanisms of PAD2 using up-to-date computational techniques is essential. We have designed five different PAD2–substrate complex systems based on varying protonation states of the active site residues. To search the conformational space broadly, multiple independent molecular dynamics simulations of the complexes have been performed. In total, 50 replica simulations have been performed, each of 1 μs, yielding a total simulation time of 50 μs. Our findings identify that the protonation states of Cys647, Asp473, and His471 are critical for the binding and localization of the N-α-benzoyl-l-arginine ethyl ester substrate within the active site. A novel mechanism for enzyme activation is proposed according to near attack conformers. This represents an important step in understanding the mechanism of citrullination and developing PAD2-inhibiting drugs for the treatment of breast cancer.

An Efficient Chromatin Immunoprecipitation Protocol for the Analysis of Histone Modification Distributions in the Brown Alga Ectocarpus.

The brown algae are an important but understudied group of multicellular marine organisms. A number of genetic and genomic tools have been developed for the model brown alga Ectocarpus; this includes, most recently, chromatin immunoprecipitation methodology, which allows genome-wide detection and analysis of histone post-translational modifications. Post-translational modifications of histone molecules have been shown to play an important role in gene regulation in organisms from other major eukaryotic lineages, and this methodology will therefore be a very useful tool to investigate genome function in the brown algae. This article provides a detailed, step-by-step description of the Ectocarpus ChIP protocol, which effectively addresses the difficult problem of efficiently extracting chromatin from cells protected by a highly resistant cell wall. The protocol described here will be an essential tool for the future application of chromatin analysis methodologies in brown algal research.

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Overexpression of Replication-Dependent Histone Signifies a Subset of Dedifferentiated Liposarcoma with Increased Aggressiveness.

Simple SummaryAlthough the genetic event that initiates liposarcoma is relatively well studied, understanding how the tumor progresses and displays differential severities will shed new insights into the diagnosis and prognosis determination of patients with liposarcoma. We analyzed the genome and transcriptome of liposarcoma samples and found that the overexpression of the histone gene cluster is an important determinant for dedifferentiated liposarcoma in predicting genome instability and cell proliferation. The overexpression of histone genes was caused by increased HMGA2 with amplification of chromosome 12q13-14. This dedifferentiated liposarcoma-specific genetic signature may serve as a biomarker for prognosis prediction.Liposarcoma (LPS) is an adult soft tissue malignancy that arises from fat tissue, where well-differentiated (WD) and dedifferentiated (DD) forms are the most common. DDLPS represents the progression of WDLPS into a more aggressive high-grade and metastatic form. Although a few DNA copy-number amplifications are known to be specifically found in WD- or DDLPS, systematic genetic differences that signify subtype determination between WDLPS and DDLPS remain unclear. Here, we profiled the genome and transcriptome of 38 LPS tumors to uncover the genetic signatures of subtype differences. Replication-dependent histone (RD-HIST) mRNAs were highly elevated and their regulation was disrupted in a subset of DDLPS, increasing cellular histone molecule levels, as measured using RNA-seq (the averaged fold change of 53 RD-HIST genes between the DD and WD samples was 10.9) and immunohistochemistry. The change was not observed in normal tissues. Integrated whole-exome sequencing, RNA-seq, and methylation analyses revealed that the overexpressed HMGA2 (the fold change between DD and WD samples was 7.3) was responsible for the increased RD-HIST level, leading to aberrant cell proliferation. Therefore, HMGA2-mediated elevation of RD-HISTs were crucial events in determining the aggressiveness of DDLPS, which may serve as a biomarker for prognosis prediction for liposarcoma patients.

Surface structures consisting of chromatin fibers in isolated barley (Hordeum vulgare) chromosomes revealed by helium ion microscopy.

The chromosome compaction of chromatin fibers results in the formation of the nucleosome, which consists of a DNA unit coiled around a core of histone molecules associated with linker histone. The compaction of chromatin fibers has been a topic of controversy since the discovery of chromosomes in the 19th century. Although chromatin fibers were first identified using electron microscopy, the chromatin fibers on the surface of chromosome structures in plants remain unclear due to shrinking and breaking caused by prior chromosome isolation or preparation with alcohol and acid fixation, and critical point drying occurred into dehydration and denatured chromosomal proteins. This study aimed to develop a high-quality procedure for the isolation and preparation of plant chromosomes, maintaining the native chromosome structure, to elucidate the organization of chromatin fibers on the surface of plant chromosomes by electron microscopy. A simple technique to isolate intact barley (Hordeum vulgare) chromosomes with a high yield was developed, allowing chromosomes to be observed with a high-resolution scanning ion microscopy and helium ion microscopy (HIM) imaging technology, based on a scanning helium ion beam. HIM images from the surface chromatin fibers were analyzed to determine the size and alignment of the chromatin fibers. The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Our findings indicate that compacting the surface structure of barley via a chromatin network and observation via HIM are powerful tools for investigating the structure of chromatin.

Combinations of histone post-translational modifications.

Histones are essential proteins that package the eukaryotic genome into its physiological state of nucleosomes, chromatin, and chromosomes. Post-translational modifications (PTMs) of histones are crucial to both the dynamic and persistent regulation of the genome. Histone PTMs store and convey complex signals about the state of the genome. This is often achieved by multiple variable PTM sites, occupied or unoccupied, on the same histone molecule or nucleosome functioning in concert. These mechanisms are supported by the structures of 'readers' that transduce the signal from the presence or absence of PTMs in specific cellular contexts. We provide background on PTMs and their complexes, review the known combinatorial function of PTMs, and assess the value and limitations of common approaches to measure combinatorial PTMs. This review serves as both a reference and a path forward to investigate combinatorial PTM functions, discover new synergies, and gather additional evidence supporting that combinations of histone PTMs are the central currency of chromatin-mediated regulation of the genome.

The atomic interactions between Histone and 3LPT protein using an equilibrium molecular dynamics simulation

The atomic interactions between histone molecule and 3LPT protein are reported using the molecular dynamics (MD) method in this computational work. This computational study is done for the first time. The results of the MD simulations on the dynamical manner of these structures have been reported by calculating various physical parameters. Physically, our results show that the attraction forces between the histone molecule and 3LPT protein destructed the 3LPT protein after 2 ns. Further, the temperature and pressure of these structures are important parameters in their atomic interactions. Numerically, by increasing these two parameters from 300 K to 350 K and 0 to 1 bar, respectively, the interatomic distance between these structures decreases from 7 Å to 2.54 Å and 7 Å to 2.79 Å (respectively). Further, bonding energy in these simulations varies from 23.51 eV to 31.78 eV. The diffusion coefficient of histone structure into 3LPT protein is another important parameter. Numerically, this physical coefficient changes from 0.58μm2/s to 0.89 μm2/s.

In silico and in vitro Evaluation of Mimetic Peptides as Potential Antigen Candidates for Prophylaxis of Leishmaniosis.

Antigen formulation is the main feature for the success of leishmaniosis diagnosis and vaccination, since the disease is caused by different parasite species that display particularities which determine their pathogenicity and virulence. It is desirable that the antigens are recognized by different antibodies and are immunogenic for almost all Leishmania species. To overcome this problem, we selected six potentially immunogenic peptides derived from Leishmania histones and parasite membrane molecules obtained by phage display or spot synthesis and entrapped in liposome structures. We used these peptides to immunize New Zealand rabbits and determine the immunogenic capacity of the chimeric antigen. The peptides induced the production of antibodies as a humoral immune response against L. braziliensis or L. infantum. Next, to evaluate the innate response to induce cellular activation, macrophages from the peptide mix-immunized rabbits were infected in vitro with L. braziliensis or L. infantum. The peptide mix generated the IFN-γ, IL-12, IL-4 and TGF-β that led to Th1 and Th2 cellular immune responses. Interestingly, this mix of peptides also induced high expression of iNOS. These results suggest that the mix of peptides derived from histone and parasites membrane molecules was able to mimic parasites proteins and induce cytokines important to CD4+ T cell Th1 and Th2 differentiation and effector molecule to control the parasite infection. Finally, this peptide induced an immune balance that is important to prevent immunopathological disorders, inflammatory reactions, and control the parasite infection.

Visualization of DNA Replication in Single Chromosome by Stable Isotope Labeling

Among the inheritance of cellular components during cell division, deoxyribonucleic acid (DNA) and its condensate (chromosome) are conventionally visualized using chemical tag-labeled nucleotide analogs. However, associated mutagenesis with nucleotide analogs in the visualization of chromosomes is cause for concern. This study investigated the efficiency of using stable isotope labels in visualizing the replicating cultured human cell-chromosomes, in the absence of analog labels, at a high spatial resolution of 100 nm. The distinct carbon isotope ratio between sister chromatids reflected the semi-conservative replication of individual DNA strands through cell cycles and suggested the renewal of histone molecules in daughter chromosomes. Thus, this study provides a new, powerful approach to trace and visualize cellular components with stable isotope labeling.Key words: stable isotope, chromosome replication, semi-conservative replication, imaging, mass spectrometry.

Transcriptional regulation by methyltransferases and their role in the heart: highlighting novel emerging functionality.

Methyltransferases are a superfamily of enzymes that transfer methyl groups to proteins, nucleic acids, and small molecules. Traditionally, these enzymes have been shown to carry out a specific modification (mono-, di-, or trimethylation) on a single, or limited number of, amino acid(s). The largest subgroup of this family, protein methyltransferases, target arginine and lysine side chains of histone molecules to regulate gene expression. Although there is a large number of functional studies that have been performed on individual methyltransferases describing their methylation targets and effects on biological processes, no analyses exist describing the spatial distribution across tissues or their differential expression in the diseased heart. For this review, we performed tissue profiling in protein databases of 199 confirmed or putative methyltransferases to demonstrate the unique tissue-specific expression of these individual proteins. In addition, we examined transcript data sets from human heart failure patients and murine models of heart disease to identify 40 methyltransferases in humans and 15 in mice, which are differentially regulated in the heart, although many have never been functionally interrogated. Lastly, we focused our analysis on the largest subgroup, that of protein methyltransferases, and present a newly emerging phenomenon in which 16 of these enzymes have been shown to play dual roles in regulating transcription by maintaining the ability to both activate and repress transcription through methyltransferase-dependent or -independent mechanisms. Overall, this review highlights a novel paradigm shift in our understanding of the function of histone methyltransferases and correlates their expression in heart disease.

Visualization of the dynamics of histone modifications and their crosstalk using PTM-CrossTalkMapper.

Visualization of large-scale, multi-dimensional omics data is a major challenge. Experimental study designs in proteomics research produce multiple data layers, e.g. relationships between hundreds of proteins, their interactions and abundances as a function of time or perturbation, as well as dynamics of post-translational modifications (PTMs) and allelic variants (proteoforms). These different levels and types of information of proteins and proteoforms complicate data analysis and generation of insightful and comprehensible graphic visualizations. Middle-down mass spectrometry of histone proteins now allows quantifying hundreds of histone proteoforms including co-existing methylation, acetylation and phosphorylation events at distinct amino acid residues within histone molecules. The histone PTM landscape plays a dominant role in the regulation of chromatin activity and transcriptional and epigenetic control. The dynamics of these reversible modifications are governed by reader, writer and eraser enzymes that cooperate to regulate molecular mechanisms that rely on multiple interdependent PTM marks in histones and nucleosomes in chromatin. This PTM crosstalk can be quantified and provides a detailed picture of the underlying rules for setting the histone PTM landscape and chromatin activity, and is available to the community via our CrosstalkDB platform. Here, we developed a new computational method, PTM-CrossTalkMapper, to visualize the dynamics of histone PTMs for different experimental conditions, replicates and proteoforms onto a landscape, thereby describing the crosstalk and interplay between PTMs in a more comprehensible manner. We show the power of combining different levels of information on such crosstalk maps for histone PTM dynamics in mouse organs during the aging process. The PTM-CrossTalkMapper toolkit provides flexible functions to create these maps in various scenarios of multi-dimensional experimental designs, including histone PTM patterns and PTM crosstalk. The source code is available at https://github.com/veitveit/CrossTalkMapper.

Variability in Killler Immunglobulin like Receptor Gene Expression As a Periodic Function of Gene Position on Chromosome 19

Killer Immunoglobulin Like Receptors (KIR) are expressed on natural killer (NK) and T cell surface. KIR interactions with KIR-ligands have been implicated in outcomes of hematopoietic stem cell transplantation, placental implantation, autoimmune disease and viral infections. While these KIR interactions are clearly important our understanding of what governs their expression is lacking. The KIR Locus is made up of a highly polymorphic and homologous set of genes located on Chromosome 19q13.4 within the leukocyte receptor complex. Unique to the KIR Gene Cluster, KIR haplotypes not only vary allelically, but they also differ in the number of KIR genes present in different individuals, ranging from 7 to 12 genes. KIR haplotypes have been divided into 2 broad groups; Haplotypes A & B, based on the number of activating and inhibitory KIR genes they possess; where haplotype A contains only one activating allele KIR2DS4 and haplotype B contains various combinations of activating alleles. Expression of KIR on the NK cell surface stochastic, with some KIR found more commonly then others. In this abstract we aim to explore the relationship of KIR gene expression and the organization of the KIR gene locus using available genomic and tissue expression data. KIR gene coordinates on chromosome 19 were collected using the UCSC Genome Browser GRCh38/hg38 gene assembly and the NCBI gene website gene assembly NC_000019.10. Initial KIR gene nucleotide position along chromosome 19 were obtained and converted to angular distance (A.D. in radians) from reference. This was done to account for the double helical nature of DNA molecules, using the following equation A.D. = 2px/10.4; where x is the initial KIR gene nucleotide coordinate. Haplotype A was utilized as an initial test case as gene expression data are available for the genes in this haplotype. GTEX Portal was used to collect mRNA expression for each of the haplotype A genes, KIR-2DL1, -2DL3, -2DL4, -2DP1, -2DS4, -3DL1, -3DL2, -3DL3 & -3DP1, including total (median), splenic and whole blood expression in Reads/Kilobase of transcript/million mapped reads (RPKM). Gene expression was determined via RNA-seq of 53 tissues from 570 donors. KIR haplotype A is located along chromosome 19 between nucleotide position 55,236,713 and 55,370,584 which corresponded with an A.D. of 33,354,476 to 33,435,314 radians. When the KIR gene expression was aligned with the coordinates of the corresponding gene on the KIR locus a distinct periodic pattern of variation in expression levels was observed across the KIR genes comprising Haplotype A (Figure 1). This was true for total KIR expression from across various organs, as well as in blood and splenic tissue. This is borne out by expression data on several Haplotype A KIR genes reported in the literature (Figure 1, yellow curve). These observations suggesting that KIR gene expression may be a periodic function of the gene coordinates on the chromosome are analogous to the periodicity observed in T cell receptor VDJ recombination (Meier et al, BBMT 2019 25(5);868). This observation supports the idea that the spiral structure of the helical DNA molecules coiled around the histone molecules and their arrangement on chromosomes may have a fundamental influence on the expression of different genes, perhaps in conjunction with known epigenetic influence of DNA methylation and histone acetylation. This may also point to a role for the non-coding DNA in regulation of gene expression. We posit that the helical DNA chromosome structure may have a fundamental role in determining gene expression, as exemplified by the KIR and T cell receptor gene loci. This knowledge may allow an improved understanding of variability in global gene expression. Disclosures No relevant conflicts of interest to declare.

HDAC2 inhibitor CAY10683 reduces intestinal epithelial cell apoptosis by inhibiting mitochondrial apoptosis pathway in acute liver failure.

Histone deacetylases (HDAC) inhibitor has the effect of anti-tumor and inhibiting apoptosis, and could inhibit the release of inflammatory factors, reducing the damage to liver and enterocytes in acute liver failure (ALF). HDAC2 specific inhibitor CAY10683 was used to verify the protective effect on acute liver failure through reducing intestinal epithelial cell apoptosis by inhibiting mitochondrial apoptosis pathway. Lipopolysaccharide/D-galactosamine (LPS/D-GalN) was used to induce ALF in Sprague-Dawley rats. A total of 18 healthy rats were randomly divided into three groups. Rats in ALF group and CAY10683 group were given the same amount of normal sodium or CAY10683 2 hours before ALF model protocol was conducted. NCM460 cells were given LPS/D-GalN to establish an apoptotic model. Flow cytometry analysis was used to determine the apoptosis of enterocytes, and TUNEL assay was used to observe the apoptosis of NCM460 cells. The expression of bax was observed by immunofluorescence. The expression of histone proteins, HDAC2 and molecules in the apoptotic signaling pathway were determined by Western blotting and real-time PCR. CAY10683 improves histological and functional changes in ALF model. Compared with control group, LPS/D-GalN induced massive apoptosis of rat intestinal tissues and NCM460 cells (P<0.05), and the apoptosis rate was significantly reduced after CAY10683 treatment (P<0.05). The expression of bax was increased significantly in the model groups (P<0.05), and reduced with the treatment of CAY10683 (P<0.05). Compared with the model group, CAY10683 inhibits mitochondrial apoptosis in intestinal tissues and NCM460 cells (P<0.05). CAY10683 reduces the damage to liver and intestinal tissue, and plays an important role in inhibiting mitochondrial apoptosis in ALF rats and in NCM460 cells.

The Role of Acetylation/Deacetylation of Histones and Transcription Factors in Regulating Metabolism in Skeletal Muscles

Two types of enzymes control the reversibility of the acetylation of amino acid lysine residues in histone molecules in the posttranslational modifications of proteins – histone acetyltransferases (HAT) and histone deacetylases (HDAC). HAT enzymes catalyze the transfer of an acetyl group to the amino acid lysine in the N-terminal parts of histones H2A, H2B, H3, and H4. HDAC enzymes are members of the hydrolases family and play an important role in signal transmission by deacetylating histones, inducing changes in gene expression in skeletal muscle in different functional states in humans. Of the 18 histone deacetylases, class IIa includes four enzymes (HDAC 4, 5, 7, and 9), which are tissue-specific and are involved in regulating metabolism in skeletal and cardiac muscles. Class IIa enzymes react with specific transcription factors to form multicomponent protein complexes, nuclear-cytoplasmic transfer of which in muscle cells influences the expression of regulatory and metabolic genes. Studies in recent years have provided evidence of the important role of this group of histone deacetylases in controlling intracellular metabolism and point to the need for studies of the molecular mechanisms involved in gene expression in skeletal muscles.

The histone H4 proteoform dynamics in response to SUV4-20 inhibition reveals single molecule mechanisms of inhibitor resistance

BackgroundThe dynamics of histone post-translational modifications (PTMs) are sparsely described, especially in their true physiological context of proteoforms (single histone molecules harboring combinations of PTMs).MethodsHere we time-resolve the response of cells to SUV4-20 methyltransferase inhibition and unbiasedly quantitate the dynamic response of histone H4 PTMs and proteoforms.ResultsContrary to the prevailing dogma, cells exhibit an immediate-early response with changes to histone proteoforms. Cells also recover to basal-like conditions upon removal of epigenetic inhibitors rapidly. Inhibition of SUV4-20 results in decreased H4{K20me2}; however, no effects on H4{K20me3} are observed, implying that another enzyme mediates H4K20me3. Most surprisingly, SUV4-20 inhibition results in an increase in histone H4 acetylation attributable to proteoforms containing K20me2. This led us to hypothesize that hyperacetylated proteoforms protect K20me2 from demethylation as an evolved compensatory mechanism. This concept is supported by subsequent results that pretreatment with an HDACi substantially diminishes the effects of SUV4-20 inhibition in prone cells and is further confirmed by HATi-facilitating SUV4-20 inhibition to decrease discrete H4{K20me2} in resistant cells.ConclusionsThe chromatin response of cells to sudden perturbations is significantly faster, nuanced and complex than previously described. The persistent nature of chromatin regulation may be achieved by a network of dynamic equilibria with compensatory mechanisms that operate at the proteoform level.

Sequence analysis and expression patterns of histone H2A variants in Phytophthora infestans

In eukaryotic cells, DNA is packaged inside the nucleus together with histones to form nucleosomes. Each histone molecule contains two of each core histone subunits H2A, H2B, H3 and H4. Among core histones, the H2A family is of great interest due to the high diversity of specialized variants. These variants have shown important role in critical cellular processes. Epigenetic mechanism in oomycetes is barely known. Phytophthora infestans is a severe pathogen and a model species in oomycetes. In this study, we studied the sequence and expression pattern of H2A variants of P. infestans through genome search, sequences alignment, phylogenetic analysis and realtime qPCR detection. P. infestans contains conserved genes encoding histone variants H2A.X.1, H2A.X.2 and H2A.Z, and these genes have specific expression patterns during development and infection stages. Our datasets provide useful inputs to help explore the epigenetic mechanisms of oomycetes.

Epigenetics: Impacts on Disease Susceptibility and Pharmacotherapy

Abstract: Deoxyribonucleic acid (DNA) is a hereditary material comprising of all the genetic information in humans and almost all other living creatures. It is made up of subunits called genes, which have four nucleotides. MicroRNAs comprises of the short noncoding RNA. They are non-protein coding with about 18-22 nucleotides which control gene expression and activities post-transcriptionally. Notably, the whole DNA structure usually coiled itself around a protein called histone moiety. Consequently, any addition of methyl, ethyl, phosphate, Ubiquitin or small ubiquitin-related modifier either directly on the DNA itself or the histone molecule will ultimately change its configuration and stability leading to the epigenetic modifications. Usually, epigenetic changes manifest phenotypically without changing the actual gene sequence. MicroRNAs also play a vital role in the control of the expression epigenetic regulators, such as DNA methyltransferase (DNMT), histone deacetylase, and Polycomb group genes. The implication of this may significantly affect the normal function of an organism and perhaps contribute to the wide-range of disease aetiology. As such, epigenetics linked variations and diseases are inheritable. The landmark achievement in pharmacotherapy involves the possibility of silencing the oncogenes that are cancer provoking genes and possible reactivation of cancer suppression genes that battle with and repress the tumour cells proliferation. Besides these, more genes that are linked to other diseases apart from cancer can be identified, silenced or activated using drugs or diets in order to treat the disease aetiology. This appraisal is to discuss the role of epigenetic changes in the disease susceptibility and drug treatment. Key words: Epigenetics, Gene-methylation, Histone, Methyl-group, Cancer, Pharmacotherapy.

Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation

Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12(-/-)), PRC1 (Ring1A/B(-/-)) and (Dnmt1/3a/3b(-/-)) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http://crosstalkdb.bmb.sdu.dk.

The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we suggest that it may play the same role in meiosis.

A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin

The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner.