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Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutate histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleash cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupts the deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal levels of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs. In this study, the authors mutate histone H3.3 K9 and K27 residues, demonstrating their importance in maintaining repressive chromatin states at endogenous retrovirus-derived cryptic enhancers and bivalent promoters in mouse embryonic stem cells. [ABSTRACT FROM AUTHOR]

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR–Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers. Here, the authors identify PHF6 as a dependency in SMARCB1-deficient rhabdoid cancers. Mechanistically, this study suggests a regulatory role for PHF6 in recruiting SWI/SNF complexes to enable RNA polymerase progression. [ABSTRACT FROM AUTHOR]

Dysregulated transcription due to disruption in histone lysine methylation dynamics is an established contributor to tumorigenesis1,2. However, whether analogous pathologic epigenetic mechanisms act directly on the ribosome to advance oncogenesis is unclear. Here we find that trimethylation of the core ribosomal protein L40 (rpL40) at lysine 22 (rpL40K22me3) by the lysine methyltransferase SMYD5 regulates mRNA translation output to promote malignant progression of gastric adenocarcinoma (GAC) with lethal peritoneal ascites. A biochemical–proteomics strategy identifies the monoubiquitin fusion protein partner rpL40 (ref. 3) as the principal physiological substrate of SMYD5 across diverse samples. Inhibiting the SMYD5–rpL40K22me3 axis in GAC cell lines reprogrammes protein synthesis to attenuate oncogenic gene expression signatures. SMYD5 and rpL40K22me3 are upregulated in samples from patients with GAC and negatively correlate with clinical outcomes. SMYD5 ablation in vivo in familial and sporadic mouse models of malignant GAC blocks metastatic disease, including peritoneal carcinomatosis. Suppressing SMYD5 methylation of rpL40 inhibits human cancer cell and patient-derived GAC xenograft growth and renders them hypersensitive to inhibitors of PI3K and mTOR. Finally, combining SMYD5 depletion with PI3K–mTOR inhibition and chimeric antigen receptor T cell administration cures an otherwise lethal in vivo mouse model of aggressive GAC-derived peritoneal carcinomatosis. Together, our work uncovers a ribosome-based epigenetic mechanism that facilitates the evolution of malignant GAC and proposes SMYD5 targeting as part of a potential combination therapy to treat this cancer.The lysine methyltransferase SMYD5 and its newly identified substrate ribosomal protein L40 are implicated in the progression of gastric adenocarcinoma, and ablation of SMYD5 prevents metastatic disease in mouse models of this cancer. [ABSTRACT FROM AUTHOR]

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype. Missense mutations in histones can drive oncogenesis and disrupt chromatin, but the associated mechanisms for many such mutations remain poorly understood. Here, the authors show that cancer-associated histone mutations at arginines in the H3 N-terminal tail disrupt repressive chromatin domains, alter gene expression, and in one case impair differentiation via reduction of PRC2 function. [ABSTRACT FROM AUTHOR]

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes. Trypanosoma brucei DOT1A and DOT1B methylate H3K76 without H2B-ubiquitin. Based on structural and enzymatic data, Frisbie et al. reveal a mechanism of how these enzymes cooperatively and efficiently tri-methylate H3K76 in a ubiquitin-independent way. [ABSTRACT FROM AUTHOR]

Arboleda-Tham Syndrome (ARTHS) is a rare genetic disorder caused by heterozygous, de novo mutations in Lysine(K) acetyltransferase 6A (KAT6A). ARTHS is clinically heterogeneous and characterized by several common features, including intellectual disability, developmental and speech delay, and hypotonia, and affects multiple organ systems. KAT6A is the enzymatic core of a histone–acetylation protein complex; however, the direct histone targets and gene regulatory effects remain unknown. In this study, we use ARTHS patient (n = 8) and control (n = 14) dermal fibroblasts and perform comprehensive profiling of the epigenome and transcriptome caused by KAT6A mutations. We identified differential chromatin accessibility within the promoter or gene body of 23% (14/60) of genes that were differentially expressed between ARTHS and controls. Within fibroblasts, we show a distinct set of genes from the posterior HOXC gene cluster (HOXC10, HOXC11, HOXC-AS3, HOXC-AS2, and HOTAIR) that are overexpressed in ARTHS and are transcription factors critical for early development body segment patterning. The genomic loci harboring HOXC genes are epigenetically regulated with increased chromatin accessibility, high levels of H3K23ac, and increased gene–body DNA methylation compared to controls, all of which are consistent with transcriptomic overexpression. Finally, we used unbiased proteomic mass spectrometry and identified two new histone post-translational modifications (PTMs) that are disrupted in ARTHS: H2A and H3K56 acetylation. Our multi-omics assays have identified novel histone and gene regulatory roles of KAT6A in a large group of ARTHS patients harboring diverse pathogenic mutations. This work provides insight into the role of KAT6A on the epigenomic regulation in somatic cell types. [ABSTRACT FROM AUTHOR]

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2–4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.Cellular lysine residues can be both methylated and acetylated on the same sidechain to form Nε-acetyl-Nε-methyllysine (Kacme), which is found on histone H4 across a range of species and across mammalian tissues and is associated with active chromatin. [ABSTRACT FROM AUTHOR]

Purpose: Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates. Methods: Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson's disease (PD) and Alzheimer's disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21. Results: In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aβ42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.). Conclusion: Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aβ and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies. [ABSTRACT FROM AUTHOR]

Glioblastoma is a lethal malignant brain tumor, and the development of efficient chemotherapeutic agents remains an urgent need. Niclosamide, an anthelmintic drug, which has been used to treat tapeworm infections more than 50 years, has recently attracted renewed attention due to its evident anticancer activities. It has been shown that niclosamide induces cytotoxicity in human glioblastoma U-87 MG cells corresponding with increased protein ubiquitination, ER stress, and autophagy. Furthermore, niclosamide showed down regulation of multiple pro-survival signaling pathways including Wnt/β-catenin, PI3K/AKT, MAPK/ERK, and STAT3, which further caused reduction of U87-MG cell viability. However, the molecular mechanisms of niclosimide and its derivatives in cancer are not fully understood. In the present article, 12 niclosamide derivatives were synthesized by the replacement of substituents for the structure-activity relationship (SAR) study of the protein ubiquitination and related signaling pathways. Our approach is to identify which substituents of niclosamide play important roles in inducing cell apoptosis, inhibition of cell growth, and down regulation of cell survival signaling pathways. Our results indicate that phenol OH of niclosamide plays a significant role in the anti-glioblastoma activity, while missing Cl (5- or 2′-Cl) showed almost no such effect. 4′-N3 or CF3 has the similar activity to niclosamide (4′-NO2) whereas NH2 significantly decreased the cytotoxicity in U87 cells. These modified compounds can be tested to determine which are most effective on cancer treatment. These findings are important in the development of multi-functionalized niclosamide and drug design therapy in the future. [ABSTRACT FROM AUTHOR]

Appropriate timing of cervical remodeling (CR) is key to normal term parturition. To date, mechanisms behind normal and abnormal (premature or delayed) CR remain unclear. Recent studies show regional differences exist in human cervical tissue structure. While the entire cervix contains extracellular matrix (ECM), the internal os is highly cellular containing 50–60% cervical smooth muscle (CSM). The external os contains 10–20% CSM. Previously, we reported ECM rigidity and different ECM proteins influence CSM cell function, highlighting the importance of understanding not only how cervical cells orchestrate cervical ECM remodeling in pregnancy, but also how changes in specific ECM proteins can influence resident cellular function. To understand this dynamic process, we utilized a systematic proteomic approach to understand which soluble ECM and cellular proteins exist in the different regions of the human cervix and how the proteomic profiles change from the non-pregnant (NP) to the pregnant (PG) state. We found the human cervix proteome contains at least 4548 proteins and establish the types and relative abundance of cellular and soluble matrisome proteins found in the NP and PG human cervix. Further, we report the relative abundance of proteins involved with elastic fiber formation and ECM organization/degradation were significantly increased while proteins involved in RNA polymerase I/promoter opening, DNA methylation, senescence, immune system, and compliment activation were decreased in the PG compared to NP cervix. These findings establish an initial platform from which we can further comprehend how changes in the human cervix proteome results in normal and abnormal CR. [ABSTRACT FROM AUTHOR]

Schizophrenia (SZ) is a psychiatric disorder with complex genetic risk dictated by interactions between hundreds of risk variants. Epigenetic factors, such as histone posttranslational modifications (PTMs), have been shown to play critical roles in many neurodevelopmental processes, and when perturbed may also contribute to the precipitation of disease. Here, we apply an unbiased proteomics approach to evaluate combinatorial histone PTMs in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons from individuals with SZ. We observe hyperacetylation of H2A.Z and H4 in neurons derived from SZ cases, results that were confirmed in postmortem human brain. We demonstrate that the bromodomain and extraterminal (BET) protein, BRD4, is a bona fide 'reader' of H2A.Z acetylation, and further provide evidence that BET family protein inhibition ameliorates transcriptional abnormalities in patient-derived neurons. Thus, treatments aimed at alleviating BET protein interactions with hyperacetylated histones may aid in the prevention or treatment of SZ. Schizophrenia (SZ) is a severe psychiatric disorder; unfortunately, only ~1/3 of patients respond favorably to treatment. Here, the authors reveal hyperacetylation of histone H2A.Z in SZ neurons and postmortem SZ human brain tissues. They further show BRD4 is a reader of hyperacetylated H2A.Z and treatment with bromodomain inhibitor JQ1 largely rescues abnormal gene expression associated with SZ. [ABSTRACT FROM AUTHOR]

Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαβ isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein. KRAS is commonly mutated at codon 12 in several cancer types, offering a unique opportunity for the development of neoantigen-targeted immunotherapy. Here the authors present a pipeline for the prediction, identification and validation of HLA class-I restricted mutant KRAS G12 peptides, leading to the generation of mutant KRAS-specific T cell receptors for adoptive T cell immunotherapy. [ABSTRACT FROM AUTHOR]

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene. Substrate channeling can improve biosynthetic efficiency and has been implicated in the reactions of fusicoccadiene synthase. Here, the authors analyze this bifunctional enzyme complex by cryoEM, cross-linking MS and integrative modeling, providing structural insights into how substrate channeling is achieved. [ABSTRACT FROM AUTHOR]

The versatile nucleotide excision repair (NER) pathway initiates as the XPC–RAD23B–CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4–Rad23-Rad33 (yeast homologue of XPC–RAD23B–CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9–9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification. The conserved eukaryotic nucleotide excision repair (NER) pathway protects the genome from a wide variety of environmentally induced DNA lesions. Here, the authors provide insights into how NER is initiated on lesions by determining the cryo-EM structure of the yeast TFIIH/Rad4–Rad23-Rad33 complex bound to a DNA containing a single carcinogen-DNA adduct. [ABSTRACT FROM AUTHOR]

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens. It is unclear why different antibiotics vary in their ability to shorten treatment of tuberculosis. Here, the authors show that a measure based on ribosomal RNA synthesis in Mycobacterium tuberculosis correlates with treatment shortening in culture, in mice and in human studies. [ABSTRACT FROM AUTHOR]

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria. Respiratory chains generate the proton motive force used for ATP synthesis. Cryo-EM structures of functional respiratory CIII2CIV supercomplex and native CIII2 from Rhodobacter capsulatus provide insight into CIII2CIV assembly and respiratory electron transport pathways in Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Complex organisms can rapidly induce select genes in response to diverse environmental cues. This regulation occurs in the context of large genomes condensed by histone proteins into chromatin. The sensing of pathogens by macrophages engages conserved signalling pathways and transcription factors to coordinate the induction of inflammatory genes1–3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a general feature of dynamically regulated chromatin and transcription4–7. However, how chromatin is regulated at induced genes, and what features of H3.3 might enable rapid and high-level transcription, are unknown. The amino terminus of H3.3 contains a unique serine residue (Ser31) that is absent in 'canonical' H3.1 and H3.2. Here we show that this residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along rapidly induced genes in mouse macrophages. This selective mark of stimulation-responsive genes directly engages the histone methyltransferase SETD2, a component of the active transcription machinery, and 'ejects' the elongation corepressor ZMYND118,9. We propose that features of H3.3 at stimulation-induced genes, including H3.3S31ph, provide preferential access to the transcription apparatus. Our results indicate dedicated mechanisms that enable rapid transcription involving the histone variant H3.3, its phosphorylation, and both the recruitment and the ejection of chromatin regulators. The histone variant H3.3 is phosphorylated at Ser31 in induced genes, and this selective mark stimulates the histone methyltransferase SETD2 and ejects the ZMYND11 repressor, thus revealing a role for histone phosphorylation in amplifying de novo transcription. [ABSTRACT FROM AUTHOR]

Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXΔRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes. ATRX is an RNA binding protein that mediates targeting of polycomb repressive complex 2 (PRC2) to genomic sites. Here the authors identify the RNA binding region and show that the RNA binding is required for ATRX localization and for its recruitment of PRC2 to a subset of polycomb targets. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are a unique mode of intercellular communication capable of incredible specificity in transmitting signals involved in cellular function, including germ cell maturation. Spermatogenesis occurs in the testes, behind a protective barrier to ensure safeguarding of germline DNA from environmental insults. Following DNA compaction, further sperm maturation occurs in the epididymis. Here, we report reproductive tract EVs transmit information regarding stress in the paternal environment to sperm, potentially altering fetal development. Using intracytoplasmic sperm injection, we found that sperm incubated with EVs collected from stress-treated epididymal epithelial cells produced offspring with altered neurodevelopment and adult stress reactivity. Proteomic and transcriptomic assessment of these EVs showed dramatic changes in protein and miRNA content long after stress treatment had ended, supporting a lasting programmatic change in response to chronic stress. Thus, EVs as a normal process in sperm maturation, can also perform roles in intergenerational transmission of paternal environmental experience. Evidence for sperm small noncoding RNA-mediated intergenerational transmission implies communication from responsive somatic cells to sperm. Here, authors show that epididymal cells alter extracellular vesicle cargo after stress exposure, to impact offspring neurodevelopment and stress reactivity. [ABSTRACT FROM AUTHOR]

The field of epitranscriptomics continues to reveal how post-transcriptional modification of RNA affects a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. Existing software solutions lack the raw performance and statistical grounding to efficiently handle the numerous modifications found on RNA. We present a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), that addresses these shortcomings. We demonstrate the capability of NASE to reliably identify a wide range of modified RNA sequences in four original datasets of varying complexity. In human tRNA, we characterize over 20 different modification types simultaneously and find many cases of incomplete modification. Mass spectrometry (MS) enables identification of modified RNA residues, but high-throughput processing is currently a bottleneck. Here, the authors present a free and open-source database search engine for RNA MS data to facilitate reliable identification of modified RNA sequences. [ABSTRACT FROM AUTHOR]

Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection. Here, using high spatiotemporal resolution light-sheet and fluorescence microscopy, the authors investigate the role of cytoskeletal components on the intracellular transport of Rab11A and influenza virus (IAV) vRNP), and show a preference for Rab11A movement along microtubules that is not essential for IAV vRNP transport. [ABSTRACT FROM AUTHOR]

Purpose: Alternations to the paternal epigenome, specifically the components of sperm chromatin, can lead to infertility in humans and potentially transmit aberrant information to the embryo. One key component of sperm chromatin is the post-translational modification of histones (PTMs). We previously identified a comprehensive profile of histone PTMs in normozoospermic sperm; however, only specific histone PTMs have been identified in abnormal sperm by antibody-based approaches and comprehensive changes to histone PTM profiles remain unknown. Here, we investigate if sperm with abnormalities of total motility, progressive motility, and morphology have altered histone PTM profiles compared to normozoospermic sperm samples.Methods: Discarded semen samples from 31 men with normal or abnormal semen parameters were analyzed for relative abundance of PTMs on histone H3 and H4 by "bottom-up" nano-liquid chromatography-tandem mass spectrometry.Results: Asthenoteratozoospermic samples (abnormal motility, forward progression, and morphology, n = 6) displayed overall decreased H4 acetylation (p = 0.001) as well as alterations in H4K20 (p = 0.003) and H3K9 methylation (p < 0.04) when compared to normozoospermic samples (n = 8). Asthenozoospermic samples (abnormal motility and progression, n = 5) also demonstrated decreased H4 acetylation (p = 0.04) and altered H4K20 (p = 0.005) and H3K9 methylation (p < 0.04). Samples with isolated abnormal progression (n = 6) primarily demonstrated decreased acetylation on H4 (p < 0.02), and teratozoospermic samples (n = 6) appeared similar to normozoospermic samples (n = 8).Conclusion: Sperm samples with combined and isolated abnormalities of total motility, progressive motility, and morphology display distinct and altered histone PTM signatures compared to normozoospermic sperm. This provides evidence that alterations in histone PTMs may be important for normal sperm function and fertility. [ABSTRACT FROM AUTHOR]

Over the last decade, numerous histone acyl post-translational modifications (acyl-PTMs) have been discovered, of which the functional significance is still under intense study. Here, we use high-resolution mass spectrometry to accurately quantify eight acyl-PTMs in vivo and after in vitro enzymatic assays. We assess the ability of seven histone acetyltransferases (HATs) to catalyze acylations on histones in vitro using short-chain acyl-CoA donors, proving that they are less efficient towards larger acyl-CoAs. We also observe that acyl-CoAs can acylate histones through non-enzymatic mechanisms. Using integrated metabolomic and proteomic approaches, we achieve high correlation (R² > 0.99) between the abundance of acyl-CoAs and their corresponding acyl-PTMs. Moreover, we observe a dose-dependent increase in histone acyl-PTM abundances in response to acyl-CoA supplementation in in nucleo reactions. This study represents a comprehensive profiling of scarcely investigated low-abundance histone marks, revealing that concentrations of acyl-CoAs affect histone acyl-PTM abundances by both enzymatic and non-enzymatic mechanisms. [ABSTRACT FROM AUTHOR]

The histone deacetylase HDAC3 is a critical mediator of hepatic lipid metabolism, and liver-specific deletion of HDAC3 leads to fatty liver. To elucidate the underlying mechanism, here we report a method of cross-linking followed by mass spectrometry to define a high-confidence HDAC3 interactome in vivo that includes the canonical NCoR–HDAC3 complex as well as Prospero-related homeobox 1 protein (PROX1). HDAC3 and PROX1 co-localize extensively on the mouse liver genome, and are co-recruited by hepatocyte nuclear factor 4α (HNF4α). The HDAC3–PROX1 module controls the expression of a gene program regulating lipid homeostasis, and hepatic-specific ablation of either component increases triglyceride content in liver. These findings underscore the importance of specific combinations of transcription factors and coregulators in the fine tuning of organismal metabolism. [ABSTRACT FROM AUTHOR]

Heterochromatic DNA domains have important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway. Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3, respectively) and the formation of heterochromatin. H3K9me is in turn required for the recruitment of RNAi to chromatin to promote the amplification of sRNA. Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast Schizosaccharomyces pombe homologue of mammalian SUV39H H3K9 methyltransferases, we design active-site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, the transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for the formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defence. [ABSTRACT FROM AUTHOR]

Insulin resistance is a cardinal feature of Type 2 diabetes (T2D) and a frequent complication of multiple clinical conditions, including obesity, ageing and steroid use, among others. How such a panoply of insults can result in a common phenotype is incompletely understood. Furthermore, very little is known about the transcriptional and epigenetic basis of this disorder, despite evidence that such pathways are likely to play a fundamental role. Here, we compare cell autonomous models of insulin resistance induced by the cytokine tumour necrosis factor-α or by the steroid dexamethasone to construct detailed transcriptional and epigenomic maps associated with cellular insulin resistance. These data predict that the glucocorticoid receptor and vitamin D receptor are common mediators of insulin resistance, which we validate using gain- and loss-of-function studies. These studies define a common transcriptional and epigenomic signature in cellular insulin resistance enabling the identification of pathogenic mechanisms. [ABSTRACT FROM AUTHOR]

Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb and has been proposed to release RNA polymerase II (Pol II) from promoter-proximal pausing. We show that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function. BRD4 interacts with elongating Pol II complexes and assists Pol II in progression through hyperacetylated nucleosomes by interacting with acetylated histones via bromodomains. On active enhancers, the BET inhibitor JQ1 antagonizes BRD4-associated eRNA synthesis. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by facilitating transcript elongation both at enhancers and on gene bodies independently of P-TEFb. [ABSTRACT FROM AUTHOR]

Over the past decade, rapid advances in epigenomics research have extensively characterized critical roles for chromatin regulatory events during normal periods of eukaryotic cell development and plasticity, as well as part of aberrant processes implicated in human disease. Application of such approaches to studies of the CNS, however, is more recent. Here we provide a comprehensive overview of available tools for analyzing neuroepigenomics data, as well as a discussion of pending challenges specific to the field of neuroscience. Integration of numerous unbiased genome-wide and proteomic approaches will be necessary to fully understand the neuroepigenome and the extraordinarily complex nature of the human brain. This will be critical to the development of future diagnostic and therapeutic strategies aimed at alleviating the vast array of heterogeneous and genetically distinct disorders of the CNS. [ABSTRACT FROM AUTHOR]

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling. [ABSTRACT FROM AUTHOR]

The chromatin regulatory factor SIRT6 plays pivotal roles in metabolism, tumor suppression, and aging biology. Despite the fundamental roles of SIRT6 in physiology and disease, only a handful of molecular and functional interactions of SIRT6 have been reported. Here, we characterize the SIRT6 interactome and identify 801 novel SIRT6-interacting proteins. The discovery of these SIRT6-associations considerably expands knowledge of the SIRT6 interaction network, and suggests previously unknown functional interactions of SIRT6 in fundamental cellular processes. These include chromatin remodeling, mitotic chromosome segregation, protein homeostasis, and transcriptional elongation. Extended analysis of the SIRT6 interaction with G3BP1, a master stress response factor, uncovers an unexpected role and mechanism of SIRT6 in regulating stress granule assembly and cellular stress resistance. [ABSTRACT FROM AUTHOR]

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming. [ABSTRACT FROM AUTHOR]

X-chromosome dosage compensation by the MSL (male-specific lethal) complex is required in Drosophila melanogaster to increase gene expression from the single male X to equal that of both female X chromosomes. Instead of focusing solely on protein complexes released from DNA, here we used chromatin-interacting protein MS (ChIP-MS) to identify MSL interactions on cross-linked chromatin. We identified MSL-enriched histone modifications, including histone H4 Lys16 acetylation and histone H3 Lys36 methylation, and CG4747, a putative Lys36-trimethylated histone H3 (H3K36me3)-binding protein. CG4747 is associated with the bodies of active genes, coincident with H3K36me3, and is mislocalized in the Set2 mutant lacking H3K36me3. CG4747 loss of function in vivo results in partial mislocalization of the MSL complex to autosomes, and RNA interference experiments confirm that CG4747 and Set2 function together to facilitate targeting of the MSL complex to active genes, validating the ChIP-MS approach. [ABSTRACT FROM AUTHOR]

Bone is the one of the most common sites of distant metastasis of solid tumors. Secreted proteins are known to influence pathological interactions between metastatic cancer cells and the bone stroma. To comprehensively profile secreted proteins associated with bone metastasis, we used quantitative and non-quantitative mass spectrometry to globally analyze the secretomes of nine cell lines of varying bone metastatic ability from multiple species and cancer types. By comparing the secretomes of parental cells and their bone metastatic derivatives, we identified the secreted proteins that were uniquely associated with bone metastasis in these cell lines. We then incorporated bioinformatic analyses of large clinical metastasis datasets to obtain a list of candidate novel bone metastasis proteins of several functional classes that were strongly associated with both clinical and experimental bone metastasis. Functional validation of selected proteins indicated that in vivo bone metastasis can be promoted by high expression of (1) the salivary cystatins CST1, CST2, and CST4; (2) the plasminogen activators PLAT and PLAU; or (3) the collagen functionality proteins PLOD2 and COL6A1. Overall, our study has uncovered several new secreted mediators of bone metastasis and therefore demonstrated that secretome analysis is a powerful method for identification of novel biomarkers and candidate therapeutic targets. [ABSTRACT FROM AUTHOR]

Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD+-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo. [ABSTRACT FROM AUTHOR]

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45?C-KITlow/+TIE2+ bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process. [ABSTRACT FROM AUTHOR]

Although the role of miR-200s in regulating E-cadherin expression and epithelial-to-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here we have used clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. miR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis-suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin-dependent epithelial traits and Sec23a-mediated tumor cell secretome. [ABSTRACT FROM AUTHOR]

Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs. [ABSTRACT FROM AUTHOR]

Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm. [ABSTRACT FROM AUTHOR]

The physiological state of eukaryotic DNA is chromatin. Nucleosomes, which consist of DNA in complex with histones, are the fundamental unit of chromatin. The post-translational modifications (PTMs) of histones play a critical role in the control of gene transcription, epigenetics and other DNA-templated processes. It has been known for several years that these PTMs function in concert to allow for the storage and transduction of highly specific signals through combinations of modifications. This code, the combinatorial histone code, functions much like a bar code or combination lock providing the potential for massive information content. The capacity to directly measure these combinatorial histone codes has mostly been laborious and challenging, thus limiting efforts often to one or two samples. Recently, progress has been made in determining such information quickly, quantitatively and sensitively. Here we review both the historical and recent progress toward routine and rapid combinatorial histone code analysis. [ABSTRACT FROM AUTHOR]

A central hub of carbon metabolism is the tricarboxylic acid cycle, which serves to connect the processes of glycolysis, gluconeogenesis, respiration, amino acid synthesis and other biosynthetic pathways. The protozoan intracellular malaria parasites (Plasmodium spp.), however, have long been suspected of possessing a significantly streamlined carbon metabolic network in which tricarboxylic acid metabolism plays a minor role. Blood-stage Plasmodium parasites rely almost entirely on glucose fermentation for energy and consume minimal amounts of oxygen, yet the parasite genome encodes all of the enzymes necessary for a complete tricarboxylic acid cycle. Here, by tracing 13C-labelled compounds using mass spectrometry we show that tricarboxylic acid metabolism in the human malaria parasite Plasmodium falciparum is largely disconnected from glycolysis and is organized along a fundamentally different architecture from the canonical textbook pathway. We find that this pathway is not cyclic, but rather is a branched structure in which the major carbon sources are the amino acids glutamate and glutamine. As a consequence of this branched architecture, several reactions must run in the reverse of the standard direction, thereby generating two-carbon units in the form of acetyl-coenzyme A. We further show that glutamine-derived acetyl-coenzyme A is used for histone acetylation, whereas glucose-derived acetyl-coenzyme A is used to acetylate amino sugars. Thus, the parasite has evolved two independent production mechanisms for acetyl-coenzyme A with different biological functions. These results significantly clarify our understanding of the Plasmodium metabolic network and highlight the ability of altered variants of central carbon metabolism to arise in response to unique environments. [ABSTRACT FROM AUTHOR]

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability. Causal mutations have been found in approximately 90 X-linked genes; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of ∼7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR. [ABSTRACT FROM AUTHOR]

Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a 'histone code'. Here, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found that Thr3 phosphorylation, Arg2 methylation and Thr6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multisite PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent 'reader' specificity. [ABSTRACT FROM AUTHOR]

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we show that HP1α, -β, and -γ are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein–protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark. [ABSTRACT FROM AUTHOR]

Methylation of histones is central to chromatin regulation, and thus previously unknown mechanisms regulating genome function can be revealed through the discovery of new histone methyl marks. Here we identify Set5 as the first histone H4 methyltransferase, which monomethylates the critical H4 lysine residues 5, 8 and 12 in budding yeast. Set5's enzymatic activity functions together with the global chromatin-modifying complexes COMPASS and NuA4 to regulate cell growth and stress responses. [ABSTRACT FROM AUTHOR]

We developed a platform using hydrophilic interaction chromatography and high-resolution tandem mass spectrometry (MS) for analyses of histone H3 that allows comprehensive characterization of 'histone codes' at the molecular level. We identified over 150 differentially modified forms of histone H3.2 in asynchronously grown and butyrate-treated HeLa cells, revealing pervasive combinatorial modification previously unaccounted for by other techniques and providing a clarified estimate of the molecular diversity of histone H3 in mammals. [ABSTRACT FROM AUTHOR]