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In this study, Ma¨tlik et al. used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in progenitor cerebellar granule cells. Inhibition of H3K27 methyltransferases EZH1/2 perturbed glial-guided migration while accelerating neuronal maturation, and this study demonstrates that temporally regulated H3K4me3/H3K27me3 bivalency influences neurodevelopmental gene expression, lineage specification, migration, and differentiation.

The genetic information of human cells is stored in the context of chromatin, which is subjected to DNA methylation and various histone modifications. Such a ‘language’ of chromatin modification constitutes a fundamental means of gene and (epi)genome regulation, underlying a myriad of cellular and developmental processes. In recent years, mounting evidence has demonstrated that miswriting, misreading or mis-erasing of the modification language embedded in chromatin represents a common, sometimes early and pivotal, event across a wide range of human cancers, contributing to oncogenesis through the induction of epigenetic, transcriptomic and phenotypic alterations. It is increasingly clear that cancer-related metabolic perturbations and oncohistone mutations also directly impact chromatin modification, thereby promoting cancerous transformation. Phase separation-based deregulation of chromatin modulators and chromatin structure is also emerging to be an important underpinning of tumorigenesis. Understanding the various molecular pathways that underscore a misregulated chromatin language in cancer, together with discovery and development of more effective drugs to target these chromatin-related vulnerabilities, will enhance treatment of human malignancies.

Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The localization of de novo DNA methyltransferase DNMT3A is facilitated by its PWWP domain recognizing histone H3 lysine 36 (H3K36) methylation1,2and is normally depleted at CpG islands (CGIs)3. However, methylation of CGIs regulated by Polycomb repressive complexes (PRCs) has also been observed4–8. Here, we report that DNMT3A PWWP domain mutations identified in paragangliomas9and microcephalic dwarfism10promote aberrant localization of DNMT3A to CGIs in a PRC1-dependent manner. DNMT3A PWWP mutants accumulate at regions containing PRC1-mediated formation of monoubiquitylated histone H2A lysine 119 (H2AK119ub), irrespective of the amounts of PRC2-catalyzed formation of trimethylated histone H3 lysine 27 (H3K27me3). DNMT3A interacts with H2AK119ub-modified nucleosomes through a putative amino-terminal ubiquitin-dependent recruitment region, providing an alternative form of DNMT3A genomic targeting that is augmented by the loss of PWWP reader function. Ablation of PRC1 abrogates localization of DNMT3A PWWP mutants to CGIs and prevents aberrant DNA hypermethylation. Our study implies that a balance between DNMT3A recruitment by distinct reader domains guides de novo CpG methylation and may underlie the abnormal DNA methylation landscapes observed in select human cancer subtypes and developmental disorders.

Whole-genome sequencing data mining efforts have revealed numerous histone mutations in a wide range of cancer types. These occur in all four core histones in both the tail and globular domains and remain largely uncharacterized. Here we used two high-throughput approaches, a DNA-barcoded mononucleosome library and a humanized yeast library, to profile the biochemical and cellular effects of these mutations. We identified cancer-associated mutations in the histone globular domains that enhance fundamental chromatin remodeling processes, histone exchange and nucleosome sliding, and are lethal in yeast. In mammalian cells, these mutations upregulate cancer-associated gene pathways and inhibit cellular differentiation by altering expression of lineage-specific transcription factors. This work represents a comprehensive functional analysis of the histone mutational landscape in human cancers and leads to a model in which histone mutations that perturb nucleosome remodeling may contribute to disease development and/or progression.

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B–E (H1B, H1C, H1Dand H1E; also known as H1-5, H1-2, H1-3and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1cand H1e(also known as H1f2and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.

Epigenetic regulation is critical to physiological control of development, cell fate, cell proliferation, genomic integrity and, fundamentally, transcriptional regulation. This epigenetic control occurs at multiple levels including through DNA methylation, histone modification, nucleosome remodelling and modulation of the 3D chromatin structure. Alterations in genes that encode chromatin regulators are common among mesenchymal neoplasms, a collection of more than 160 tumour types including over 60 malignant variants (sarcomas) that have unique and varied genetic, biological and clinical characteristics. Herein, we review those sarcomas in which chromatin pathway alterations drive disease biology. Specifically, we emphasize examples of dysregulation of each level of epigenetic control though mechanisms that include alterations in metabolic enzymes that regulate DNA methylation and histone post-translational modifications, mutations in histone genes, subunit loss or fusions in chromatin remodelling and modifying complexes, and disruption of higher-order chromatin structure. Epigenetic mechanisms of tumorigenesis have been implicated in mesenchymal tumours ranging from chondroblastoma and giant cell tumour of bone to chondrosarcoma, malignant peripheral nerve sheath tumour, synovial sarcoma, epithelioid sarcoma and Ewing sarcoma — all diseases that present in a younger patient population than most cancers. Finally, we review current and potential future approaches for the development of sarcoma therapies based on this emerging understanding of chromatin dysregulation.

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.

Elucidating the physiological binding partners of histone post-translational modifications (hPTMs) is key to understanding fundamental epigenetic regulatory pathways. Determining such interactomes will enable the study of how perturbations of these interactions affect disease. Here we use a synthetic biology approach to set a series of hPTM-controlled photo-affinity traps in native chromatin. Using quantitative proteomics, the local interactomes of these chemically customized chromatin landscapes are determined. We show that the approach captures transiently interacting factors such as methyltransferases and demethylases, as well as previously reported and novel hPTM reader proteins. We also apply this in situ proteomics approach to a recently disclosed cancer-associated histone mutation, H3K4M, revealing a number of perturbed interactions with the mutated tail. Collectively our studies demonstrate that modifying and interrogating native chromatin with chemical precision is a powerful tool for exploring epigenetic regulation and dysregulation at the molecular level.

Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by ‘reader’ proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia1. Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour2,3, the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.

Mutations in epigenetic pathways are common oncogenic drivers. Histones, the fundamental substrates for chromatin-modifying and remodelling enzymes, are mutated in tumours including gliomas, sarcomas, head and neck cancers, and carcinosarcomas. Classical ‘oncohistone’ mutations occur in the N-terminal tail of histone H3 and affect the function of polycomb repressor complexes 1 and 2 (PRC1 and PRC2). However, the prevalence and function of histone mutations in other tumour contexts is unknown. Here we show that somatic histone mutations occur in approximately 4% (at a conservative estimate) of diverse tumour types and in crucial regions of histone proteins. Mutations occur in all four core histones, in both the N-terminal tails and globular histone fold domains, and at or near residues that contain important post-translational modifications. Many globular domain mutations are homologous to yeast mutants that abrogate the need for SWI/SNF function, occur in the key regulatory ‘acidic patch’ of histones H2A and H2B, or are predicted to disrupt the H2B–H4 interface. The histone mutation dataset and the hypotheses presented here on the effect of the mutations on important chromatin functions should serve as a resource and starting point for the chromatin and cancer biology fields in exploring an expanding role of histone mutations in cancer.

Chemical probes of epigenetic ‘readers’ of histone post-translational modifications (PTMs) have become powerful tools for mechanistic and functional studies of their target proteins in normal physiology and disease pathogenesis. Here we report the development of the first class of chemical probes of YEATS domains, newly identified ‘readers’ of histone lysine acetylation (Kac) and crotonylation (Kcr). Guided by the structural analysis of a YEATS–Kcr complex, we developed a series of peptide-based inhibitors of YEATS domains by targeting a unique π-π-π stacking interaction at the proteins’ Kcr recognition site. Further structure optimization resulted in the selective inhibitors preferentially binding to individual YEATS-containing proteins including AF9 and ENL with submicromolar affinities. We demonstrate that one of the ENL YEATS-selective inhibitors, XL-13m, engages with endogenous ENL, perturbs the recruitment of ENL onto chromatin, and synergizes the BET and DOT1L inhibition-induced downregulation of oncogenes in MLL-rearranged acute leukemia.

Peripheral nerve lesions provoke apoptosis in the dorsal horn of the spinal cord. The cause of cell death, the involvement of neurons, and the relevance for the processing of somatosensory information are controversial. Here, we demonstrate in a mouse model of sciatic nerve injury that glutamate-induced neurodegeneration and loss of γ-aminobutyric acid (GABA)ergic interneurons in the superficial dorsal horn promote the transition from acute to chronic neuropathic pain. Conditional deletion of Grin1, the essential subunit of N-methyl-d-aspartate-type glutamate receptors (NMDARs), protects dorsal horn neurons from excitotoxicity and preserves GABAergic inhibition. Mice deficient in functional NMDARs exhibit normal nociceptive responses and acute pain after nerve injury, but this initial increase in pain sensitivity is reversible. Eliminating NMDARs fully prevents persistent pain-like behavior. Reduced pain in mice lacking proapoptotic Baxconfirmed the significance of neurodegeneration. We conclude that NMDAR-mediated neuron death contributes to the development of chronic neuropathic pain.

ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved protein domains that interact with nucleosomal features. Here we used a quantitative high-throughput approach, based on the use of a DNA-barcoded mononucleosome library, to profile the biochemical activity of human ISWI family remodellers in response to a diverse set of nucleosome modifications. We show that accessory (non-ATPase) subunits of ISWI remodellers can distinguish between differentially modified nucleosomes, directing remodelling activity towards specific nucleosome substrates according to their modification state. Unexpectedly, we show that the nucleosome acidic patch is necessary for maximum activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface.

Cancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs. Recognition of modified histones by ‘reader’ proteins constitutes a key mechanism underlying these processes; therefore, targeting such pathways holds clinical promise, as exemplified by the development of bromodomain and extra-terminal (BET) inhibitors. We recently identified the YEATS domain as an acetyl-lysine-binding module, but its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralogue AF9, is required for disease maintenance in acute myeloid leukaemia. CRISPR–Cas9-mediated depletion of ENL led to anti-leukaemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies and chromatin-immunoprecipitation followed by sequencing analyses revealed that ENL binds to acetylated histone H3, and co-localizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemia. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced the recruitment of RNA polymerase II to ENL-target genes, leading to the suppression of oncogenic gene expression programs. Notably, disrupting the functionality of ENL further sensitized leukaemia cells to BET inhibitors. Together, our data identify ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in acute myeloid leukaemia, and suggest that displacement of ENL from chromatin may be a promising epigenetic therapy, alone or in combination with BET inhibitors, for aggressive leukaemia.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs) are deadly and common cancers. Recent genomic studies implicate multiple genetic pathways, including cell signaling, cell cycle and immune evasion, in their development. Here we analyze public data sets and uncover a previously unappreciated role of epigenome deregulation in the genesis of 13% of HPV-negative HNSCCs. Specifically, we identify novel recurrent mutations encoding p.Lys36Met (K36M) alterations in multiple H3 histone genes. histones. We further validate the presence of these alterations in multiple independent HNSCC data sets and show that, along with previously described NSD1 mutations, they correspond to a specific DNA methylation cluster. The K36M substitution and NSD1 defects converge on altering methylation of histone H3 at K36 (H3K36), subsequently blocking cellular differentiation and promoting oncogenesis. Our data further indicate limited redundancy for NSD family members in HPV-negative HNSCCs and suggest a potential role for impaired H3K36 methylation in their development. Further investigation of drugs targeting chromatin regulators is warranted in HPV-negative HNSCCs driven by aberrant H3K36 methylation.

Recognition of histone covalent modifications by 'reader' modules constitutes a major mechanism for epigenetic regulation. A recent upsurge of newly discovered histone lysine acylations, such as crotonylation (Kcr), butyrylation (Kbu), and propionylation (Kpr), greatly expands the coding potential of histone lysine modifications. Here we demonstrate that the histone acetylation-binding double PHD finger (DPF) domains of human MOZ (also known as KAT6A) and DPF2 (also known as BAF45d) accommodate a wide range of histone lysine acylations with the strongest preference for Kcr. Crystal structures of the DPF domain of MOZ in complex with H3K14cr, H3K14bu, and H3K14pr peptides reveal that these non-acetyl acylations are anchored in a hydrophobic 'dead-end' pocket with selectivity for crotonylation arising from intimate encapsulation and an amide-sensing hydrogen bonding network. Immunofluorescence and chromatin immunoprecipitation (ChIP)–quantitative PCR (qPCR) showed that MOZ and H3K14cr colocalize in a DPF-dependent manner. Our studies call attention to a new regulatory mechanism centered on histone crotonylation readout by DPF family members.

The gut microbiota influences acetylation on host histones by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-coenzyme A (CoA), a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivowith 13C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis.

Over the past 20 years, breakthrough discoveries of chromatin-modifying enzymes and associated mechanisms that alter chromatin in response to physiological or pathological signals have transformed our knowledge of epigenetics from a collection of curious biological phenomena to a functionally dissected research field. Here, we provide a personal perspective on the development of epigenetics, from its historical origins to what we define as 'the modern era of epigenetic research'. We primarily highlight key molecular mechanisms of and conceptual advances in epigenetic control that have changed our understanding of normal and perturbed development.

Covalent modifications of both DNA and histones act in concert to define the landscape of our epigenome. In this review, we explore the interconnections between histone and DNA modifications by focusing on a conserved chromatin-binding regulatory domain, the ATRX-DNMT3-DNMT3L (ADD) domain. New studies show that the ADD domain is capable of sensing, and therefore integrating, the status of multiple histone modifications. This in turn dictates the in vivolocalization or allosteric regulation of the full-length ADD-containing protein and its ability to function in downstream chromatin remodeling events. Strategies to re-engineer the ADD “reader pocket” in the de novoDNA methyltransferase DNMT3A such that it redirects this “writer” to new genomic loci proved useful in understanding important biological downstream consequences of mis-targeting of DNA methylation via altered reading of histone marks. Combined with genome-editing tools, this approach stands as a poof-of-principle and will be broadly applicable to the elucidation of epigenetic networks that have been altered by “reader” mutations, either artificially or as naturally occurs in some human diseases.

Transfer of a somatic nucleus into an enucleated oocyte is the most efficient approach for somatic cell reprogramming. While this process is known to involve extensive chromatin remodeling of the donor nucleus, the maternal factors responsible and the underlying chromatin-based mechanisms remain largely unknown. Here we discuss our recent findings demonstrating that the histone variant H3.3 plays an essential role in reprogramming and is required for reactivation of key pluripotency genes in somatic cell nuclear transfer (SCNT) embryos. Maternal-derived H3.3 replaces H3 in the donor nucleus shortly after oocyte activation, with the amount of replacement directly related to the differentiation status of the donor nucleus in SCNT embryos. We provide additional evidence to suggest that de novo synthesized H3.3 replaces histone H3 carrying repressive modifications in the donor nuclei of SCNT embryos, and hypothesize that replacement may occur at specific loci that must be reprogrammed for gene reactivation.

The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLLchromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product.

Histone covalent modifications regulate many, if not all, DNA-templated processes, including gene expression and DNA damage response. The biological consequences of histone modifications are mediated partially by evolutionarily conserved “reader/effector” modules that bind to histone marks in a modification- and context-specific fashion and subsequently enact chromatin changes or recruit other proteins to do so. Recently, the Plant Homeodomain (PHD) finger has emerged as a class of specialized “reader” modules that, in some instances, recognize the methylation status of histone lysine residues, such as histone H3 lysine 4 (H3K4). While mutations in catalytic enzymes that mediate the addition or removal of histone modifications (i.e., “writers” and “erasers”) are already known to be involved in various human diseases, mutations in the modification-specific “reader” proteins are only beginning to be recognized as contributing to human diseases. For instance, point mutations, deletions or chromosomal translocations that target PHD fingers encoded by many genes (such as recombination activating gene 2 (RAG2), Inhibitor of Growth (ING), nuclear receptor-binding SET domain-containing 1 (NSD1) and Alpha Thalassaemia and Mental Retardation Syndrome, X-linked (ATRX)) have been associated with a wide range of human pathologies including immunological disorders, cancers, and neurological diseases. In this review, we will discuss the structural features of PHD fingers as well as the diseases for which direct mutation or dysregulation of the PHD finger has been reported. We propose that misinterpretation of the epigenetic marks may serve as a general mechanism for human diseases of this category. Determining the regulatory roles of histone covalent modifications in the context of human disease will allow for a more thorough understanding of normal and pathological development, and may provide innovative therapeutic strategies wherein “chromatin readers” stand as potential drug targets.

Histone proteins are the major protein components of chromatin, the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. Chromatin is the substrate of many biological processes, such as gene regulation and transcription, replication, mitosis and apoptosis. Since histones are extensively post-translationally modified, the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Many different biochemical techniques have been developed to purify and separate histone proteins. Here, we present standard protocols for acid extraction and salt extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modification-specific antibodies.

Post-translational modifications of histone proteins, the basic building blocks around which eukaryotic DNA is organized, are crucially involved in the regulation of genome activity as they control chromatin structure and dynamics. The recruitment of specific binding proteins that recognize and interact with particular histone modifications is thought to constitute a fundamental mechanism by which histone marks mediate biological function. For instance, tri-methylation of histone H3 lysine 9 (H3K9me3) is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Until now, little was known about the regulation of effector-histone mark interactions, and in particular, of the binding of HP1 to H3K9me3. Recently, we and others presented evidence that a "binary methylation-phosphorylation switch" mechanism controls the dynamic release of HP1 from H3K9me3 during the cell cycle: phosphorylation of histone H3 serine 10 (H3S10ph) occurs at the onset of mitosis, interferes with HP1-H3K9me3 interaction, and therefore, ejects HP1 from its binding site. Here, we discuss the biological function of HP1 release from chromatin during mitosis, consider implications why the cell controls HP1 binding by such a methylation-phosphorylation switching mechanism, and reflect on other cellular pathways where binary switching of HP1 might occur.

Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophilabut becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation “signature” for this linker histone. Histone H1 from both growing and starved Tetrahymenawas analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymenawere obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.

The nucleosome, composed of an octamer of highly conserved histone proteins and associated DNA, is the fundamental unit of eukaryotic chromatin. How arrays of nucleosomes are folded into higher-order structures, and how the dynamics of such compaction are regulated, are questions that remain largely unanswered. Our recent studies demonstrated that phosphorylation of histone H2B is necessary to induce cell death that exhibits phenotypic hallmarks of apoptosis including DNA fragmentation and chromatin condensation in yeast (serine 10)1 and in mammalian cells (serine 14).2 In this article, we extend these findings by uncovering a role for H2B phosphorylation at serine 10 (Ser10) in another biological event that is associated with dramatic alterations in higher-order chromatin structure, meiosis. Our data show strong staining, indicative of H2B (Ser10) phosphorylation, during the pachytene stage of yeast meiotic prophase. These data broaden the use of this phosphorylation mark in chromatin remodeling that closely correlates with chromatin compaction. How phosphorylation marks are translated into meaningful downstream events during processes as diverse as apoptosis and meiosis remains a challenge for future studies.

Posttranslational modifications of histone tails regulate numerous biological processes including transcription, DNA repair, and apoptosis. Although recent studies suggest that structural alterations in chromatin are critical for triggering the DNA damage response, very little is known about the nature of DNA damage-induced chromatin perturbations. Here we show that the serine 14 residue in the NH2-terminal tail of histone H2B is rapidly phosphorylated at sites of DNA double-strand breaks. At late time points after irradiation, the phosphorylated form of H2B, H2B-Ser14P, accumulates into irradiation-induced foci. H2B-Ser14P foci formation is not associated with the apoptotic phosphorylation of H2B but is strictly dependent on the phosphorylated isoform of H2AX. Our results broaden the spectrum of histone modifications that constitute the DNA damage “histone code” and suggest a model for the underlying chromatin structure within damage-induced foci.

We describe distinct patterns of histone methylation during human cell cycle progression. Histone H4 methyltransferase activity was found to be cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely followed the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. These data suggest that H4 Lys 20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16 acetylation and to establish a mechanism by which this mark is epigenetically transmitted.

ABSTRACTPrevious studies have established an important role of histone acetylation in transcriptional control by nuclear hormone receptors. With chromatin immunoprecipitation assays, we have now investigated whether histone methylation and phosphorylation are also involved in transcriptional regulation by thyroid hormone receptor (TR). We found that repression by unliganded TR is associated with a substantial increase in methylation of H3 lysine 9 (H3-K9) and a decrease in methylation of H3 lysine 4 (H3-K4), methylation of H3 arginine 17 (H3-R17), and a dual modification of phosphorylation of H3 serine 10 and acetylation of lysine 14 (pS10/acK14). On the other hand, transcriptional activation by liganded TR is coupled with a substantial decrease in both H3-K4 and H3-K9 methylation and a robust increase in H3-R17 methylation and the dual modification of pS10/acK14. Trichostatin A treatment results in not only histone hyperacetylation but also an increase in methylation of H3-K4, increase in dual modification of pS10/acK14, and reduction in methylation of H3-K9, revealing an extensive interplay between histone acetylation, methylation, and phosphorylation. In an effort to understand the underlying mechanism for an increase in H3-K9 methylation during repression by unliganded TR, we demonstrated that TR interacts in vitro with an H3-K9-specific histone methyltransferase (HMT), SUV39H1. Functional analysis indicates that SUV39H1 can facilitate repression by unliganded TR and in so doing requires its HMT activity. Together, our data uncover a novel role of H3-K9 methylation in repression by unliganded TR and provide strong evidence for the involvement of multiple distinct histone covalent modifications (acetylation, methylation, and phosphorylation) in transcriptional control by nuclear hormone receptors.

Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae. Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail. Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo. Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain. These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription.

A novel histone methyltransferase, termed Set9, was isolated from human cells. Set9 contains a SET domain, but lacks the pre- and post-SET domains. Set9 methylates specifically lysine 4 (K4) of histone H3 (H3-K4) and potentiates transcription activation. The histone H3 tail interacts specifically with the histone deacetylase NuRD complex. Methylation of histone H3-K4 by Set9 precludes the association of NuRD with the H3 tail. Moreover, methylation of H3-K4 impairs Suv39h1-mediated methylation at K9 of H3 (H3-K9). The interplay between the Set9 and Suv39h1 histone methyltransferases is specific, as the methylation of H3-K9 by the histone methyltransferase G9a was not affected by Set9 methylation of H3-K4. Our studies suggest that Set9-mediated methylation of H3-K4 functions in transcription activation by competing with histone deacetylases and by precluding H3-K9 methylation by Suv39h1. Our results suggest that the methylation of histone tails can have distinct effects on transcription, depending on its chromosomal location, the combination of posttranslational modifications, and the enzyme (or protein complex) involved in the particular modification.

Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G2phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G2/M transition. During the G2phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system.

We examined the phosphorylation and acetylation of histone H3 in ovarian granulosa cells stimulated to differentiate by follicle-stimulating hormone (FSH). We found that protein kinase A (PKA) mediates H3 phosphorylation on serine 10, based on inhibition exclusively by PKA inhibitors. FSH-stimulated H3 phosphorylation in granulosa cells is not downstream of mitogen-activated protein kinase/extracellular signal-regulated kinase, ribosomal S6 kinase-2, mitogen- and stress-activated protein kinase-1, p38 MAPK, phosphatidylinositol-3 kinase, or protein kinase C. Transcriptional activation-associated H3 phosphorylation on serine 10 and acetylation of lysine 14 leads to activation of serum glucocorticoid kinase, inhibin α, and c-fosgenes. We propose that phosphorylation of histone H3 on serine 10 by PKA in coordination with acetylation of H3 on lysine 14 results in reorganization of the promoters of select FSH responsive genes into a more accessible configuration for activation. The unique role of PKA as the physiological histone H3 kinase is consistent with the central role of PKA in initiating granulosa cell differentiation.

Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an in vitrochromosome assembly system based on Xenopusegg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser10kinase activity associated with isolated mitotic chromosomes. The histone H3 kinase was not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90rsk, or cAMP-dependent protein kinase. The activity could be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity was regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibited kinase activity. The presence of PP1 on chromatin suggested that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generated an H3 kinase activity that was also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, we found that stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser10kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1.

After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that around H3 Ser10, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, we generated peptide antibodies against the putative phosphorylation site of CENP-A. ELISA, western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression.

ABSTRACTA clear relationship exists between histone acetylation and transcriptional output, the balance of which is conferred by opposing histone acetyltransferases (HATs) and histone deacetylases (HDACs). To explore the role of HDAC activity in determining the transcriptional competency of chromatin, we have exploited the biological features ofTetrahymenaas a model. Each vegetative cell contains two nuclei: a somatic, transcriptionally active macronucleus containing hyperacetylated chromatin and a transcriptionally silent, germ line micronucleus containing hypoacetylated histones. Using a PCR-based strategy, a deacetylase gene (named THD1) encoding a homolog of the yeast HDAC Rpd3p was cloned. Thd1p deacetylates all four core histones in vitro. It resides exclusively in the macronucleus during vegetative growth and is asymmetrically distributed to developing new macronuclei early in their differentiation during the sexual pathway. Together, these data are most consistent with a potential role for Thd1p in transcriptional regulation and suggest that histone deacetylation may be important for the differentiation of micronuclei into macronuclei during development.

A striking feature of early embryogenesis in a number of organisms is the use of embryonic linker histones or high mobility group proteins in place of somatic histone H1. The transition in chromatin composition towards somatic H1 appears to be correlated with a major increase in transcription at the activation of the zygotic genome. Previous studies have supported the idea that the mouse embryo essentially follows this pattern, with the significant difference that the substitute linker histone might be the differentiation variant H1°, rather than an embryonic variant. We show that histone H1° is not a major linker histone during early mouse development. Instead, somatic H1 was present throughout this period. Though present in mature oocytes, somatic H1 was not found on maternal metaphase II chromatin. Upon formation of pronuclear envelopes, somatic H1 was rapidly incorporated onto maternal and paternal chromatin, and the amount of somatic H1 steadily increased on embryonic chromatin through to the 8-cell stage. Microinjection of somatic H1 into oocytes, and nuclear transfer experiments, demonstrated that factors in the oocyte cytoplasm and the nuclear envelope, played central roles in regulating the loading of H1 onto chromatin. Exchange of H1 from transferred nuclei onto maternal chromatin required breakdown of the nuclear envelope and the extent of exchange was inversely correlated with the developmental advancement of the donor nucleus.

ABSTRACTConversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophilarequires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

In Drosophila, dosage compensation—the equalization of most X-linked gene products in males and females—is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophilamales.

Description

The centromere‐kinetochore complex can be divided into distinct domains based on structure and function. Previous work has used CREST auto‐antibodies with various microscopic techniques to map the locations of proteins within the centromere‐kinetochore complex and to analyze the maturation of prekinetochores before mitosis. Here we have focused on the centromere‐specific histone Centromere Protein (CENP)‐A and its spatial relationship to other histones and histone modifications found in condensed chromatin. We demonstrate that the phosphorylation of histone H3 is essentially excluded from a specific region of centromeric chromatin, defined by the presence of CENP‐A. Interspersion of CENP‐B with phosphorylated H3 in the inner centromere indicates that the exclusion of H3 modification is not a general property of α‐satellite DNA. We also demonstrate that these regions are functionally distinct by fragmenting mitotic chromatin into motile centromere‐kinetochore fragments that contain CENP‐A with little or no phosphorylated H3 and nonmotile fragments that contain exclusively phosphorylated H3. The sequence of CENP‐A diverges from H3 in a number of key residues involved in chromosome condensation and in transcription, potentially allowing a more specialized chromatin structure within centromeric heterochromatin, on which kinetochore plates may nucleate and mature. This specialized centromere subdomain would be predicted to have a very tight and static nucleosome structure as a result of the absence of H3 phosphorylation and acetylation.—Van Hooser, A. A., Mancini, M. A., Allis, C. D., Sullivan, K. F., Brinkley, B. R. The mammalian centromere: structural domains and the attenuation of chromatin modeling. FASEB J.13 (Suppl.), S216–S220 (1999)

The temporal and spatial patterns of histone H3 phosphorylation implicate a specific role for this modification in mammalian chromosome condensation. Cells arrest in late G2 when H3 phosphorylation is competitively inhibited by microinjecting excess substrate at mid-S-phase, suggesting a requirement for activity of the kinase that phosphorylates H3 during the initiation of chromosome condensation and entry into mitosis. Basal levels of phosphorylated H3 increase primarily in late-replicating/early-condensing heterochromatin both during G2 and when premature chromosome condensation is induced. The prematurely condensed state induced by okadaic acid treatment during S-phase culminates with H3 phosphorylation throughout the chromatin, but in an absence of mitotic chromosome morphology, indicating that the phosphorylation of H3 is not sufficient for complete condensation. Mild hypotonic treatment of cells arrested in mitosis results in the dephosphorylation of H3 without a cytological loss of chromosome compaction. Hypotonic-treated cells, however, complete mitosis only when H3 is phosphorylated. These observations suggest that H3 phosphorylation is required for cell cycle progression and specifically for the changes in chromatin structure incurred during chromosome condensation.

It has recently been demonstrated that bromodomains — motifs found in several eukaryotic transcription factors — bind to acetyl-lysine, a modification of histones that is important for transcription. This finding suggests that the regulatory effects of histone acetylation may be exerted by bromodomain-containing proteins.

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fosand c-mycare rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fosand c-mycgenes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of rasexpression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

ABSTRACTHeterochromatin represents a specialized chromatin environment vital to both the repression and expression of certain eukaryotic genes. One of the best-studied heterochromatin-associated proteins isDrosophilaHP1. In this report, we have disrupted all somatic copies of the Tetrahymena HHP1gene, which encodes an HP1-like protein, Hhp1p, in macronuclei (H. Huang, E. A. Wiley, R. C. Lending, and C. D. Allis, Proc. Natl. Acad. Sci. USA 95:13624–13629, 1998). Unlike the DrosophilaHP1 gene,HHP1is not essential in Tetrahymenaspp., and during vegetative growth no clear phenotype is observed in cells lacking Hhp1p (?HHP1). However, during a shift to nongrowth conditions, the survival rate of ?HHP1cells is reduced compared to that of wild-type cells. Upon starvation, Hhp1p becomes hyperphosphorylated concomitant with a reduction in macronuclear volume and an increase in the size of electron-dense chromatin bodies; neither of these morphological changes occurs in the absence of Hhp1p. Activation of two starvation-induced genes (ngoAand CyP) is significantly reduced in ?HHP1cells while, in contrast, the expression of several growth-related or constitutively expressed genes is comparable to that in wild-type cells. These results suggest that Hhp1p functions in the establishment and/or maintenance of a specialized condensed chromatin environment that facilitates the expression of certain genes linked to a starvation-induced response.

ABSTRACTRecently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymenamacronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymenamacronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymenaactivities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymenaactivity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be ~80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena.

Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34cdc2kinase consensus sequences. Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34cdc2site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymenaand demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena.