Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation.

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

Single-Cell Chromatin Modification Profiling Reveals Increased Epigenetic Variations with Aging.

Post-translational modifications of histone proteins and exchanges of histone variants of chromatin are central to the regulation of nearly all DNA-templated biological processes. However, the degree and variability of chromatin modifications in specific human immune cells remain largely unknown. Here, we employ a highly multiplexed mass cytometry analysis to profile the global levels of a broad array of chromatin modifications in primary human immune cells at the single-cell level. Our data reveal markedly different cell-type- and hematopoietic-lineage-specific chromatin modification patterns. Differential analysis between younger and older adults shows that aging is associated with increased heterogeneity between individuals and elevated cell-to-cell variability in chromatin modifications. Analysis of a twin cohort unveils heritability of chromatin modifications and demonstrates that aging-related chromatin alterations are predominantly driven by non-heritable influences. Together, we present a powerful platform for chromatin and immunology research. Our discoveries highlight the profound impacts of aging on chromatin modifications.

Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling.

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.

A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36.

The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Here, structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni(2+) and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, we postulate that steric constraints could prevent α-ketoglutarate from undergoing an "off-line"-to-"in-line" transition necessary for the demethylation reaction. Furthermore, structure-guided substitutions of residues in the KDM2A catalytic pocket abrogate KDM2A-mediated functions important for suppression of cancer cell phenotypes. Together, our results deduce insights into the molecular basis underlying KDM2A regulation of the biologically important methylated H3K36 mark.

NSD2 links dimethylation of histone H3 at lysine 36 to oncogenic programming.

The histone lysine methyltransferase NSD2 (MMSET/WHSC1) is implicated in diverse diseases and commonly overexpressed in multiple myeloma due to a recurrent t(4;14) chromosomal translocation. However, the precise catalytic activity of NSD2 is obscure, preventing progress in understanding how this enzyme influences chromatin biology and myeloma pathogenesis. Here, we show that dimethylation of histone H3 at lysine 36 (H3K36me2) is the principal chromatin-regulatory activity of NSD2. Catalysis of H3K36me2 by NSD2 is sufficient for gene activation. In t(4;14)-positive myeloma cells, the normal genome-wide and gene-specific distribution of H3K36me2 is obliterated, creating a chromatin landscape that selects for a transcription profile favorable for myelomagenesis. Catalytically active NSD2 confers xenograft tumor formation upon t(4;14)-negative cells and promotes oncogenic transformation of primary cells in an H3K36me2-dependent manner. Together, our findings establish H3K36me2 as the primary product generated by NSD2 and demonstrate that genomic disorganization of this canonical chromatin mark by NSD2 initiates oncogenic programming.

Single-cell epigenetics - Chromatin modification atlas unveiled by mass cytometry.

Modifications of histone proteins are fundamental to the regulation of epigenetic phenotypes. Dysregulations of histone modifications have been linked to the pathogenesis of diverse human diseases. However, identifying differential histone modifications in patients with immune-mediated diseases has been challenging, in part due to the lack of a powerful analytic platform to study histone modifications in the complex human immune system. We recently developed a highly multiplexed platform, Epigenetic landscape profiling using cytometry by Time-Of-Flight (EpiTOF), to analyze the global levels of a broad array of histone modifications in single cells using mass cytometry. In this review, we summarize the development of EpiTOF and discuss its potential applications in biomedical research. We anticipate that this platform will provide new insights into the roles of epigenetic regulation in hematopoiesis, immune cell functions, and immune system aging, and reveal aberrant epigenetic patterns associated with immune-mediated diseases.

The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome.

The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1--a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing--contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

ING4 mediates crosstalk between histone H3 K4 trimethylation and H3 acetylation to attenuate cellular transformation.

Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little is known of the molecular mechanisms linking histone lysine methylation to neoplastic disease. ING4 (Inhibitor of Growth 4) is a native subunit of an HBO1 histone acetyltransferase (HAT) complex and a tumor suppressor protein. Here we show a critical role for specific recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions. The interaction between ING4 and H3K4me3 augments HBO1 acetylation activity on H3 tails and drives H3 acetylation at ING4 target promoters. Further, ING4 facilitates apoptosis in response to genotoxic stress and inhibits anchorage-independent cell growth, and these functions depend on ING4 interactions with H3K4me3. Together, our results demonstrate a mechanism for brokering crosstalk between H3K4 methylation and H3 acetylation and reveal a molecular link between chromatin modulation and tumor suppressor mechanisms.

RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination.

Nuclear processes such as transcription, DNA replication and recombination are dynamically regulated by chromatin structure. Eukaryotic transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent post-translational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here we show that RAG2--an essential component of the RAG1/2 V(D)J recombinase, which mediates antigen-receptor gene assembly--contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the mouse RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together, our results identify a new function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence indicating that disrupting the read-out of histone modifications can cause an inherited human disease.

Aire employs a histone-binding module to mediate immunological tolerance, linking chromatin regulation with organ-specific autoimmunity.

Aire induces ectopic expression of peripheral tissue antigens (PTAs) in thymic medullary epithelial cells, which promotes immunological tolerance. Beginning with a broad screen of histone peptides, we demonstrate that the mechanism by which this single factor controls the transcription of thousands of genes involves recognition of the amino-terminal tail of histone H3, but not of other histones, by one of Aire's plant homeodomain (PHD) fingers. Certain posttranslational modifications of H3 tails, notably dimethylation or trimethylation at H3K4, abrogated binding by Aire, whereas others were tolerated. Similar PHD finger-H3 tail-binding properties were recently reported for BRAF-histone deacetylase complex 80 and DNA methyltransferase 3L; sequence alignment, molecular modeling, and biochemical analyses showed these factors and Aire to have structure-function relationships in common. In addition, certain PHD1 mutations underlying the polyendocrine disorder autoimmune polyendocrinopathy-candidiases-ectodermaldystrophy compromised Aire recognition of H3. In vitro binding assays demonstrated direct physical interaction between Aire and nucleosomes, which was in part buttressed by its affinity to DNA. In vivo Aire interactions with chromosomal regions depleted of H3K4me3 were dependent on its H3 tail-binding activity, and this binding was necessary but not sufficient for the up-regulation of genes encoding PTAs. Thus, Aire's activity as a histone-binding module mediates the thymic display of PTAs that promotes self-tolerance and prevents organ-specific autoimmunity.

The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2.

Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatin-dependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications.

NMR assignments and histone specificity of the ING2 PHD finger.

The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding to histone H3 trimethylated at lysine 4 (H3K4me3). Here, we describe backbone and side chain assignments of the ING2 PHD finger, analyze its binding to the unmodified and modified histone and p53 peptides, and map the histone H3 and H3K4me3 binding sites based on chemical shift perturbation analysis.

Single-cell technologies - studying rheumatic diseases one cell at a time.

Cells, the basic units of life, have striking differences at transcriptomic, proteomic and epigenomic levels across tissues, organs, organ systems and organisms. The coordination of individual immune cells is essential for the generation of effective immune responses to pathogens while immune tolerance is maintained to protect the host. In rheumatic diseases, when immune responses are dysregulated, pathologically important cells might represent only a small fraction of the immune system. Interrogation of the contributions of individual immune cells to pathogenesis and disease progression should therefore reveal important insights into the complicated aetiology of rheumatic diseases. Technological advances are enabling the high-dimensional dissection of single cells at multiple omics levels, which could facilitate the identification of dysregulated molecular mechanisms in patients with rheumatic diseases and the discovery of new therapeutic targets and biomarkers. The single-cell technologies that have been developed over the past decade and the experimental platforms that enable multi-omics integrative analyses have already made inroads into immunology-related fields of study and have potential for use in rheumatology. Layers of omics data derived from single cells are likely to fundamentally change our understanding of the molecular pathways that underpin the pathogenesis of rheumatic diseases.

Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks.

Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.