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Background and Aims Current understanding of histone post-translational modifications [histone modifications] across immune cell types in patients with inflammatory bowel disease [IBD] during remission and flare is limited. The present study aimed to quantify histone modifications at a single-cell resolution in IBD patients during remission and flare and how they differ compared to healthy controls. Methods We performed a case-control study of 94 subjects [83 IBD patients and 11 healthy controls]. IBD patients had either ulcerative colitis [n = 38] or Crohn’s disease [n = 45] in clinical remission or flare. We used epigenetic profiling by time-of-flight [EpiTOF] to investigate changes in histone modifications within peripheral blood mononuclear cells from IBD patients. Results We discovered substantial heterogeneity in histone modifications across multiple immune cell types in IBD patients. They had a higher proportion of less differentiated CD34+ haematopoietic progenitors, and a subset of CD56bright natural killer [NK] cells and γδ T cells characterized by distinct histone modifications associated with gene transcription. The subset of CD56bright NK cells had increases in several histone acetylations. An epigenetically defined subset of NK cells was associated with higher levels of C-reactive protein in peripheral blood. CD34+ monocytes from IBD patients had significantly decreased cleaved H3T22, suggesting they were epigenetically primed for macrophage differentiation. Conclusion We describe the first systems-level quantification of histone modifications across immune cells from IBD patients at a single-cell resolution, revealing the increased epigenetic heterogeneity that is not possible with traditional ChIP-seq profiling. Our data open new directions in investigating the association between histone modifications and IBD pathology using other epigenomic tools.

Chromatin remodeling through post-translational modifications of histone tails (HPTM) is fundamental for regulation and maintenance of DNA-centered processes. Systems level understanding of coordination and interactions between HPTMs and their impact on the functional state of the immune cells remain unexplored due to the technical reasons. We leveraged large biologically heterogeneous data (>27 million cells), comprising of primary human immune cells profiled for 33 HPTMs and 4 histone variants at the single-cell level using high-dimensional mass cytometry (EpiTOF), to discover and map relations between HPTMs at the systems level. Briefly, we elucidated a comprehensive epigenetic network of HPTM interactions, discovered a novel subset of hematopoietic progenitors with distinct epigenetic profile, and revealed hitherto undescribed associations between a decrease in global methylations, modulation of one-carbon metabolism, and immune cell life span. Ultimately our work lays a foundation for future studies aimed at understanding complexity of HPTM interactions in immune response in infectious or autoimmune diseases, cancers, and vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we used a systems biology approach to map the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza resulted in persistently reduced H3K27ac in monocytes and myeloid dendritic cells, which was associated with impaired cytokine responses to TLR stimulation. Single cell ATAC-seq analysis of 120,305 single cells revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were also observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at loci targeted by interferon response factors (IRFs). This was associated with elevated expression of antiviral genes and type 1 IFN production and heightened resistance to infection with the heterologous viruses Zika and Dengue. These results demonstrate that influenza vaccines stimulate persistent epigenomic remodeling of the innate immune system. Notably, AS03-adjuvanted vaccination remodeled the epigenome of myeloid cells to confer heightened resistance against heterologous viruses, revealing its potentially unappreciated role as an epigenetic adjuvant.

SummaryTelomerase is intimately associated with stem cells and upregulated in cancer, where it serves essential roles through its catalytic action in elongating telomeres, nucleoprotein caps that protect chromosome ends1. Overexpression of the telomerase reverse transcriptase (TERT) enhances cell proliferation through telomere-independent means, yet definitive evidence for such a direct role in stem cell function has yet to be revealed through loss-of-function studies. Here, we show that conditional deletion of TERT in spermatogonial stem cells (SSCs) markedly impairs competitive clone formation. Using lineage-tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that selective TERT-inactivation in SSCs causes accelerated stem cell differentiation thereby disrupting clone formation. This requirement for TERT in clone formation is bypassed by expression of a catalytically inactive TERT transgene and occurs independently of the canonical telomerase complex. TERT inactivation induces a genome-wide reduction in open chromatin evident in purified SSCs, but not in committed progenitor cells. Loss of TERT causes reduced activity of the MYC oncogene and transgenic expression of MYC in TERT-deleted SSCs efficiently rescues clone formation. These data reveal a required catalytic activity-independent role for TERT in preventing stem cell differentiation, forge a genetic link between TERT and MYC and suggest new means by which TERT may promote tumorigenesis.

COVID-19 is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. Here we develop three protein arrays to measure IgG autoantibodies associated with connective tissue diseases, anti-cytokine antibodies, and anti-viral antibody responses in serum from 147 hospitalized COVID-19 patients. Autoantibodies are identified in approximately 50% of patients but in less than 15% of healthy controls. When present, autoantibodies largely target autoantigens associated with rare disorders such as myositis, systemic sclerosis and overlap syndromes. A subset of autoantibodies targeting traditional autoantigens or cytokines develop de novo following SARS-CoV-2 infection. Autoantibodies track with longitudinal development of IgG antibodies recognizing SARS-CoV-2 structural proteins and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. We conclude that SARS-CoV-2 causes development of new-onset IgG autoantibodies in a significant proportion of hospitalized COVID-19 patients and are positively correlated with immune responses to SARS-CoV-2 proteins., Infection with SARS-CoV2 and the development of Coronavirus disease 2019 (COVID-19) has been linked to induction of autoimmunity and autoantibody production. Here the authors characterise the new-onset IgG autoantibody response in hospitalised patients with COVID-19 which they correlate to the magnitude of the SARS-CoV2 response.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

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.

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. Chromatin undergoes extensive reprogramming during immune cell differentiation. Here Kuo and colleagues uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

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. Single-cell sequencing technologies enable transcriptomic, epigenomic and proteomic analysis of rare or heterogeneous populations of cells. In this Review, Kuo and colleagues discuss current and future uses of single-cell sequencing technologies for rheumatology research.

Gene expression analysis at the point-of-care is important for rapid disease diagnosis, but traditional techniques are limited by multiplexing capabilities, bulky equipment, and cost. We present a gene expression analysis platform using a giant magnetoresistive (GMR) biosensor array, which allows multiplexed transcript detection and quantification through cost-effective magnetic detection. In this work, we have characterized the sensitivity, dynamic range, and quantification accuracy of Polymerase chain reaction (PCR)-amplified complementary DNA (cDNA) on the GMR for the reference gene GAPDH. A synthetic GAPDH single-stranded DNA (ssDNA) standard was used to calibrate the detection, and ssDNA dilutions were qPCR-amplified to obtain a standard curve. We demonstrate that the GMR platform provides a dynamic range of 4 orders of magnitude and a limit of detection of 1 pM and 0.1 pM respectively for 15 and 18-cycle amplified synthetic GAPDH PCR products. The quantitative results of GMR analysis of cell-line RNA were confirmed by qPCR.

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.

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 Ni2+ 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.

Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or di-methylated lysine. The domain is broadly specific for methylated lysine (“pan-specific”) and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of novel methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferse GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.

Systemic sclerosis (SSc) is a rare autoimmune disease with the highest case-fatality rate of all connective tissue diseases. Current efforts to determine patient response to a given treatment using the modified Rodnan skin score (mRSS) are complicated by interclinician variability, confounding, and the time required between sequential mRSS measurements to observe meaningful change. There is an unmet critical need for an objective metric of SSc disease severity. Here, we performed an integrated, multicohort analysis of SSc transcriptome data across 7 datasets from 6 centers composed of 515 samples. Using 158 skin samples from SSc patients and healthy controls recruited at 2 centers as a discovery cohort, we identified a 415-gene expression signature specific for SSc, and validated its ability to distinguish SSc patients from healthy controls in an additional 357 skin samples from 5 independent cohorts. Next, we defined the SSc skin severity score (4S). In every SSc cohort of skin biopsy samples analyzed in our study, 4S correlated significantly with mRSS, allowing objective quantification of SSc disease severity. Using transcriptome data from the largest longitudinal trial of SSc patients to date, we showed that 4S allowed us to objectively monitor individual SSc patients over time, as (a) the change in 4S of a patient is significantly correlated with change in the mRSS, and (b) the change in 4S at 12 months of treatment could predict the change in mRSS at 24 months. Our results suggest that 4S could be used to distinguish treatment responders from nonresponders prior to mRSS change. Our results demonstrate the potential clinical utility of a novel robust molecular signature and a computational approach to SSc disease severity quantification.

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.

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.

Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have previously demonstrated that double-strand breaks near telomeres in mammalian cells result in either the addition of a new telomere at the site of the break, termed chromosome healing, or sister chromatid fusion that initiates chromosome instability. In the present study, we have investigated the role of telomerase in chromosome healing and the importance of chromosome healing in preventing chromosome instability. In embryonic stem cell lines that are wild type for the catalytic subunit of telomerase ( TERT ), chromosome healing at I- Sce I-induced double-strand breaks near telomeres accounted for 22 of 35 rearrangements, with the new telomeres added directly at the site of the break in all but one instance. In contrast, in two TERT -knockout embryonic stem cell lines, chromosome healing accounted for only 1 of 62 rearrangements, with a 23 bp insertion at the site of the sole chromosome-healing event. However, in a third TERT -knockout embryonic stem cell line, 10PTKO-A, chromosome healing was a common event that accounted for 20 of 34 rearrangements. Although this chromosome healing also occurred at the I- Sce I site, differences in the microhomology at the site of telomere addition demonstrated that the mechanism was distinct from that in wild-type embryonic stem cell lines. In addition, the newly added telomeres in 10PTKO-A shortened with time in culture, eventually resulting in either telomere elongation through a telomerase-independent mechanism or loss of the subtelomeric plasmid sequences entirely. The combined results demonstrate that chromosome healing can occur through both telomerase-dependent and -independent mechanisms, and that although both mechanisms can prevent degradation and sister chromatid fusion, neither mechanism is efficient enough to prevent sister chromatid fusion from occurring in many cells experiencing double-strand breaks near telomeres.

The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae1,2. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype3. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein3, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder4,5. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome4,5. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.

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.

Telomerase maintains cell viability and chromosomal stability through the addition of telomere repeats to chromosome ends. The reactivation of telomerase through the upregulation of TERT, the telomerase protein subunit, is an important step during cancer development, yet TERT protein function and regulation remain incompletely understood. Despite its close sequence similarity to human TERT (hTERT), we find that mouse TERT (mTERT) does not immortalize primary human fibroblasts. Here we exploit these differences in activity to understand TERT protein function by creating chimeric mouse-human TERT proteins. Through the analysis of these chimeric TERT proteins, we find that sequences in the human carboxy-terminal domain are critical for telomere maintenance in human fibroblasts. The substitution of the human carboxy-terminal sequences into the mouse TERT protein is sufficient to confer immortalization and maintenance of telomere length and function. Strikingly, we find that hTERT protein accumulates to markedly higher levels than does mTERT protein and that the sequences governing this difference in protein regulation also reside in the carboxy-terminal domain. These elevated protein levels, which are characteristic of hTERT, are necessary but not sufficient for telomere maintenance because stabilized mTERT mutants cannot immortalize human cells. Thus, the TERT carboxy terminus contains sequences that regulate TERT protein levels and determinants that are required for productive action on telomere ends.

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.

Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes--differentiated epithelial cells crucial for filtration--are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN results in marked normalization of podocytes, including rapid cell-cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt-β-catenin pathways in podocyte proliferation and disease.

The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.

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.

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/beta-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/beta-catenin signalling by serving as a cofactor in a beta-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior-posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert(-/-) mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/beta-catenin signalling pathway.

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.

Telomeres protect chromosome ends and serve as a substrate for telomerase, a reverse transcriptase that adds DNA repeats to the telomere terminus. In the absence of telomerase, telomeres progressively shorten, ultimately leading to telomere uncapping, a structural change at the telomere that activates DNA damage responses and leads to ligation of chromosome ends. Telomere uncapping has been implicated in aging and cancer, yet the precise mechanism of uncapping and its relationship to cell cycle remain to be defined. Here, we show that telomeres uncap in an S-phase-dependent manner in gastrointestinal progenitors of TERT −/− mice. We develop an in vivo assay that allows a quantitative kinetic assessment of telomere dysfunction-induced apoptosis and its relationship to cell cycle. By exploiting the mathematical relationship between rates of generation and clearance of apoptotic cells, we show that 86.2 ± 8.8% of apoptotic gastrointestinal cells undergo programmed cell death either late in S-phase or in G 2 . Apoptosis is primarily triggered via a signaling cascade from newly uncapped telomeres to the tumor suppressor p53, rather than by chromosome fusion-bridge breakage, because mitotic blockade did not alter the rate of newly generated apoptotic bodies. These data support a model in which rapidly dividing progenitor cells within a tissue with short telomeres are vulnerable to telomere uncapping during or shortly after telomere replication.

TERT, the protein component of telomerase1,2, serves to maintain telomere function through the de novo addition of telomere repeats to chromosome ends and is reactivated in 90% of human cancers. In normal tissues, TERT is expressed in stem cells and in progenitor cells3, but its role in these compartments is not fully understood. Here, we show that conditional transgenic induction of TERT in mouse skin epithelium causes a rapid transition from telogen, the resting phase of the hair follicle cycle, to anagen, the active phase, thereby facilitating robust hair growth. TERT overexpression promotes this developmental transition by causing proliferation of quiescent, multipotent stem cells in the hair follicle bulge region. This new function for TERT does not require the telomerase RNA component (TERC), which encodes the template for telomere addition, and therefore operates through a novel mechanism independent of its activity in synthesizing telomere repeats. These data indicate that, in addition to its established role in extending telomeres, TERT can promote proliferation of resting stem cells through a non-canonical pathway.

While selective binding of the tandem tudor domains of p53 binding protein 1 (53BP1) to dimethylated histone H4 lysine 20 (H4K20me2) at DNA double strand breaks (DSBs) is well-documented to be an essential event for proficient DSB repair, the enzyme(s) responsible for H4K20 methylation at DSBs remained unknown. Contrary to previous reports implicating the MMSET/NSD2 methyltransferase in this pathway, we demonstrate that MMSET is dispensable for H4K20 methylation, 53BP1 accumulation and DSB repair. Alternatively, we discovered that the PR-Set7 H4K20 monomethyltransferase (H4K20me1) is an essential component of nonhomologous end joining (NHEJ) which functions early and directly in DSB repair. PR-Set7 was found to mobilize immediately to DSBs and PR-Set7 enzymatic activity was necessary for H4K20 methylation, 53BP1 accumulation and proficient DSB repair. Identification of PR-Set7-associated proteins revealed interactions between PR-Set7 and several NHEJ repair proteins. Consistent with these findings, the rapid recruitment of PR-Set7 to induced DSBs was dependent on direct binding to the NHEJ Ku70/Ku86 heterodimer. PR-Set7-mediated H4K20me1 was found to be necessary, but not sufficient, for H4K20me2 and 53BP1 recruitment at induced DSBs and an undamaged ectopic locus. In addition, we also discovered that the Suv4-20 H4K20 dimethyltransferases mobilize to DSBs and were required for H4K20me2 and 53BP1 accumulation. Importantly, Suv4-20 was not sufficient for H4K20me2 and 53BP1 localization at induced DSBs or an undamaged ectopic locus in cells lacking a catalytically competent PR-Set7. Therefore, these results illuminate an orchestrated molecular pathway whereby PR-Set7-mediated H4K20me1 “primes” the DSB to expedite subsequent catalysis by Suv4-20 for the generation of H4K20me2 at the lesion necessary for 53BP1 binding. The collective findings identify PR-Set7 as an essential early component of NHEJ and reveal a novel temporal PR-Set7-dependent pathway that facilitates H4K20me2 and 53BP1 accumulation at DSBs necessary for proficient DSB repair. Furthermore, these results and preliminary studies indicate that small molecule inhibitors targeting PR-Set7 and/or Suv4-20 will likely be clinically effective radio- and chemo-sensitizing agents in cancer treatment. Citation Format: Creighton T. Tuzon, Tanya M. Spektor, Lauren M. Congdon, Xiaodong Kong, Peggie Cheung, Alex J. Kuo, Kyoko Yokomori, Or Gozani, Judd C. Rice. Orchestrated recruitment of histone methyltransferases to DNA double strand breaks facilitates 53BP1 binding and proficient repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-133. doi:10.1158/1538-7445.AM2014-LB-133

Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERTci) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERTci retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways., Author Summary Stem cells and progenitor cells within a tissue are required to maintain tissue homeostasis and to repair tissues after injury by giving rise to differentiated daughter cells. Many progenitor cells express telomerase, a reverse transcriptase enzyme that adds DNA repeats to telomeres, the protective structures that cap chromosome ends. Telomere addition by telomerase is important for normal progenitor cell function and is crucial for enabling cancer cells to divide an unlimited number of times. In addition to its telomere-lengthening function, telomerase reverse transcriptase (TERT) can directly activate quiescent epidermal stem cells. However, the mechanism underlying this novel function for TERT is still not understood. In this study, we demonstrate that the catalytic activity of TERT is dispensable for its ability to activate tissue progenitor cells in vivo. Furthermore, using gene microarrays, we show that TERT controls a developmental program that overlaps the natural transcriptional program of hair follicle cycling in mouse skin. Using pattern-matching algorithms, we find that the TERT-controlled genetic program significantly resembles programs regulated by Myc and Wnt, two pathways critical for stem cell function and tumorigenesis. This paper reveals critical new insights into novel mechanisms of non-telomerase functions of TERT, identifying TERT as a developmental regulator linked to control of transcriptional responses.