Blimp-1 molds the epigenetic architecture of IL-21-mediated autoimmune diseases through an autoregulatory circuit.

Positive regulatory domain 1 (PRDM1) encodes B lymphocyte-induced maturation protein 1 (BLIMP1), also known as a master regulator of T cell homeostasis. We observed a negative relationship between Blimp-1 and IL-21 based on our previous data that Blimp-1 overexpression in T cells suppresses autoimmune diabetes while Blimp-1-deficient T cells contribute to colitis in NOD mice. Reanalysis of published data sets also revealed an inverse correlation between PRDM1 and IL21 in Crohn's disease. Here, we illustrate that Blimp-1 repressed IL-21 by reducing chromatin accessibility and evicting an IL-21 activator, c-Maf, from the Il21 promoter. Moreover, Blimp-1 overexpression-mediated reduction in permissive chromatin structures at the Il21 promoter could override IL-21-accelerated autoimmune diabetogenesis in small ubiquitin-like modifier-defective c-Maf-transgenic mice. An autoregulatory feedback loop to harness IL-21 expression was unveiled by the evidence that IL-21 addition induced time-dependent Blimp-1 expression and subsequently enriched its binding to the Il21 promoter to suppress IL-21 overproduction. Furthermore, intervention of this feedback loop by IL-21 blockade, with IL-21R.Fc administration or IL-21 receptor deletion, attenuated Blimp-1 deficiency-mediated colitis and reinforced the circuit between Blimp-1 and IL-21 in the regulation of autoimmunity. We highlight the translation of Blimp-1-based epigenetic and transcriptomic profiles applicable to a personalized medicine approach in autoimmune diseases.

Integrated Multiomic Profiling Identifies the Epigenetic Regulator PRC2 as a Therapeutic Target to Counteract Leukemia Immune Escape and Relapse.

Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse. SIGNIFICANCE: Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397.

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Functional dissection of inherited non-coding variation influencing multiple myeloma risk.

Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy.

Clinical advances in targeting epigenetics for cancer therapy.

The appropriate coordination between epigenetic regulators is essential for spatial and temporal regulation of gene expression and maintenance of cell identity. Cancer is a disease driven by both genetic and epigenetic alterations. The widespread dysregulation and reversible nature of epigenetic alterations confer cancer cells with vulnerabilities for therapeutic interventions. Over the past decades, remarkable progress has been made in developing drugs that target epigenetic regulators, with many drugs under evaluation in clinical trials. Here, we summarize the epigenetic drugs currently in clinical investigations and highlight the potentials and challenges in their implication to treat cancer. We also discuss the preclinical and clinical results of combination therapies with epigenetic drugs and other therapies such as targeted and immune-based therapies.

Hierarchical regulation of the resting and activated T cell epigenome by major transcription factor families.

T cell activation, a key early event in the adaptive immune response, is subject to elaborate transcriptional control. In the present study, we examined how the activities of eight major transcription factor (TF) families are integrated to shape the epigenome of naive and activated CD4 and CD8 T cells. By leveraging extensive polymorphisms in evolutionarily divergent mice, we identified the 'heavy lifters' positively influencing chromatin accessibility. Members of Ets, Runx and TCF/Lef TF families occupied the vast majority of accessible chromatin regions, acting as 'housekeepers', 'universal amplifiers' and 'placeholders', respectively, at sites that maintained or gained accessibility upon T cell activation. In addition, a small subset of strongly induced immune response genes displayed a noncanonical TF recruitment pattern. Our study provides a key resource and foundation for the understanding of transcriptional and epigenetic regulation in T cells and offers a new perspective on the hierarchical interactions between critical TFs.

Lupus band test can be used in combination with anti-chromatin antibodies and complement analysis to predict transition from cutaneous to systemic lupus.

The lupus band test (LBT) is frequently performed for patients with lupus erythematosus (LE) but its capacity to discriminate cutaneous (C)LE from systemic (S)LE is debated, as well as its association with serum antinuclear antibodies (ANA) and complement reduction. Among 158 patients, 56 received retrospectively a diagnosis of CLE, 37 have SLE and 65 other skin disorders. Considering 29 clinical, histopathologic, LBT, and serological parameters: 5 parameters were effective in distinguishing LE from other skin disorders (e.g. skin photosensitivity, LBT positivity, basal vacuolar changes, thickening of the basement membrane, and anti-SSA-60 kDa); and 8 parameters were able to separate SLE from CLE (e.g. arthritis, lupus nephritis, hematological manifestations, Raynaud/sicca manifestations, anti-chromatin, anti-dsDNA, and low levels of C3/4). A positive LBT was further determined to be associated with systemic manifestations when associated with anti-chromatin response and complement reduction in the profile of patients evolving to a systemic form of lupus.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection.

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system.

Early and Late Processes Driving NET Formation, and the Autocrine/Paracrine Role of Endogenous RAGE Ligands.

Neutrophil extracellular trap (NET) formation has emerged as an important response against various pathogens; it also plays a role in chronic inflammation, autoimmunity, and cancer. Despite a growing understanding of the mechanisms underlying NET formation, much remains to be elucidated. We previously showed that in human neutrophils activated with different classes of physiological stimuli, NET formation features both early and late events that are controlled by discrete signaling pathways. However, the nature of these events has remained elusive. We now report that PAD4 inhibition only affects the early phase of NET generation, as do distinct signaling intermediates (TAK1, MEK, p38 MAPK). Accordingly, the inducible citrullination of residue R2 on histone H3 is an early neutrophil response that is regulated by these kinases; other arginine residues on histones H3 and H4 do not seem to be citrullinated. Conversely, elastase blockade did not affect NET formation by several physiological stimuli, though it did so in PMA-activated cells. Among belated events in NET formation, we found that chromatin decondensation is impaired by the inhibition of signaling pathways controlling both early and late stages of the phenomenon. In addition to chromatin decondensation, other late processes were uncovered. For instance, unstimulated neutrophils can condition themselves to be poised for rapid NET induction. Similarly, activated neutrophils release endogenous proteic factors that promote and largely mediate NET generation. Several such factors are known RAGE ligands and accordingly, RAGE inbibition largely prevents both NET formation and the conditioning of neutrophils to rapidly generate NETs upon stimulation. Our data shed new light on the cellular processes underlying NET formation, and unveil unsuspected facets of the phenomenon that could serve as therapeutic targets. In view of the involvement of NETs in both homeostasis and several pathologies, our findings are of broad relevance.

OCT2 pre-positioning facilitates cell fate transition and chromatin architecture changes in humoral immunity.

During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE 'seeds' featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCAB in GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in accessibility and architectural interactivity of these regions were dependent on recruitment of OCAB. Pre-positioning key regulators at SEs may represent a broadly used strategy for facilitating rapid cell fate transitions.

Altered regulation of DPF3, a member of the SWI/SNF complexes, underlies the 14q24 renal cancer susceptibility locus.

Our study investigated the underlying mechanism for the 14q24 renal cell carcinoma (RCC) susceptibility risk locus identified by a genome-wide association study (GWAS). The sentinel single-nucleotide polymorphism (SNP), rs4903064, at 14q24 confers an allele-specific effect on expression of the double PHD fingers 3 (DPF3) of the BAF SWI/SNF complex as assessed by massively parallel reporter assay, confirmatory luciferase assays, and eQTL analyses. Overexpression of DPF3 in renal cell lines increases growth rates and alters chromatin accessibility and gene expression, leading to inhibition of apoptosis and activation of oncogenic pathways. siRNA interference of multiple DPF3-deregulated genes reduces growth. Our results indicate that germline variation in DPF3, a component of the BAF complex, part of the SWI/SNF complexes, can lead to reduced apoptosis and activation of the STAT3 pathway, both critical in RCC carcinogenesis. In addition, we show that altered DPF3 expression in the 14q24 RCC locus could influence the effectiveness of immunotherapy treatment for RCC by regulating tumor cytokine secretion and immune cell activation.

The Chromatin Accessibility Landscape of Peripheral Blood Mononuclear Cells in Patients With Systemic Lupus Erythematosus at Single-Cell Resolution.

Objective: Systemic lupus erythematosus (SLE) is a complex autoimmune disease, and various immune cells are involved in the initiation, progression, and regulation of SLE. Our goal was to reveal the chromatin accessibility landscape of peripheral blood mononuclear cells (PBMCs) in SLE patients at single-cell resolution and identify the transcription factors (TFs) that may drive abnormal immune responses. Methods: The assay for transposase accessible chromatin in single-cell sequencing (scATAC-seq) method was applied to map the landscape of active regulatory DNA in immune cells from SLE patients at single-cell resolution, followed by clustering, peak annotation and motif analysis of PBMCs in SLE. Results: Peripheral blood mononuclear cells were robustly clustered based on their types without using antibodies. We identified twenty patterns of TF activation that drive abnormal immune responses in SLE patients. Then, we observed ten genes that were highly associated with SLE pathogenesis by altering T cell activity. Finally, we found 12 key TFs regulating the above six genes (CD83, ELF4, ITPKB, RAB27A, RUNX3, and ZMIZ1) that may be related to SLE disease pathogenesis and were significantly enriched in SLE patients (p <0.05, FC >2). With qPCR experiments on CD83, ELF4, RUNX3, and ZMIZ1 in B cells, we observed a significant difference in the expression of genes (ELF4, RUNX3, and ZMIZ1), which were regulated by seven TFs (EWSR1-FLI1, MAF, MAFA, NFIB, NR2C2 (var. 2), TBX4, and TBX5). Meanwhile, the seven TFs showed highly accessible binding sites in SLE patients. Conclusions: These results confirm the importance of using single-cell sequencing to uncover the real features of immune cells in SLE patients, reveal key TFs in SLE-PBMCs, and provide foundational insights relevant for epigenetic therapy.

B cell subset composition segments clinically and serologically distinct groups in chronic cutaneous lupus erythematosus.

OBJECTIVE: While the contribution of B-cells to SLE is well established, its role in chronic cutaneous lupus erythematosus (CCLE) remains unclear. Here, we compare B-cell and serum auto-antibody profiles between patients with systemic lupus erythematosus (SLE), CCLE, and overlap conditions. METHODS: B-cells were compared by flow cytometry amongst healthy controls, CCLE without systemic lupus (CCLE+/SLE-) and SLE patients with (SLE+/CCLE+) or without CCLE (SLE+/CCLE-). Serum was analyed for autoreactive 9G4+, anti-double-stranded DNA, anti-chromatin and anti-RNA antibodies by ELISA and for anti-RNA binding proteins (RBP) by luciferase immunoprecipitation. RESULTS: Patients with CCLE+/SLE- share B-cell abnormalities with SLE including decreased unswitched memory and increased effector B-cells albeit at a lower level than SLE patients. Similarly, both SLE and CCLE+/SLE- patients have elevated 9G4+ IgG autoantibodies despite lower levels of anti-nucleic acid and anti-RBP antibodies in CCLE+/SLE-. CCLE+/SLE- patients could be stratified into those with SLE-like B-cell profiles and a separate group with normal B-cell profiles. The former group was more serologically active and more likely to have disseminated skin lesions. CONCLUSION: CCLE displays perturbations in B-cell homeostasis and partial B-cell tolerance breakdown. Our study demonstrates that this entity is immunologically heterogeneous and includes a disease segment whose B-cell compartment resembles SLE and is clinically associated with enhanced serological activity and more extensive skin disease. This picture suggests that SLE-like B-cell changes in primary CCLE may help identify patients at risk for subsequent development of SLE. B-cell profiling in CCLE might also indentify candidates who would benefit from B-cell targeted therapies.

Epigenetic regulation in antiviral innate immunity.

Emerging life-threatening viruses have posed great challenges to public health. It is now increasingly clear that epigenetics plays a role in shaping host-virus interactions and there is a great need for a more thorough understanding of these intricate interactions through the epigenetic lens, which may represent potential therapeutic opportunities in the clinic. In this review, we highlight the current understanding of the roles of key epigenetic regulators - chromatin remodeling and histone modification - in modulating chromatin openness during host defense against virus. We also discuss how the RNA modification m6A (N6-methyladenosine) affects fundamental aspects of host-virus interactions. We conclude with future directions for uncovering more detailed functions that epigenetic regulation exerts on both host cells and viruses during infection.

NFAT5 Amplifies Antipathogen Responses by Enhancing Chromatin Accessibility, H3K27 Demethylation, and Transcription Factor Recruitment.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.

The Interplay Between Chromatin Architecture and Lineage-Specific Transcription Factors and the Regulation of Rag Gene Expression.

Cell type-specific gene expression is driven through the interplay between lineage-specific transcription factors (TFs) and the chromatin architecture, such as topologically associating domains (TADs), and enhancer-promoter interactions. To elucidate the molecular mechanisms of the cell fate decisions and cell type-specific functions, it is important to understand the interplay between chromatin architectures and TFs. Among enhancers, super-enhancers (SEs) play key roles in establishing cell identity. Adaptive immunity depends on the RAG-mediated assembly of antigen recognition receptors. Hence, regulation of the Rag1 and Rag2 (Rag1/2) genes is a hallmark of adaptive lymphoid lineage commitment. Here, we review the current knowledge of 3D genome organization, SE formation, and Rag1/2 gene regulation during B cell and T cell differentiation.

Kdm6b Regulates the Generation of Effector CD8+ T Cells by Inducing Chromatin Accessibility in Effector-Associated Genes.

The transcriptional and epigenetic regulation of CD8+ T cell differentiation is critical for balancing pathogen eradication and long-term immunity by effector and memory CTLs, respectively. In this study, we demonstrate that the lysine demethylase 6b (Kdm6b) is essential for the proper generation and function of effector CD8+ T cells during acute infection and tumor eradication. We found that cells lacking Kdm6b (by either T cell-specific knockout mice or knockdown using short hairpin RNA strategies) show an enhanced generation of memory precursor and early effector cells upon acute viral infection in a cell-intrinsic manner. We also demonstrate that Kdm6b is indispensable for proper effector functions and tumor protection, and that memory CD8+ T cells lacking Kdm6b displayed a defective recall response. Mechanistically, we identified that Kdm6b, through induction of chromatin accessibility in key effector-associated gene loci, allows for the proper generation of effector CTLs. Our results pinpoint the essential function of Kdm6b in allowing chromatin accessibility in effector-associated genes, and identify Kdm6b as a potential target for therapeutics in diseases with dysregulated effector responses.

A unified atlas of CD8 T cell dysfunctional states in cancer and infection.

CD8 T cells play an essential role in defense against viral and bacterial infections and in tumor immunity. Deciphering T cell loss of functionality is complicated by the conspicuous heterogeneity of CD8 T cell states described across experimental and clinical settings. By carrying out a unified analysis of over 300 assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) experiments from 12 studies of CD8 T cells in cancer and infection, we defined a shared differentiation trajectory toward dysfunction and its underlying transcriptional drivers and revealed a universal early bifurcation of functional and dysfunctional T cell states across models. Experimental dissection of acute and chronic viral infection using single-cell ATAC (scATAC)-seq and allele-specific single-cell RNA (scRNA)-seq identified state-specific drivers and captured the emergence of similar TCF1+ progenitor-like populations at an early branch point, at which functional and dysfunctional T cells diverge. Our atlas of CD8 T cell states will facilitate mechanistic studies of T cell immunity and translational efforts.

Spatial Organization of Chromatin: Transcriptional Control of Adaptive Immune Cell Development.

Higher-order spatial organization of the genome into chromatin compartments (permissive and repressive), self-associating domains (TADs), and regulatory loops provides structural integrity and offers diverse gene regulatory controls. In particular, chromatin regulatory loops, which bring enhancer and associated transcription factors in close spatial proximity to target gene promoters, play essential roles in regulating gene expression. The establishment and maintenance of such chromatin loops are predominantly mediated involving CTCF and the cohesin machinery. In recent years, significant progress has been made in revealing how loops are assembled and how they modulate patterns of gene expression. Here we will discuss the mechanistic principles that underpin the establishment of three-dimensional (3D) chromatin structure and how changes in chromatin structure relate to alterations in gene programs that establish immune cell fate.

Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells.

Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.