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The relatively quiet mutational landscape of rhabdomyosarcoma (RMS) suggests that epigenetic deregulation could be central to oncogenesis and tumour aggressiveness. Histone variants have long been recognised as important epigenetic regulators of gene expression. However, the role of histone variants in RMS has not been studied hitherto. In this study, we show that histone variant H3.3 is overexpressed in alveolar rhabdomyosarcoma (ARMS), an aggressive subtype of RMS. Functionally, knockdown of H3F3A, which encodes for H3.3, significantly impairs the ability of ARMS cells to undertake migration and invasion and reduces Rho activation. In addition, a striking reduction in metastatic tumour burden and improved survival is apparent in vivo. Using RNA-sequencing and ChIP-sequencing analyses, we identified Melanoma Cell Adhesion Molecule (MCAM/CD146) as a direct downstream target of H3.3. Loss of H3.3 resulted in a reduction in the presence of active marks, and an increase in the occupancy of H1 at the MCAM promoter. Cell migration and invasion were rescued in H3F3A-depleted cells through MCAM overexpression. Moreover, we identified G9a, a lysine methyltransferase encoded by EHMT2, as an upstream regulator of H3F3A. Therefore, this study identifies a novel H3.3 dependent axis involved in ARMS metastasis. These findings establish the potential of MCAM as a therapeutic target for high-risk ARMS patients. This article is protected by copyright. All rights reserved.

Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell–dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti–PD-1 and anti–CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen–specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti–PD-1 and anti–CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome—tumor outgrowth—several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target.

Supplementary Figure from BHLHE40 Regulates the T-Cell Effector Function Required for Tumor Microenvironment Remodeling and Immune Checkpoint Therapy Efficacy

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. As transient and stable modifications to chromatin have emerged as critical mechanisms in oncogenic signaling, efforts to target epigenetic modifiers as a therapeutic strategy have accelerated in recent years. To identify chromatin modifiers that sustain tumor growth, we performed an epigenetic screen and found that inhibition of lysine methyltransferase G9a significantly affected the viability of ARMS cell lines. Targeting expression or activity of G9a reduced cellular proliferation and motility in vitro and tumor growth in vivo. Transcriptome and chromatin immunoprecipitation–sequencing analysis provided mechanistic evidence that the tumor-suppressor PTEN was a direct target gene of G9a. G9a repressed PTEN expression in a methyltransferase activity–dependent manner, resulting in increased AKT and RAC1 activity. Re-expression of constitutively active RAC1 in G9a-deficient tumor cells restored oncogenic phenotypes, demonstrating its critical functions downstream of G9a. Collectively, our study provides evidence for a G9a-dependent epigenetic program that regulates tumor growth and suggests targeting G9a as a therapeutic strategy in ARMS.Significance:These findings demonstrate that RAC1 is an effector of G9a oncogenic functions and highlight the potential of G9a inhibitors in the treatment of ARMS.

Supplementary Figure S1. Inhibition of G9a activity reduces colony formation. Supplementary Figure S2. Inhibition of G9a expression or activity in RH30 cells impacts proliferation, differentiation and motility Supplementary Figure S3. Inhibition of G9a expression or activity in RH41 cells impacts proliferation, differentiation and motility Supplementary Figure S4. Validation of transcriptome analysis with G9a knockdown cells Supplementary Figure S5. Validation of ChIP-Seq analysis by CHIP-PCR Supplementary Figure S6. Inhibition of RAC1 activity reduces proliferation, migration and invasion Supplementary Table 1. Nanostring PanCancer pathway analysis in siG9a cells

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. Cancer stem cells (CSCs) are seeds for tumor relapse and metastasis. However, pathways that maintain stemness genes are not fully understood. Here, we report that the enzyme euchromatic histone lysine methyltransferase 1 (EHMT1) is expressed in primary and relapse ARMS tumors. EHMT1 suppression impaired motility and induced differentiation in ARMS cell lines and reduced tumor progression in a mouse xenograft model in vivo. RNA sequencing of EHMT1-depleted cells revealed downregulation of ALDH1A1 that is associated with CSCs. Consistent with this, inhibition of ALDH1A1 expression and activity mimicked EHMT1 depletion phenotypes and reduced tumorsphere formation. Mechanistically, we demonstrate that EHMT1 does not bind to the ALDH1A1 promoter but activates it by stabilizing C/EBPβ, a known regulator of ALDH1A1 expression. Our findings identify a role for EHMT1 in maintenance of stemness by regulating ALDH1A1 expression and suggest that targeting ALDH

Memory-phenotype (MP) CD4+ T cells are a substantial population of conventional T cells that exist in steady-state mice, and their immunologic functions in autoimmune disease have not yet been studied. In this work, we unveil a unique phenotype of MP CD4+ T cells by analyzing single-cell transcriptomics and T cell receptor (TCR) repertoires. We found that steady-state MP CD4+ T cells exist regardless of germ and food-antigen which are composed of heterogenous effector subpopulations. Distinct subpopulations of MP CD4+ T cells are specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells have TCR-independent bystander effector functions like innate lymphoid cell. Especially, CCR6high MP CD4+ T cells are major responders to IL-1β and IL-23 without MOG35 − 55 antigen reactivity, which gives them pathogenic-Th17 characteristics and allows them to contribute to autoimmune encephalomyelitis. We identified Bhlhe40 in CCR6high MP CD4+ T cells drives the expression of GM-CSF through IL-1β and IL-23 signaling, contributing to CNS pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal clearly distinct effector-like heterogeneity of MP CD4+ T cells in steady state and CCR6high MP CD4+ T cells exacerbate autoimmune neuroinflammation by Bhlhe40/GM-CSF axis in bystander manner synergistically with antigen-specific T cells.

Metabolic deregulation is a key factor in cancer progression. Epigenetic changes and metabolic rewiring are intertwined in cancer. Deregulated epigenetic modifiers cause metabolic aberrations by targeting the expression of metabolic enzymes. Conversely, metabolites and cofactors affect the expression and activity of epigenetic regulators. Small molecules are promising therapeutic approaches to target the epigenetic-metabolomic crosstalk in cancer. Here, we focus on the interplay between metabolic rewiring and epigenetic landscape in the context of tumourigenesis and highlight recent advances in the use of small-molecule drug targets for therapy.

Cancer cachexia (CC) is a multifactorial syndrome characterized by a significant reduction in body weight that is predominantly caused by the loss of skeletal muscle and adipose tissue. Although the ill effects of cachexia are well known, the condition has been largely overlooked, in part due to its complex etiology, heterogeneity in mediators, and the involvement of diverse signaling pathways. For a long time, inflammatory factors have been the focus when developing therapeutics for the treatment of CC. Despite promising pre-clinical results, they have not yet advanced to the clinic. Developing new therapies requires a comprehensive understanding of how deregulated signaling leads to catabolic gene expression that underlies muscle wasting. Here, we review CC-associated signaling pathways and the transcriptional cascade triggered by inflammatory cytokines. Further, we highlight epigenetic factors involved in the transcription of catabolic genes in muscle wasting. We conclude with reflections on the directions that might pave the way for new therapeutic approaches to treat CC.

Memory-phenotype (MP) CD4+T cells are a substantial population of conventional T cells that exist in steady-state mice, and their immunologic functions in autoimmune disease have not yet been studied. In this work, we unveil a unique phenotype of MP CD4+T cells by analyzing single-cell transcriptomics and T cell receptor (TCR) repertoires. We found that steady-state MP CD4+T cells exist regardless of germ and food-antigen which are composed of heterogenous effector subpopulations. Distinct subpopulations of MP CD4+T cells are specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells have TCR-independent effector functions. Especially, CCR6highMP CD4+T cells are major responders to IL-1β and IL-23 without MOG35-55antigen reactivity, which gives them pathogenic-Th17 characteristics and allows them to contribute to autoimmune encephalomyelitis. We identified Bhlhe40 in CCR6highMP CD4+T cells drives the expression of GM-CSF, contributing to CNS pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal heterogeneity of MP CD4+T cells that can contribute to autoimmune neuroinflammation in bystander manner synergistically with antigen-specific T cells.

Embryonal rhabdomyosarcoma (ERMS) is characterized by a failure of cells to complete skeletal muscle differentiation. Although ERMS cells are vulnerable to oxidative stress, the relevance of mitochondrial calcium homeostasis in oncogenesis is unclear. Here, we show that ERMS cell lines as well as primary tumours exhibit elevated expression of the Mitochondrial Calcium Uniporter (MCU). MCU knockdown resulted in impaired mitochondrial calcium uptake and a reduction in mitochondrial reactive oxygen species (mROS) levels. Phenotypically, MCU knockdown cells exhibited reduced cellular proliferation and motility, with an increased propensity to differentiate in vitro and in vivo. RNA-sequencing of MCU knockdown cells revealed a significant reduction in genes involved in TGFβ signalling that play prominent roles in oncogenesis and inhibition of myogenic differentiation. Interestingly, modulation of mROS production impacted TGFβ signalling. Our study elucidates mechanisms by which mitochondrial calcium dysregulation promotes tumour progression and suggests that targeting the MCU complex to restore mitochondrial calcium homeostasis could be a therapeutic avenue in ERMS.

Significance: The epigenomic/metabolic landscape in cancer has been studied extensively in the past decade and forms the basis of various drug targets. Yet, cancer treatment remains a challenge, with clinical trials exhibiting limited efficacy and high relapse rates. Patients respond differently to therapy, which is fundamentally attributed to tumor heterogeneity, both across and within tumors. This review focuses on the interactions between the heterogeneous tumor microenvironment (TME) and the epigenomic/metabolic axis in cancer, as well as the emerging technologies under development to aid heterogeneity studies. Recent Advances: Interlinks between epigenetics and metabolism in cancer have been reported. Emerging studies have unveiled interactions between the TME and cancer cells that play a critical role in regulating epigenetics and reprogramming cancer metabolism, suggesting a three-way cross talk. Critical Issues: This cross talk accentuates the multiplex nature of cancer, and the importance of considering tumor heterogeneity in various epigenomic/metabolic cancer studies. Future Directions: With the advancement in single-cell profiling, it may be possible to identify cancer subclones and their unique vulnerabilities to develop a multimodal therapy. Drugs targeting the TME are currently being studied, and a better understanding of the TME in regulating cancer epigenetics and metabolism may hold the key to identifying novel therapeutic targets.

Histone variants regulate chromatin accessibility and gene transcription. Given their distinct properties and functions, histone varint substitutions allow for profound alteration of nucleosomal architecture and local chromatin landscape. Skeletal myogenesis driven by the key transcription factor MyoD is characterized by precise temporal regulation of myogenic genes. Timed substitution of variants within the nucleosomes provides a powerful means to ensure sequential expression of myogenic genes. Indeed, growing evidence has shown H3.3, H2A.Z, macroH2A, and H1b to be critical for skeletal myogenesis. However, the relative importance of various histone variants and their associated chaperones in myogenesis is not fully appreciated. In this review, we summarize the role that histone variants play in altering chromatin landscape to ensure proper muscle differentiation. The temporal regulation and cross talk between histones variants and their chaperones in conjunction with other forms of epigenetic regulation could be critical to understanding myogenesis and their involvement in myopathies.

Gastrointestinal (GI) tract involvement is the major cause of morbidity and mortality in acute graft-versus-host disease (GVHD), and pathological damage is largely attributable to inflammatory cytokine production. Recently, granulocyte-macrophage colony stimulating factor (GM-CSF) has been identified as a cytokine that mediates inflammation in the GI tract, but the transcriptional program that governs GM-CSF production and the mechanism by which GM-CSF links adaptive to innate immunity within this tissue site have not been defined. In the current study, we identified Bhlhe40 as a key transcriptional regulator that governs GM-CSF production by CD4+ T cells and mediates pathological damage in the GI tract during GVHD. In addition, we observed that GM-CSF was not regulated by either interleukin 6 (IL-6) or IL-23, which are both potent inducers of GVHD-induced colonic pathology, indicating that GM-CSF constitutes a nonredundant inflammatory pathway in the GI tract. Mechanistically, GM-CSF had no adverse effect on regulatory T-cell reconstitution, but linked adaptive to innate immunity by enhancing the activation of donor-derived dendritic cells in the colon and subsequent accumulation of these cells in the mLNs. In addition, GM-CSF promoted indirect alloantigen presentation, resulting in the accumulation of donor-derived T cells with a proinflammatory cytokine phenotype in the colon. Thus, Bhlhe40+ GM-CSF+ CD4+ T cells constitute a colitogenic T-cell population that promotes indirect alloantigen presentation and pathological damage within the GI tract, positioning GM-CSF as a key regulator of GVHD in the colon and a potential therapeutic target for amelioration of this disease.

Significance: Mitochondria undergo constant morphological changes through fusion, fission, and mitophagy. As the key organelle in cells, mitochondria are responsible for numerous essential cellular functions such as metabolism, regulation of calcium (Ca2+), generation of reactive oxygen species, and initiation of apoptosis. Unsurprisingly, mitochondrial dysfunctions underlie many pathologies including cancer. Recent Advances: Currently, the gold standard for cancer treatment is chemotherapy, radiation, and surgery. However, the efficacy of these treatments varies across different cancer cells. It has been suggested that mitochondria may be at the center of these diverse responses. In the past decade, significant advances have been made in understanding distinct types of mitochondrial dysfunctions in cancer. Through investigations of underlying mechanisms, more effective treatment options are developed. Critical Issues: We summarize various mitochondria dysfunctions in cancer progression that have led to the development of therapeutic options. Current mitochondrial-targeted therapies and challenges are discussed. Future Directions: To address the "root" of cancer, utilization of mitochondrial-targeted therapy to target cancer stem cells may be valuable. Investigation of other areas such as mitochondrial trafficking may offer new insights into cancer therapy. Moreover, common antibiotics could be explored as mitocans, and synthetic lethality screens can be utilized to overcome the plasticity of cancer cells.

The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction, which relies on a complex interplay between specialized effector B and CD4 T lymphocytes, the GC B cells and T follicular helper (TFH) cells. Intriguingly, several positive key regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation, thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by the accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.

PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.

Aurora kinases are Ser/Thr-directed protein kinases which play pivotal roles in mitosis. Recent evidences highlight the importance of these kinases in multiple biological events including skeletal muscle differentiation. Our earlier study identified the transcription factor POU6F1 (or mPOU) as a novel Aurora kinase (Aurk) A substrate. Here, we report that Aurora kinase A phosphorylates mPOU at Ser197 and inhibit its DNA-binding ability. Delving into mPOU physiology, we find that the phospho-mimic (S197D) mPOU mutant exhibits enhancement, while the wild type or the phospho-deficient mutant shows retardation in C2C12 myoblast differentiation. Interestingly, POU6F1 depletion phenocopies S197D-mPOU overexpression in the differentiation context. Collectively, our results signify mPOU as a negative regulator of skeletal muscle differentiation and strengthen the importance of AurkA in skeletal myogenesis.

We show that both supplemental and ambient magnetic fields modulate myogenesis. A lone 10 min exposure of myoblasts to 1.5 mT amplitude supplemental pulsed magnetic fields (PEMFs) accentuated in vi...

Most tissue-resident macrophage populations develop during embryogenesis, self-renew in the steady state and expand during type 2 immunity. Whether shared mechanisms regulate the proliferation of macrophages in homeostasis and disease is unclear. Here we found that the transcription factor Bhlhe40 was required in a cell-intrinsic manner for the self-renewal and maintenance of large peritoneal macrophages (LPMs), but not that of other tissue-resident macrophages. Bhlhe40 was necessary for the proliferation, but not the polarization, of LPMs in response to the cytokine IL-4. During infection with the helminth Heligmosomoides polygyrus bakeri, Bhlhe40 was required for cell cycling of LPMs. Bhlhe40 repressed the expression of genes encoding the transcription factors c-Maf and Mafb and directly promoted expression of transcripts encoding cell cycle-related proteins to enable the proliferation of LPMs. In LPMs, Bhlhe40 bound to genomic sites co-bound by the macrophage lineage-determining factor PU.1 and to unique sites, including Maf and loci encoding cell-cycle-related proteins. Our findings demonstrate a tissue-specific control mechanism that regulates the proliferation of resident macrophages in homeostasis and type 2 immunity. Edelson and colleagues show that the transcription factor Bhlhe40 is required in a cell-intrinsic manner for the self-renewal and maintenance of large peritoneal macrophages, but not that of other tissue-resident macrophages.

Euchromatin histone lysine methyltransferases (EHMTs) are epigenetic regulators responsible for silencing gene transcription by catalyzing H3K9 dimethylation. Dysregulation of EHMT1/2 has been reported in multiple cancers and is associated with poor clinical outcomes. Although substantial insights have been gleaned into the downstream targets and pathways regulated by EHMT1/2, few studies have uncovered mechanisms responsible for their dysregulated expression. Moreover, EHMT1/2 interacting partners, which can influence their function and, therefore, the expression of target genes, have not been extensively explored. As none of the currently available EHMT inhibitors have made it past clinical trials, understanding upstream regulators and EHMT protein complexes may provide unique insights into novel therapeutic avenues in EHMT-overexpressing cancers. Here, we review our current understanding of the regulators and interacting partners of EHMTs. We also discuss available therapeutic drugs that target the upstream regulators and binding partners of EHMTs and could potentially modulate EHMT function in cancer progression.

The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction – a process that relies on a complex interplay between specialized effector subsets of B and CD4 T lymphocytes – GC B cells and T follicular helper (TFH) cells. Intriguingly, several key positive regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Conditional Bhlhe40 inactivation confirmed cell-autonomous functions of Bhlhe40 in both GC B and TFH cells, while the GC phenotype was further enhanced upon loss of Bhlhe40 in both cell types. This negative regulation of the GC reaction by Bhlhe40 was of crucial importance, as Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.

Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell–dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti–PD-1 and anti–CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen–specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti–PD-1 and anti–CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome—tumor outgrowth—several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target.

Wnt signaling is downregulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way toward novel therapeutic modalities. We demonstrate that EHMT2 suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of EHMT2 expression or activity in human ERMS cell lines reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in mouse xenograft models in vivo. Mechanistically, EHMT2 impacted Sp1 and p300 enrichment at the DKK1 promoter. The reduced tumor growth upon EHMT2 deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among 13 drugs targeting chromatin modifiers, EHMT2 inhibitors were highly effective in reducing ERMS cell viability. Our study demonstrates that ERMS cells are vulnerable to EHMT2 inhibitors and suggest that targeting the EHMT2-DKK1-β-catenin node holds promise for differentiation therapy.

This study demonstrates that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is essential during Mycobacterium tuberculosis infection to specifically regulate Il10 expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection., The cytokine IL-10 antagonizes pathways that control Mycobacterium tuberculosis (Mtb) infection. Nevertheless, the impact of IL-10 during Mtb infection has been difficult to decipher because loss-of-function studies in animal models have yielded only mild phenotypes. We have discovered that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is required to repress Il10 expression during Mtb infection. Loss of Bhlhe40 in mice results in higher Il10 expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-γ. Deletion of Il10 in Bhlhe40−/− mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c+ cells is sufficient to cause susceptibility to Mtb. Bhlhe40 represents the first transcription factor found to be essential during Mtb infection to specifically regulate Il10 expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection. Our findings uncover a previously elusive but significant role for IL-10 in Mtb pathogenesis., Graphical Abstract

Myogenic differentiation is accompanied by alterations in the chromatin states, which permit or restrict the transcriptional machinery and thus impact distinctive gene expression profiles. The mechanisms by which higher-order chromatin remodeling is associated with gene activation and silencing during differentiation is not fully understood. In this study, we provide evidence that the euchromatic lysine methyltransferase GLP regulates heterochromatin organization and myogenic differentiation. Interestingly, GLP represses expression of the methyl-binding protein MeCP2 that induces heterochromatin clustering during differentiation. Consequently, MeCP2 and HP1γ localization at major satellites are altered upon modulation of GLP expression. In GLP knockdown cells, depletion of MeCP2 restored both chromatin organization and myogenic differentiation. These results identify a novel regulatory axis between a histone methylation writer and DNA methylation reader, which is important for heterochromatin organization during differentiation.

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41-/-Bhlhe40-/- B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12Vκ4 BCR. Expression of a pre-rearranged VH12Vκ4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

Summary Tissue-resident memory CD8+ T (Trm) cells share core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic, and epigenetic regulation of Trm cell and TIL development and function is largely undefined. Here, we found that the transcription factor Bhlhe40 was specifically required for Trm cell and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TIL reinvigoration following anti-PD-L1 blockade. Mechanistically, Bhlhe40 sustained Trm cell and TIL mitochondrial fitness and a functional epigenetic state. Building on these findings, we identified an epigenetic and metabolic regimen that promoted Trm cell and TIL gene signatures associated with tissue residency and polyfunctionality. This regimen empowered the anti-tumor activity of CD8+ T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights into the local regulation of Trm cell and TIL function.

We show that both supplemental and ambient magnetic fields modulate myogenesis. A lone 10 min exposure of myoblasts to 1.5 mT amplitude supplemental pulsed magnetic fields (PEMFs) accentuated in vitro myogenesis by stimulating transient receptor potential (TRP)-C1–mediated calcium entry and downstream nuclear factor of activated T cells (NFAT)-transcriptional and P300/CBP-associated factor (PCAF)-epigenetic cascades, whereas depriving myoblasts of ambient magnetic fields slowed myogenesis, reduced TRPC1 expression, and silenced NFAT-transcriptional and PCAF-epigenetic cascades. The expression levels of peroxisome proliferator–activated receptor γ coactivator 1α, the master regulator of mitochondriogenesis, was also enhanced by brief PEMF exposure. Accordingly, mitochondriogenesis and respiratory capacity were both enhanced with PEMF exposure, paralleling TRPC1 expression and pharmacological sensitivity. Clustered regularly interspaced short palindromic repeats–Cas9 knockdown of TRPC1 precluded proliferative and mitochondrial responses to supplemental PEMFs, whereas small interfering RNA gene silencing of TRPM7 did not, coinciding with data that magnetoreception did not coincide with the expression or function of other TRP channels. The aminoglycoside antibiotics antagonized and down-regulated TRPC1 expression and, when applied concomitantly with PEMF exposure, attenuated PEMF-stimulated calcium entry, mitochondrial respiration, proliferation, differentiation, and epigenetic directive in myoblasts, elucidating why the developmental potential of magnetic fields may have previously escaped detection. Mitochondrial-based survival adaptations were also activated upon PEMF stimulation. Magnetism thus deploys an authentic myogenic directive that relies on an interplay between mitochondria and TRPC1 to reach fruition.—Yap, J. L. Y., Tai, Y. K., Fröhlich, J., Fong, C. H. H., Yin, J. N., Foo, Z. L., Ramanan, S., Beyer, C., Toh, S. J., Casarosa, M., Bharathy, N., Kala, M. P., Egli, M., Taneja, R., Lee, C. N., Franco-Obregón, A. Ambient and supplemental magnetic fields promote myogenesis via a TRPC1-mitochondrial axis: evidence of a magnetic mitohormetic mechanism.

Tissues in multicellular organisms are populated by resident macrophages, which perform both generic and tissue-specific functions. The latter are induced by signals from the microenvironment and rely on unique tissue-specific molecular programs requiring the combinatorial action of tissue-specific and broadly expressed transcriptional regulators. Here, we identify the transcription factors Bhlhe40 and Bhlhe41 as novel regulators of alveolar macrophages (AMs)-a population that provides the first line of immune defense and executes homeostatic functions in lung alveoli. In the absence of these factors, AMs exhibited decreased proliferation that resulted in a severe disadvantage of knockout AMs in a competitive setting. Gene expression analyses revealed a broad cell-intrinsic footprint of Bhlhe40/Bhlhe41 deficiency manifested by a downregulation of AM signature genes and induction of signature genes of other macrophage lineages. Genome-wide characterization of Bhlhe40 DNA binding suggested that these transcription factors directly repress the expression of lineage-inappropriate genes in AMs. Taken together, these results identify Bhlhe40 and Bhlhe41 as key regulators of AM self-renewal and guardians of their identity.

The transcription factor BHLHE40 is an emerging regulator of the immune system. Recent studies suggest that BHLHE40 regulates type 2 immunity, but this has not been demonstrated in vivo. We found that BHLHE40 is required in T cells for a protective T(H)2 cell response in mice infected with the helminth Heligmosomoides polygyrus bakeri (H. polygyrus). H. polygyrus elicited changes in gene and cytokine expression by lamina propria CD4(+) T cells, many of which were BHLHE40-dependent, including production of the common beta (β(C), CSF2RB) chain family cytokines GM-CSF and IL-5. In contrast to deficiency in GM-CSF or IL-5 alone, loss of both GM-CSF and IL-5 signaling impaired protection against H. polygyrus. Overall, we show that BHLHE40 regulates the T(H)2 cell transcriptional program during helminth infection to support normal expression of Csf2, Il5, and other genes required for protection and reveal unexpected redundancy of β(C) chain-dependent cytokines previously thought to possess substantially divergent functions.