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Mouse embryonic stem cells (mESCs) can be directed to acquire cell-lineage-specific genetic programs and phenotypes by stepwise exposure to defined factors, allowing the development of in vitro models for studying disease and tissue generation. In this protocol, we describe the use of cultured mESCs to generate presomitic-like mesoderm cells undergoing further specification towards myogenic progenitors (MPs). Further, we describe here a procedure to obtain, dissect, and fluorescence-activated cell sorting (FACS)-isolate somitic cells from genetically labeled Pax7

The discovery of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway arose from investigations of how cells respond to interferons (IFNs), revealing a paradigm in cell signaling conserved from slime molds to mammals. These discoveries revealed mechanisms underlying rapid gene expression mediated by a wide variety of extracellular polypeptides including cytokines, interleukins, and related factors. This knowledge has provided numerous insights into human disease, from immune deficiencies to cancer, and was rapidly translated to new drugs for autoimmune, allergic, and infectious diseases, including COVID-19. Despite these advances, major challenges and opportunities remain.

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34High, with stemness properties (genuine state), and CD34Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle., The authors acknowledge funding from MINECO-Spain (grant no. RTI2018-096068), ERC2016-AdG-741966, LaCaixa-HEALTH-HR17-00040, MDA, UPGRADE-H2020-825825, AFM and DPP-Spain to P.M.-C; María-de-Maeztu-Program for Units of Excellence to UPF (grant no. MDM-2014-0370) and the Severo-Ochoa-Program for Centers of Excellence to CNIC (grant no. SEV-2015-0505). This work was also supported by NIAMS IRP through NIH grants nos AR041126 and AR041164 to V.S. and utilized computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov); ASI, Ricerca Finalizzata, Ateneo Sapienza to A.M.; AIRC (grant no. 23257); ASI (grant no. MARS-PRE, DC-VUM-2017-006); H2020-MSCA-RISE-2014 (645648) to M.S. and a FNR core grant (grant no. C15/BM/10397420) to A.d.S. L.G.P. was partially supported by an FPI fellowship and an EMBO fellowship (grant no. ALTF 420-2017); and S.C., X.H. and V.M. by FI, Severo-Ochoa and PFI Fellowships (Spain), respectively.

Noncoding RNAs (ncRNAs) direct a remarkable number of diverse functions in development and disease through their regulation of transcription, RNA processing and translation. Leading the charge in the RNA revolution is a class of ncRNAs that are synthesized at active enhancers, called enhancer RNAs (eRNAs). Here, we review recent insights into the biogenesis of eRNAs and the mechanisms underlying their multifaceted functions and consider how these findings could inform future investigations into enhancer transcription and eRNA function.

Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.

SUMMARYESCs can adopt lineage-specific gene expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single cell-based assays were employed to catalogue gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further lineage specification. These assays identified cis-regulatory regions and transcription factors presiding gene expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs, and ESCs-derived cell lineages.

How transcriptional enhancers function to activate distant genes has been the subject of lively investigation for decades. “Enhancers, gene regulation, and genome organization” was the subject of a virtual meeting held November 16–17, 2020, under sponsorship of the National Cancer Institute (NCI), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH). The goal of the meeting was to advance an understanding of how transcriptional enhancers function within the framework of the folded genome as we understand it, emphasizing how levels of organization may influence each other and may contribute to the spatiotemporal specification of transcription. Here we focus on broad questions about enhancer function that remain unsettled and that we anticipate will be central to work in this field going forward. Perforce, we cover contributions of only some speakers and apologize to other contributors in vital areas that we could not include because of space constraints.

Summary It remains challenging to generate reproducible, high-quality cDNA libraries from RNA derived from rare cell populations. Here, we describe a protocol for high-throughput RNA-seq library preparation, including isolation of 200 skeletal muscle stem cells from mouse tibialis anterior muscle by fluorescence-activated cell sorting and cDNA preparation. We also describe RNA extraction and cDNA preparation from differentiating mouse embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Juan et al. (2016) and Garcia-Prat et al. (2016)., Graphical abstract, Highlights • FACS isolation of 200 muscle stem cells (MuSCs) from one mouse tibialis anterior muscle • cDNA library construction for deep sequencing by direct lysis of 200 MuSCs • cDNA library construction for deep sequencing from 5,000–10,000 embryonic stem cells, It remains challenging to generate reproducible, high-quality cDNA libraries from RNA derived from rare cell populations. Here, we describe a protocol for high-throughput RNA-seq library preparation, including isolation of 200 skeletal muscle stem cells from mouse tibialis anterior muscle by fluorescence-activated cell sorting and cDNA preparation. We also describe RNA extraction and cDNA preparation from differentiating mouse embryonic stem cells.

RUNX1 is essential for the generation of hematopoietic stem cells (HSCs).Runx1null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, even though zebrafish embryos with arunx1W84X mutation have defects in early definitive hematopoiesis, somerunx1W84X/W84Xembryos can develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. Here, using three new zebrafishrunx1-/-lines we uncovered the compensatory mechanism forrunx1-independent hematopoiesis. We show that, in the absence of a functionalrunx1, acd41-GFP+population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of thecd41-GFP+cells identified a set ofrunx1-/--specific signature genes during hematopoiesis. Significantly,gata2b, which normally acts upstream ofrunx1for the generation of HSCs, was increased in thecd41-GFP+cells inrunx1- /-embryos. Interestingly, genetic inactivation of bothgata2band its paralog,gata2a, did not affect hematopoiesis. However, knocking outrunx1and any three of the four alleles ofgata2aandgata2babolished definitive hematopoiesis.Gata2expression was also upregulated in hematopoietic cells inRunx1-/-mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other’s safeguard.Key pointsExistence of RUNX1-independent mechanisms for the generation of HSCs and the development of functional definitive hematopoietic cellsGATA2 and RUNX1 functionally complement each other for their respective roles during hematopoiesis

Summary Innate immune responses rely on rapid and precise gene regulation mediated by accessibility of regulatory regions to transcription factors (TFs). In natural killer (NK) cells and other innate lymphoid cells, competent enhancers are primed during lineage acquisition, and formation of de novo enhancers characterizes the acquisition of innate memory in activated NK cells and macrophages. Here, we investigated how primed and de novo enhancers coordinate to facilitate high-magnitude gene induction during acute activation. Epigenomic and transcriptomic analyses of regions near highly induced genes (HIGs) in NK cells both in vitro and in a model of Toxoplasma gondii infection revealed de novo chromatin accessibility and enhancer remodeling controlled by signal-regulated TFs STATs. Acute NK cell activation redeployed the lineage-determining TF T-bet to de novo enhancers, independent of DNA-sequence-specific motif recognition. Thus, acute stimulation reshapes enhancer function through the combinatorial usage and repurposing of both lineage-determining and signal-regulated TFs to ensure an effective response.

Given their copy number differences and unique modes of inheritance, the evolved gene content and expression of sex chromosomes is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4th) that re-acquired autosomal status. Here we use single cell RNA-Seq on fly larvae to demonstrate that the single X and pair of 4th chromosomes are specifically inactivated in primary spermatocytes, based on measuring all genes or a set of broadly expressed genes in testis we identified. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5., Sex chromosome gene content and expression is unusual. Here the authors use single cell RNA-Seq on Drosophila larvae to demonstrate that the single X and pair of 4th chromosomes are specifically inactivated in primary spermatocytes, while genes on the single Y chromosome become maximally active in primary spermatocytes.

Dedicated stem cells ensure postnatal growth, repair and homeostasis of skeletal muscle. Following injury, muscle stem cells (MuSCs) exit from quiescence and divide to reconstitute the stem cell pool and give rise to muscle progenitors. The transcriptomes of pooled MuSCs have provided a rich source of information for describing the genetic programs of distinct static cell states; however, bulk microarray and RNA sequencing provide only averaged gene expression profiles, blurring the heterogeneity and developmental dynamics of asynchronous MuSC populations. Instead, the granularity required to identify distinct cell types, states, and their dynamics can be afforded by single cell analysis. We were able to compare the transcriptomes of thousands of MuSCs and primary myoblasts isolated from homeostatic or regenerating muscles by single cell RNA sequencing. Using computational approaches, we could reconstruct dynamic trajectories and place, in a pseudotemporal manner, the transcriptomes of individual MuSC within these trajectories. This approach allowed for the identification of distinct clusters of MuSCs and primary myoblasts with partially overlapping but distinct transcriptional signatures, as well as the description of metabolic pathways associated with defined MuSC states.

SummaryMyoD is a key regulator of skeletal myogenesis that directs contractile protein synthesis, but whether this transcription factor also regulates skeletal muscle metabolism has not been explored. In a genome-wide ChIP-seq analysis of skeletal muscle cells, we unexpectedly observed that MyoD directly binds to numerous metabolic genes, including those associated with mitochondrial biogenesis, fatty acid oxidation, and the electron transport chain. Results in cultured cells and adult skeletal muscle confirmed that MyoD regulates oxidative metabolism through multiple transcriptional targets, including PGC-1β, a master regulator of mitochondrial biogenesis. We find that PGC-1β expression is cooperatively regulated by MyoD and the alternative NF-κB signaling pathway. Bioinformatics evidence suggests that this cooperativity between MyoD and NF-κB extends to other metabolic genes as well. Together, these data identify MyoD as a regulator of the metabolic capacity of mature skeletal muscle to ensure that sufficient energy is available to support muscle contraction.

Dedicated stem cells ensure post-natal growth, repair, and homeostasis of skeletal muscle. Following injury, muscle stem cells (MuSCs) exit from quiescence and divide to reconstitute the stem cell pool and give rise to muscle progenitors. The transcriptomes of pooled MuSCs have provided a rich source of information for describing the genetic programs of distinct static cell states; however, bulk microarray and RNA-seq provide only averaged gene expression profiles, blurring the heterogeneity and developmental dynamics of asynchronous MuSC populations. Instead, the granularity required to identify distinct cell types, states, and their dynamics can be afforded by single-cell analysis. We were able to compare the transcriptomes of thousands of MuSCs and primary myoblasts isolated from homeostatic or regenerating muscles by single-cell RNA- sequencing. Using computational approaches, we could reconstruct dynamic trajectories and place, in a pseudotemporal manner, the transcriptomes of individual MuSC within these trajectories. This approach allowed for the identification of distinct clusters of MuSCs and primary myoblasts with partially overlapping but distinct transcriptional signatures, as well as the description of metabolic pathways associated with defined MuSC states.

In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.

Since its discovery as a skeletal muscle-specific transcription factor able to reprogram somatic cells into differentiated myofibers, MyoD has provided an instructive model to understand how transcription factors regulate gene expression. Reciprocally, studies of other transcriptional regulators have provided testable hypotheses to further understand how MyoD activates transcription. Using MyoD as a reference, in this review, we discuss the similarities and differences in the regulatory mechanisms employed by tissue-specific transcription factors to access DNA and regulate gene expression by cooperatively shaping the chromatin landscape within the context of cellular differentiation.

For many years, stem cell metabolism was viewed as a byproduct of cell fate status rather than an active regulatory mechanism; however, there is now a growing appreciation that metabolic pathways influence epigenetic changes associated with lineage commitment, specification, and self-renewal. Here we review how metabolites generated during glycolytic and oxidative processes are utilized in enzymatic reactions leading to epigenetic modifications and transcriptional regulation. We discuss how "metabolic reprogramming" contributes to global epigenetic changes in the context of naive and primed pluripotent states, somatic reprogramming, and hematopoietic and skeletal muscle tissue stem cells, and we discuss the implications for regenerative medicine.

Intermediates generated in several metabolic processes are used to regulate transcription through covalent histone and DNA modifications. In Cell Reports, Das et al. show that acetyl-coenzyme A (acetyl-CoA) generated by ATP citrate lyase (ACL) is utilized to acetylate histone H3 at MyoD regulatory regions, resulting in increased MyoD expression and improved muscle regeneration after injury.

Enhancers regulate spatiotemporal gene expression and impart cell-specific transcriptional outputs that drive cell identity. Super-enhancers (SEs), also known as stretch-enhancers, are a subset of enhancers especially important for genes associated with cell identity and genetic risk of disease. CD4(+) T cells are critical for host defence and autoimmunity. Here we analysed maps of mouse T-cell SEs as a non-biased means of identifying key regulatory nodes involved in cell specification. We found that cytokines and cytokine receptors were the dominant class of genes exhibiting SE architecture in T cells. Nonetheless, the locus encoding Bach2, a key negative regulator of effector differentiation, emerged as the most prominent T-cell SE, revealing a network in which SE-associated genes critical for T-cell biology are repressed by BACH2. Disease-associated single-nucleotide polymorphisms for immune-mediated disorders, including rheumatoid arthritis, were highly enriched for T-cell SEs versus typical enhancers or SEs in other cell lineages. Intriguingly, treatment of T cells with the Janus kinase (JAK) inhibitor tofacitinib disproportionately altered the expression of rheumatoid arthritis risk genes with SE structures. Together, these results indicate that genes with SE architecture in T cells encompass a variety of cytokines and cytokine receptors but are controlled by a 'guardian' transcription factor, itself endowed with an SE. Thus, enumeration of SEs allows the unbiased determination of key regulatory nodes in T cells, which are preferentially modulated by pharmacological intervention.

SummaryStem cells undergo a shift in metabolic substrate utilization during specification and/or differentiation, a process that has been termed metabolic reprogramming. Here, we report that during the transition from quiescence to proliferation, skeletal muscle stem cells experience a metabolic switch from fatty acid oxidation to glycolysis. This reprogramming of cellular metabolism decreases intracellular NAD+ levels and the activity of the histone deacetylase SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in SCs. Moreover, mice with muscle-specific inactivation of the SIRT1 deacetylase domain display reduced myofiber size, impaired muscle regeneration, and derepression of muscle developmental genes. Overall, these findings reveal how metabolic cues can be mechanistically translated into epigenetic modifications that regulate skeletal muscle stem cell biology.

Cancer cachexia is a devastating metabolic syndrome characterized by systemic inflammation and massive muscle and adipose tissue wasting. Although it is responsible for approximately one-third of cancer deaths, no effective therapies are available and the underlying mechanisms have not been fully elucidated. We previously identified the bromodomain and extra-terminal domain (BET) protein BRD4 as an epigenetic regulator of muscle mass. Here we show that the pan-BET inhibitor (+)-JQ1 protects tumor-bearing mice from body weight loss and muscle and adipose tissue wasting. Remarkably, in C26-tumor-bearing mice (+)-JQ1 administration dramatically prolongs survival, without directly affecting tumor growth. By ChIP-seq and ChIP analyses, we unveil that BET proteins directly promote the muscle atrophy program during cachexia. In addition, BET proteins are required to coordinate an IL6-dependent AMPK nuclear signaling pathway converging on FoxO3 transcription factor. Overall, these findings indicate that BET proteins may represent a promising therapeutic target in the management of cancer cachexia., Cachexia is a metabolic syndrome leading to muscle and adipose tissue loss in majority of cancer patients. Here the authors show that, in a mouse model, BET inhibitor JQ1 counteracts muscle and adipose tissue wasting tempering cachexia and prolonging survival through a mechanism unrelated to tumour growth.

Summary Spt6 coordinates nucleosome dis- and re-assembly, transcriptional elongation, and mRNA processing. Here, we report that depleting Spt6 in embryonic stem cells (ESCs) reduced expression of pluripotency factors, increased expression of cell-lineage-affiliated developmental regulators, and induced cell morphological and biochemical changes indicative of ESC differentiation. Selective downregulation of pluripotency factors upon Spt6 depletion may be mechanistically explained by its enrichment at ESC super-enhancers, where Spt6 controls histone H3K27 acetylation and methylation and super-enhancer RNA transcription. In ESCs, Spt6 interacted with the PRC2 core subunit Suz12 and prevented H3K27me3 accumulation at ESC super-enhancers and associated promoters. Biochemical as well as functional experiments revealed that Spt6 could compete for binding of the PRC2 methyltransferase Ezh2 to Suz12 and reduce PRC2 chromatin engagement. Thus, in addition to serving as a histone chaperone and transcription elongation factor, Spt6 counteracts repression by opposing H3K27me3 deposition at critical genomic regulatory regions.

The current notion about how hematopoietic stem cells (HSCs) are generated identifies the transcription factor RUNX1 as an essential factor for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium. Consequently, Runx1knockout mice fail to develop definitive hematopoiesis and lack all definitive blood lineages and cannot survive past embryonic day 12. However, even though zebrafish with arunx1stop codon mutation (runx1W84X/W84X) presented defects in definitive hematopoiesis during embryogenesis, runx1W84X/W84Xembryos could develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. In order to determine if a RUNX1-independent mechanism can support the generation of HSCs we have generated three new zebrafish runx1-/- with engineered deletions of the runx1gene using TALEN and CRISPR-Cas9. Our analysis shows that all three mutants have identical phenotypei.e., failure to develop definitive hematopoiesis during early embryogenesis, with later reemergence of hematopoietic cells and survivalof therunx1 mutants to adulthood, further confirming the existence of a RUNX1-independent mechanism for the emergence of HSCs. In the absence of a functional runx1, a cd41-GFP+population of hematopoietic precursors can still be detected in the aorta-gonad-mesonephros (AGM) region and in the hematopoietic tissues of the mutant embryos. Single cell RNA sequencing of the wild type and mutant HSC/HSPC at embryonic and larval stages confirmed the presence of a population of runx1- /-cd41:GFPlow cells expressing HSC signature genes at 2.5 days post fertilization. At larval stages the runx1-/-HSCs maintain their ability to generate erythroid and myeloid lineage progenitors but they present a different expression profile compared to the wild type. In order to uncover the compensatory mechanism that drives the repopulation of the hematopoietic compartment in the absence of runx1we identified the molecular signatures that separate the runx1-/-HSC/HSPCs from the wild type and subsequently focused our attention on the transcription factors differentially expressed in the runx1-/-HSC/HSPCs. Our analysis shows that the master transcription factor gata2b is strongly upregulated in the runx1- /-HSCs during the recovery of hematopoiesis and it is also upregulated in the kidney marrow of the surviving runx1-/-adults. Given the key role of GATA2 in the HSC development and maintenance in both mouse and zebrafish, gata2b represented a strong candidate gene with the potential ability to drive the rescue of the runx1-/-phenotype. Indeed, a loss of function mutation or knock-down of gata2b can significantly reduce or abolish the survivability of the runx1-/-fish, indicating that gata2bis responsible for rescuing hematopoiesis in the runx1 mutant fish. Overall our results show that even though runx1 is necessary for the normal emergence of definitive HSCs in the embryos, in the absence of runx1the transcription factor gata2 is able to support definitive hematopoiesis that is sufficient for the embryos to develop to functional adults in the zebrafish. The current notion about how hematopoietic stem cells (HSCs) are generated identifies the transcription factor RUNX1 as an essential factor for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium. Consequently, Runx1knockout mice fail to develop definitive hematopoiesis and lack all definitive blood lineages and cannot survive past embryonic day 12. However, even though zebrafish with arunx1stop codon mutation (runx1W84X/W84X) presented defects in definitive hematopoiesis during embryogenesis, runx1W84X/W84Xembryos could develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. In order to determine if a RUNX1-independent mechanism can support the generation of HSCs we have generated three new zebrafish runx1-/- with engineered deletions of the runx1gene using TALEN and CRISPR-Cas9. Our analysis shows that all three mutants have identical phenotypei.e., failure to develop definitive hematopoiesis during early embryogenesis, with later reemergence of hematopoietic cells and survivalof therunx1 mutants to adulthood, further confirming the existence of a RUNX1-independent mechanism for the emergence of HSCs. In the absence of a functional runx1, a cd41-GFP+population of hematopoietic precursors can still be detected in the aorta-gonad-mesonephros (AGM) region and in the hematopoietic tissues of the mutant embryos. Single cell RNA sequencing of the wild type and mutant HSC/HSPC at embryonic and larval stages confirmed the presence of a population of runx1- /-cd41:GFPlow cells expressing HSC signature genes at 2.5 days post fertilization. At larval stages the runx1-/-HSCs maintain their ability to generate erythroid and myeloid lineage progenitors but they present a different expression profile compared to the wild type. In order to uncover the compensatory mechanism that drives the repopulation of the hematopoietic compartment in the absence of runx1we identified the molecular signatures that separate the runx1-/-HSC/HSPCs from the wild type and subsequently focused our attention on the transcription factors differentially expressed in the runx1-/-HSC/HSPCs. Our analysis shows that the master transcription factor gata2b is strongly upregulated in the runx1- /-HSCs during the recovery of hematopoiesis and it is also upregulated in the kidney marrow of the surviving runx1-/-adults. Given the key role of GATA2 in the HSC development and maintenance in both mouse and zebrafish, gata2b represented a strong candidate gene with the potential ability to drive the rescue of the runx1-/-phenotype. Indeed, a loss of function mutation or knock-down of gata2b can significantly reduce or abolish the survivability of the runx1-/-fish, indicating that gata2bis responsible for rescuing hematopoiesis in the runx1 mutant fish. Overall our results show that even though runx1 is necessary for the normal emergence of definitive HSCs in the embryos, in the absence of runx1the transcription factor gata2 is able to support definitive hematopoiesis that is sufficient for the embryos to develop to functional adults in the zebrafish. Disclosures No relevant conflicts of interest to declare.

Sirtuins (SIRTs), class III histone deacetylases, are well characterized for their control of cellular physiology in peripheral tissues, but their influence in brain under normal and pathological conditions remains poorly understood. Here, we establish an essential role for SIRT1 and SIRT2 in regulating behavioral responses to cocaine and morphine through actions in the nucleus accumbens (NAc), a key brain reward region. We show that chronic cocaine administration increases SIRT1 and SIRT2 expression in the mouse NAc, while chronic morphine administration induces SIRT1 expression alone, with no regulation of all other sirtuin family members observed. Drug induction of SIRT1 and SIRT2 is mediated in part at the transcriptional level via the drug-induced transcription factor ΔFosB and is associated with robust histone modifications at theSirt1andSirt2genes. Viral-mediated overexpression of SIRT1 or SIRT2 in the NAc enhances the rewarding effects of both cocaine and morphine. In contrast, the local knockdown of SIRT1 from the NAc of floxedSirt1mice decreases drug reward. Such behavioral effects of SIRT1 occur in concert with its regulation of numerous synaptic proteins in NAc as well as with SIRT1-mediated induction of dendritic spines on NAc medium spiny neurons. These studies establish sirtuins as key mediators of the molecular and cellular plasticity induced by drugs of abuse in NAc, and of the associated behavioral adaptations, and point toward novel signaling pathways involved in drug action.

Elucidating the epigenetic mechanisms underlying muscle mass determination and skeletal muscle wasting holds the potential of identifying molecular pathways that constitute possible drug targets. Here, we report that the methyltransferase SMYD3 modulates myostatin and c-Met transcription in primary skeletal muscle cells and C2C12 myogenic cells. SMYD3 targets the myostatin and c-Met genes and participates in the recruitment of the bromodomain protein BRD4 to their regulatory regions through protein–protein interaction. By recruiting BRD4, SMYD3 favors chromatin engagement of the pause–release factor p-TEFb (positive transcription elongation factor) and elongation of Ser2-phosphorylated RNA polymerase II (PolIISer2P). Reducing SMYD3 decreases myostatin and c-Met transcription, thus protecting from glucocorticoid-induced myotube atrophy. Supporting functional relevance of the SMYD3/BRD4 interaction, BRD4 pharmacological blockade by the small molecule JQ1 prevents dexamethasone-induced myostatin and atrogene up-regulation and spares myotube atrophy. Importantly, in a mouse model of dexamethasone-induced skeletal muscle atrophy, SMYD3 depletion prevents muscle loss and fiber size decrease. These findings reveal a mechanistic link between SMYD3/BRD4-dependent transcriptional regulation, muscle mass determination, and skeletal muscle atrophy and further encourage testing of small molecules targeting specific epigenetic regulators in animal models of muscle wasting.

Diverse autoimmune and allergic diseases are associated with polymorphisms in a locus encoding the transcription factor BACH2; here, BACH2 is shown to be a broad regulator of immune activation that stabilizes the differentiation of Treg cells by repressing commitment of CD4+ T cells to alternate cell fates. Polymorphisms within a locus encoding the transcription factor BACH2 are associated with a number of allergic and autoimmune diseases including asthma, multiple sclerosis, Crohn's disease, coeliac disease and type 1 diabetes. This paper identifies a mechanism by which BACH2 might contribute to autoimmunity. Roychoudhuri et al. show how BACH2 limits autoimmunity by repressing alternative cell fates through the stabilization of the differentiation of regulatory T cells. These findings suggest a role for BACH2 as a regulator of CD4+ T-cell differentiation, preventing inflammatory disease by controlling the balance between tolerance and immunity. Through their functional diversification, distinct lineages of CD4+ T cells can act to either drive or constrain immune-mediated pathology. Transcription factors are critical in the generation of cellular diversity, and negative regulators antagonistic to alternate fates often act in conjunction with positive regulators to stabilize lineage commitment1. Genetic polymorphisms within a single locus encoding the transcription factor BACH2 are associated with numerous autoimmune and allergic diseases including asthma2, Crohn’s disease3,4, coeliac disease5, vitiligo6, multiple sclerosis7 and type 1 diabetes8. Although these associations point to a shared mechanism underlying susceptibility to diverse immune-mediated diseases, a function for BACH2 in the maintenance of immune homeostasis has not been established. Here, by studying mice in which the Bach2 gene is disrupted, we define BACH2 as a broad regulator of immune activation that stabilizes immunoregulatory capacity while repressing the differentiation programs of multiple effector lineages in CD4+ T cells. BACH2 was required for efficient formation of regulatory (Treg) cells and consequently for suppression of lethal inflammation in a manner that was Treg-cell-dependent. Assessment of the genome-wide function of BACH2, however, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, BACH2 constrained full effector differentiation within TH1, TH2 and TH17 cell lineages. These findings identify BACH2 as a key regulator of CD4+ T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity.

Histone chaperones affect chromatin structure and gene expression through interaction with histones and RNA polymerase II (PolII). Here, we report that the histone chaperone Spt6 counteracts H3K27me3, an epigenetic mark deposited by the Polycomb Repressive Complex 2 (PRC2) and associated with transcriptional repression. By regulating proper engagement and function of the H3K27 demethylase KDM6A (UTX), Spt6 effectively promotes H3K27 demethylation, muscle gene expression, and cell differentiation. ChIP-Seq experiments reveal an extensive genome-wide overlap of Spt6, PolII, and KDM6A at transcribed regions that are devoid of H3K27me3. Mammalian cells and zebrafish embryos with reduced Spt6 display increased H3K27me3 and diminished expression of the master regulator MyoD, resulting in myogenic differentiation defects. As a confirmation for an antagonistic relationship between Spt6 and H3K27me3, inhibition of PRC2 permits MyoD re-expression in myogenic cells with reduced Spt6. Our data indicate that, through cooperation with PolII and KDM6A, Spt6 orchestrates removal of H3K27me3, thus controlling developmental gene expression and cell differentiation.

The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK), an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE) in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.

The transforming growth factor-β (TGF-β) signaling network is a critical regulator of skeletal muscle mass and function and, thus, is an attractive therapeutic target for combating muscle disease, but the underlying mechanisms of action remain undetermined. We report that follistatin-based interventions (which modulate TGF-β network activity) can promote muscle hypertrophy that ameliorates aging-associated muscle wasting. However, the muscles of old sarcopenic mice demonstrate reduced response to follistatin compared with healthy young-adult musculature. Quantitative proteomic and transcriptomic analyses of young-adult muscles identified a transcription/translation signature elicited by follistatin exposure, which included repression of ankyrin repeat and SOCS box protein 2 (Asb2). Increasing expression of ASB2 reduced muscle mass, thereby demonstrating that Asb2 is a TGF-β network-responsive negative regulator of muscle mass. In contrast to young-adult muscles, sarcopenic muscles do not exhibit reduced ASB2 abundance with follistatin exposure. Moreover, preventing repression of ASB2 in young-adult muscles diminished follistatin-induced muscle hypertrophy. These findings provide insight into the program of transcription and translation events governing follistatin-mediated adaptation of skeletal muscle attributes and identify Asb2 as a regulator of muscle mass implicated in the potential mechanistic dysfunction between follistatin-mediated muscle growth in young and old muscles.

The nuclear envelope protein lamin A is encoded by the lamin A/C (LMNA) gene, which can contain missense mutations that cause Emery-Dreifuss muscular dystrophy (EDMD) (p.R453W). We fused mutated forms of the lamin A protein to bacterial DNA adenine methyltransferase (Dam) to define euchromatic-heterochromatin (epigenomic) transitions at the nuclear envelope during myogenesis (using DamID-seq). Lamin A missense mutations disrupted appropriate formation of lamin A–associated heterochromatin domains in an allele-specific manner—findings that were confirmed by chromatin immunoprecipitation–DNA sequencing (ChIP-seq) in murine H2K cells and DNA methylation studies in fibroblasts from muscular dystrophy patient who carried a distinct LMNA mutation (p.H222P). Observed perturbations of the epigenomic transitions included exit from pluripotency and cell cycle programs [euchromatin (open, transcribed) to heterochromatin (closed, silent)], as well as induction of myogenic loci (heterochromatin to euchromatin). In muscle biopsies from patients with either a gain- or change-of-function LMNA gene mutation or a loss-of-function mutation in the emerin gene, both of which cause EDMD, we observed inappropriate loss of heterochromatin formation at the Sox2 pluripotency locus, which was associated with persistent mRNA expression of Sox2. Overexpression of Sox2 inhibited myogenic differentiation in human immortalized myoblasts. Our findings suggest that nuclear envelopathies are disorders of developmental epigenetic programming that result from altered formation of lamina-associated domains.