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Social interaction is a fundamental behavior in all animal species, but the developmental timing of the social neural circuit formation and the cellular and molecular mechanisms governing its formation are poorly understood. We generated a mouse model with mutations in two Disheveled genes, Dvl1 and Dvl3, that displays adult social and repetitive behavioral abnormalities associated with transient embryonic brain enlargement during deep layer cortical neuron formation. These phenotypes were mediated by the embryonic expansion of basal neural progenitor cells (NPCs) via deregulation of a β-catenin/Brn2/Tbr2 transcriptional cascade. Transient pharmacological activation of the canonical Wnt pathway during this period of early corticogenesis rescued the β-catenin/Brn2/Tbr2 transcriptional cascade and the embryonic brain phenotypes. Remarkably, this embryonic treatment prevented adult behavioral deficits and partially rescued abnormal brain structure in Dvl mutant mice. Our findings define a mechanism that links fetal brain development and adult behavior, demonstrating a fetal origin for social and repetitive behavior deficits seen in disorders such as autism.

Glaucoma, a blinding neurodegenerative disease, whose risk factors include elevated intraocular pressure (IOP), age, and genetics, is characterized by accelerated and progressive retinal ganglion cell (RGC) death. Despite decades of research, the mechanism of RGC death in glaucoma is still unknown. Here, we demonstrate that the genetic effect of the SIX6 risk variant (rs33912345, His141Asn) is enhanced by another major POAG risk gene, p16INK4a (cyclin-dependent kinase inhibitor 2A, isoform INK4a). We further show that the upregulation of homozygous SIX6 risk alleles (CC) leads to an increase in p16INK4a expression, with subsequent cellular senescence, as evidenced in a mouse model of elevated IOP and in human POAG eyes. Our data indicate that SIX6 and/or IOP promotes POAG by directly increasing p16INK4a expression, leading to RGC senescence in adult human retinas. Our study provides important insights linking genetic susceptibility to the underlying mechanism of RGC death and provides a unified theory of glaucoma pathogenesis. Zhang et al. report p16INK4a as a downstream integrator of diverse signals, such as inherited genetic risk, age, and intraocular pressure, in the pathogenesis of glaucoma. They demonstrate that upregulation of SIX6 upon stress directly increases p16INK4a, leading to retinal ganglion cell senescence and death.

Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA-binding protein KSRP fa

MEF2C is a critical transcription factor in neurodevelopment, whose loss-of-function mutation in humans results in MEF2C haploinsufficiency syndrome (MHS), a severe form of autism spectrum disorder (ASD)/intellectual disability (ID). Despite prior animal studies of MEF2C heterozygosity to mimic MHS, MHS-specific mutations have not been investigated previously, particularly in a human context as hiPSCs afford. Here, for the first time, we use patient hiPSC-derived cerebrocortical neurons and cerebral organoids to characterize MHS deficits. Unexpectedly, we found that decreased neurogenesis was accompanied by activation of a micro-(mi)RNA-mediated gliogenesis pathway. We also demonstrate network-level hyperexcitability in MHS neurons, as evidenced by excessive synaptic and extrasynaptic activity contributing to excitatory/inhibitory (E/I) imbalance. Notably, the predominantly extrasynaptic (e)NMDA receptor antagonist, NitroSynapsin, corrects this aberrant electrical activity associated with abnormal phenotypes. During neurodevelopment, MEF2C regulates many ASD-associated gene networks, suggesting that treatment of MHS deficits may possibly help other forms of ASD as well., (© 2024. The Author(s).)

Genome-wide association studies (GWASs) have uncovered over 75 genomic loci associated with risk for late-onset Alzheimer's disease (LOAD), but identification of the underlying causal genes remains challenging. Studies of induced pluripotent stem cell (iPSC)-derived neurons from LOAD patients have demonstrated the existence of neuronal cell-intrinsic functional defects. Here, we searched for genetic contributions to neuronal dysfunction in LOAD using an integrative systems approach that incorporated multi-evidence-based gene mapping and network-analysis-based prioritization. A systematic perturbation screening of candidate risk genes in Caenorhabditis elegans (C. elegans) revealed that neuronal knockdown of the LOAD risk gene orthologs vha-10 (ATP6V1G2), cmd-1 (CALM3), amph-1 (BIN1), ephx-1 (NGEF), and pho-5 (ACP2) alters short-/intermediate-term memory function, the cognitive domain affected earliest during LOAD progression. These results highlight the impact of LOAD risk genes on evolutionarily conserved memory function, as mediated through neuronal endosomal dysfunction, and identify new targets for further mechanistic interrogation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Although often located at a distance from their target gene promoters, enhancers are the primary genomic determinants of temporal and spatial transcriptional specificity in metazoans. Since the discovery of the first enhancer element in simian virus 40, there has been substantial interest in unraveling the mechanism(s) by which enhancers communicate with their partner promoters to ensure proper gene expression. These research efforts have benefited considerably from the application of increasingly sophisticated sequencing- and imaging-based approaches in conjunction with innovative (epi)genome-editing technologies; however, despite various proposed models, the principles of enhancer-promoter interaction have still not been fully elucidated. In this review, we provide an overview of recent progress in the eukaryotic gene transcription field pertaining to enhancer-promoter specificity. A better understanding of the mechanistic basis of lineage- and context-dependent enhancer-promoter engagement, along with the continued identification of functional enhancers, will provide key insights into the spatiotemporal control of gene expression that can reveal therapeutic opportunities for a range of enhancer-related diseases., (© 2024. The Author(s).)

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1β complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1β coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1β complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes., Competing Interests: Competing interests statement:C.K.G. is a cofounder, equity holder and member of the Scientific Advisory Board of Asteroid Therapeutics.

Growth factor receptors rank among the most important oncogenic pathways, but pharmacologic inhibitors often demonstrate limited benefit as monotherapy. Here, we show that epidermal growth factor receptor (EGFR) signaling repressed N 6 -methyladenosine (m 6 A) levels in glioblastoma stem cells (GSCs), whereas genetic or pharmacologic EGFR targeting elevated m 6 A levels. Activated EGFR induced non-receptor tyrosine kinase SRC to phosphorylate the m 6 A demethylase, AlkB homolog 5 (ALKBH5), thereby inhibiting chromosomal maintenance 1 (CRM1)-mediated nuclear export of ALKBH5 to permit sustained mRNA m 6 A demethylation in the nucleus. ALKBH5 critically regulated ferroptosis through m 6 A modulation and YTH N6-methyladenosine RNA binding protein (YTHDF2)-mediated decay of the glutamate-cysteine ligase modifier subunit (GCLM). Pharmacologic targeting of ALKBH5 augmented the anti-tumor efficacy of EGFR and GCLM inhibitors, supporting an EGFR-ALKBH5-GCLM oncogenic axis. Collectively, EGFR reprograms the epitranscriptomic landscape through nuclear retention of the ALKBH5 demethylase to protect against ferroptosis, offering therapeutic paradigms for the treatment of lethal cancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The resolution limit of chromatin conformation capture methodologies (3Cs) has restrained their application in detection of fine-level chromatin structure mediated by cis-regulatory elements (CREs). Here, we report two 3C-derived methods, Tri-4C and Tri-HiC, which utilize multirestriction enzyme digestions for ultrafine mapping of targeted and genome-wide chromatin interaction, respectively, at up to one hundred basepair resolution. Tri-4C identified CRE loop interaction networks and quantitatively revealed their alterations underlying dynamic gene control. Tri-HiC uncovered global fine-gauge regulatory interaction networks, identifying >20-fold more enhancer:promoter (E:P) loops than in situ Hi-C. In addition to vastly improved identification of subkilobase-sized E:P loops, Tri-HiC also uncovered interaction stripes and contact domain insulation from promoters and enhancers, revealing their loop extrusion behaviors resembling the topologically associating domain boundaries. Tri-4C and Tri-HiC provide robust approaches to achieve the high-resolution interactome maps required for characterizing fine-gauge regulatory chromatin interactions in analysis of development, homeostasis, and disease.

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1β with the NCoR/HDAC3 complex, resulting in the activation of PGC1β and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts., Competing Interests: Declaration of interests C.K.G. is a co-founder, equity holder, and member of the Scientific Advisory Board of Asteroid Therapeutics. J.T.K. is on the Molecular Cell advisory board., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Glaucoma is a leading cause of irreversible blindness, with advanced age being the single most significant risk factor. However, the mechanisms underlying the relationship between aging and glaucoma remain unclear. Genome-wide association studies (GWAS) have successfully identified genetic variants strongly associated with increased glaucoma risk. Understanding how these variants function in pathogenesis is crucial for translating genetic associations into molecular mechanisms and, ultimately, clinical applications. The chromosome 9p21.3 locus is among the most replicated glaucoma risk loci discovered by GWAS. Nonetheless, the absence of protein-coding genes in the locus makes interpreting the disease association challenging, leaving the causal variant and molecular mechanism elusive. In this study, we report the identification of a functional glaucoma risk variant, rs6475604. By employing computational and experimental methods, we demonstrated that rs6475604 resides in a repressive regulatory element. Risk allele of rs6475604 disrupts the binding of YY1, a transcription factor known to repress the expression of a neighboring gene in 9p21.3, p16INK4A, which plays a crucial role in cellular senescence and aging. These findings suggest that the glaucoma disease variant contributes to accelerated senescence, providing a molecular link between glaucoma risk and an essential cellular mechanism for human aging., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Enhancer activation serves as the main mechanism regulating signal-dependent transcriptional programs, ensuring cellular plasticity, yet central questions persist regarding their mechanism of activation. Here, by successfully mapping topoisomerase I-DNA covalent complexes genome-wide, we find that most, if not all, acutely activated enhancers, including those induced by 17β-estradiol, dihydrotestosterone, tumor necrosis factor alpha and neuronal depolarization, are hotspots for topoisomerase I-DNA covalent complexes, functioning as epigenomic signatures read by the classic DNA damage sensor protein, Ku70. Ku70 in turn nucleates a heterochromatin protein 1 gamma (HP1γ)-mediator subunit Med26 complex to facilitate acute, but not chronic, transcriptional activation programs. Together, our data uncover a broad, unappreciated transcriptional code, required for most, if not all, acute signal-dependent enhancer activation events in both mitotic and postmitotic cells., (© 2023. The Author(s).)

Gene regulation by transcriptional enhancers is the dominant mechanism driving cell type- and signal-specific transcriptional diversity in metazoans. However, over four decades since the original discovery, how enhancers operate in the nuclear space remains largely enigmatic. Recent multidisciplinary efforts combining real-time imaging, genome sequencing, and biophysical strategies provide insightful but conflicting models of enhancer-mediated gene control. Here, we review the discovery and progress in enhancer biology, emphasizing the recent findings that acutely activated enhancers assemble regulatory machinery as mesoscale architectural structures with distinct physical properties. These findings help formulate novel models that explain several mysterious features of the assembly of transcriptional enhancers and the mechanisms of spatial control of gene expression., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of β-globinopathies (sickle cell disease and β-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor-like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation., (© 2021 by The American Society of Hematology.)

Type 1 diabetes (T1D) is an organ-specific autoimmune disease, whereby immune cell-mediated killing leads to loss of the insulin-producing β cells in the pancreas. Genome-wide association studies (GWAS) have identified over 200 genetic variants associated with risk for T1D. The majority of the GWAS risk variants reside in the non-coding regions of the genome, suggesting that gene regulatory changes substantially contribute to T1D. However, identification of causal regulatory variants associated with T1D risk and their affected genes is challenging due to incomplete knowledge of non-coding regulatory elements and the cellular states and processes in which they function. Here, we performed a comprehensive integrated post-GWAS analysis of T1D to identify functional regulatory variants in enhancers and their cognate target genes. Starting with 1,817 candidate T1D SNPs defined from the GWAS catalog and LDlink databases, we conducted functional annotation analysis using genomic data from various public databases. These include 1) Roadmap Epigenomics, ENCODE, and RegulomeDB for epigenome data; 2) GTEx for tissue-specific gene expression and expression quantitative trait loci data; and 3) lncRNASNP2 for long non-coding RNA data. Our results indicated a prevalent enhancer-based immune dysregulation in T1D pathogenesis. We identified 26 high-probability causal enhancer SNPs associated with T1D, and 64 predicted target genes. The majority of the target genes play major roles in antigen presentation and immune response and are regulated through complex transcriptional regulatory circuits, including those in HLA (6p21) and non-HLA (16p11.2) loci. These candidate causal enhancer SNPs are supported by strong evidence and warrant functional follow-up studies., Competing Interests: The authors have declared that no competing interests exist.

The functional engagement between an enhancer and its target promoter ensures precise gene transcription 1 . Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC)., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles 1 , particularly the promoter antisense (PAS) transcripts 2,3 . Here we report the development of an assay-referred to as chromatin isolation by RNA-Cas13a complex-to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17β-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

In diseased organs, stress-activated signalling cascades alter chromatin, thereby triggering maladaptive cell state transitions. Fibroblast activation is a common stress response in tissues that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains unclear 1,2 . Pharmacological inhibition of bromodomain and extra-terminal domain (BET) proteins alleviates cardiac dysfunction 3-7 , providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here we use single-cell epigenomic analyses of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch that underlies the activation of fibroblasts. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed previously undescribed DNA elements, the accessibility of which dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer that regulated the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program and was required for TGFβ-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFβ-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction and demonstrate its upregulation after activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide previously unknown trans- and cis-targets for treating fibrotic disease., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions. Abbreviations: ACTB: actin beta; ATG: autophagy-related; 5-Aza: 5-aza-2'-deoxycytidine; BafA1: bafilomycin A 1 ; CBZ: carbamazepine; CDKN2A: cyclin dependent kinase inhibitor 2A; ChIP: chromatin immunoprecipitation; Clon.: clonidine; CpG: cytosine-guanine dinucleotide: DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DNMT: DNA methyltransferase; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase alpha; DNMT3B: DNA methyltransferase beta; dpf: days post-fertilization; EBSS: Earle's balanced salt solution; EM: Zebrafish embryo medium; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GRO-Seq: Global Run-On sequencing; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP1LC3B2: microtubule-associated protein 1 light chain 3 beta 2; MEM: minimum essential medium; MEF: mouse embryonic fibroblasts; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction; SQSTM1/p62: sequestosome 1; Starv.: starvation; Treh.: trehalose; ULK1: unc-51 like autophagy activating kinase 1.

Emerging evidence suggested that epigenetic regulators can exhibit both activator and repressor activities in gene transcriptional regulation and disease development, such as cancer. However, how these dual activities are regulated and coordinated in specific cellular contexts remains elusive. Here, it is reported that KDM5C, a repressive histone demethylase, unexpectedly activates estrogen receptor alpha (ER α )-target genes, and meanwhile suppresses type I interferons (IFNs) and IFN-stimulated genes (ISGs) to promote ER α -positive breast cancer cell growth and tumorigenesis. KDM5C-interacting protein, ZMYND8, is found to be involved in both processes. Mechanistically, KDM5C binds to active enhancers and recruits the P-TEFb complex to activate ER α -target genes, while inhibits TBK1 phosphorylation in the cytosol to repress type I IFNs and ISGs. Pharmacological inhibition of both ER α and KDM5C is effective in inhibiting cell growth and tumorigenesis. Taken together, it is revealed that the dual activator and repressor nature of an epigenetic regulator together contributes to cancer development., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background: Concentric and eccentric cardiac hypertrophy are associated with pressure and volume overload, respectively, in cardiovascular disease both conferring an increased risk of heart failure. These contrasting forms of hypertrophy are characterized by asymmetrical growth of the cardiac myocyte in mainly width or length, respectively. The molecular mechanisms determining myocyte preferential growth in width versus length remain poorly understood. Identification of the mechanisms governing asymmetrical myocyte growth could provide new therapeutic targets for the prevention or treatment of heart failure., Methods: Primary adult rat ventricular myocytes, adeno-associated virus (AAV)-mediated gene delivery in mice, and human tissue samples were used to define a regulatory pathway controlling pathological myocyte hypertrophy. Chromatin immunoprecipitation assays with sequencing and precision nuclear run-on sequencing were used to define a transcriptional mechanism., Results: We report that asymmetrical cardiac myocyte hypertrophy is modulated by SRF (serum response factor) phosphorylation, constituting an epigenomic switch balancing the growth in width versus length of adult ventricular myocytes in vitro and in vivo. SRF Ser 103 phosphorylation is bidirectionally regulated by RSK3 (p90 ribosomal S6 kinase type 3) and PP2A (protein phosphatase 2A) at signalosomes organized by the scaffold protein mAKAPβ (muscle A-kinase anchoring protein β), such that increased SRF phosphorylation activates AP-1 (activator protein-1)-dependent enhancers that direct myocyte growth in width. AAV are used to express in vivo mAKAPβ-derived RSK3 and PP2A anchoring disruptor peptides that block the association of the enzymes with the mAKAPβ scaffold. Inhibition of RSK3 signaling prevents concentric cardiac remodeling induced by pressure overload, while inhibition of PP2A signaling prevents eccentric cardiac remodeling induced by myocardial infarction, in each case improving cardiac function. SRF Ser 103 phosphorylation is significantly decreased in dilated human hearts, supporting the notion that modulation of the mAKAPβ-SRF signalosome could be a new therapeutic approach for human heart failure., Conclusions: We have identified a new molecular switch, namely mAKAPβ signalosome-regulated SRF phosphorylation, that controls a transcriptional program responsible for modulating changes in cardiac myocyte morphology that occur secondary to pathological stressors. Complementary AAV-based gene therapies constitute rationally-designed strategies for a new translational modality for heart failure.

Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function., Competing Interests: Competing interest statement: J.U.J. is a scientific adviser of Vaccine Stabilization Institute, a California corporation.

The function of enhancer RNAs (eRNAs) in transcriptional regulation remains obscure. By analyzing the genome-wide nascent transcript profiles in breast cancer cells, we identify a special group of eRNAs that are essential for estrogen-induced transcriptional repression. Using eRNAs of TM4SF1 and EFEMP1 as the paradigms, we find that these RNA molecules not only stabilize promoter-enhancer interactions but also recruit liganded estrogen receptor α (ERα) to particular enhancer regions, facilitate the formation of a functional transcriptional complex, and cause gene silencing. Interestingly, ERα is shown to directly bind with eRNAs by its DNA-binding domain. These eRNAs help with the formation of a specific ERα-centered transcriptional complex and promote the association of the histone demethylase KDM2A, which dismisses RNA polymerase II from designated enhancers and suppresses the transcription of target genes. Our work demonstrates a complete mechanism underlying the action of eRNAs in modulating and refining the locus-specific transcriptional program., Competing Interests: Declaration of Interests The authors declare no conflicts of interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited.

Three-dimensional chromatin structures undergo dynamic reorganization during mammalian spermatogenesis; however, their impacts on gene regulation remain unclear. Here, we focused on understanding the structure-function regulation of meiotic chromosomes by Hi-C and other omics techniques in mouse spermatogenesis across five stages. Beyond confirming recent reports regarding changes in compartmentalization and reorganization of topologically associating domains (TADs), we further demonstrated that chromatin loops are present prior to and after, but not at, the pachytene stage. By integrating Hi-C and RNA-seq data, we showed that the switching of A/B compartments between spermatogenic stages is tightly associated with meiosis-specific mRNAs and piRNAs expression. Moreover, our ATAC-seq data indicated that chromatin accessibility per se is not responsible for the TAD and loop diminishment at pachytene. Additionally, our ChIP-seq data demonstrated that CTCF and cohesin remain bound at TAD boundary regions throughout meiosis, suggesting that dynamic reorganization of TADs does not require CTCF and cohesin clearance., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Noncoding genetic variation is a major driver of phenotypic diversity, but functional interpretation is challenging. To better understand common genetic variation associated with brain diseases, we defined noncoding regulatory regions for major cell types of the human brain. Whereas psychiatric disorders were primarily associated with variants in transcriptional enhancers and promoters in neurons, sporadic Alzheimer's disease (AD) variants were largely confined to microglia enhancers. Interactome maps connecting disease-risk variants in cell-type-specific enhancers to promoters revealed an extended microglia gene network in AD. Deletion of a microglia-specific enhancer harboring AD-risk variants ablated BIN1 expression in microglia, but not in neurons or astrocytes. These findings revise and expand the list of genes likely to be influenced by noncoding variants in AD and suggest the probable cell types in which they function., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell-derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17β-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E 2 -induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Whereas the actions of enhancers in gene transcriptional regulation are well established, roles of JmjC-domain-containing proteins in mediating enhancer activation remain poorly understood. Here, we report that recruitment of the JmjC-domain-containing protein 6 (JMJD6) to estrogen receptor alpha (ERα)-bound active enhancers is required for RNA polymerase II recruitment and enhancer RNA production on enhancers, resulting in transcriptional pause release of cognate estrogen target genes. JMJD6 is found to interact with MED12 in the mediator complex to regulate its recruitment. Unexpectedly, JMJD6 is necessary for MED12 to interact with CARM1, which methylates MED12 at multiple arginine sites and regulates its chromatin binding. Consistent with its role in transcriptional activation, JMJD6 is required for estrogen/ERα-induced breast cancer cell growth and tumorigenesis. Our data have uncovered a critical regulator of estrogen/ERα-induced enhancer coding gene activation and breast cancer cell potency, providing a potential therapeutic target of ER-positive breast cancers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Enhancers for embryonic stem (ES) cell-expressed genes and lineage-determining factors are characterized by conventional marks of enhancer activation in ES cells 1-3 , but it remains unclear whether enhancers destined to regulate cell-type-restricted transcription units might also have distinct signatures in ES cells. Here we show that cell-type-restricted enhancers are 'premarked' and activated as transcription units by the binding of one or two ES cell transcription factors, although they do not exhibit traditional enhancer epigenetic marks in ES cells, thus uncovering the initial temporal origins of cell-type-restricted enhancers. This premarking is required for future cell-type-restricted enhancer activity in the differentiated cells, with the strength of the ES cell signature being functionally important for the subsequent robustness of cell-type-restricted enhancer activation. We have experimentally validated this model in macrophage-restricted enhancers and neural precursor cell (NPC)-restricted enhancers using ES cell-derived macrophages or NPCs, edited to contain specific ES cell transcription factor motif deletions. DNA hydroxyl-methylation of enhancers in ES cells, determined by ES cell transcription factors, may serve as a potential molecular memory for subsequent enhancer activation in mature macrophages. These findings suggest that the massive repertoire of cell-type-restricted enhancers are essentially hierarchically and obligatorily premarked by binding of a defining ES cell transcription factor in ES cells, dictating the robustness of enhancer activation in mature cells.

As most of the mitochondrial proteome is encoded in the nucleus, mitochondrial functions critically depend on nuclear gene expression and bidirectional mito-nuclear communication. However, mitochondria-to-nucleus communication pathways in mammals are incompletely understood. Here, we identify G-Protein Pathway Suppressor 2 (GPS2) as a mediator of mitochondrial retrograde signaling and a transcriptional activator of nuclear-encoded mitochondrial genes. GPS2-regulated translocation from mitochondria to nucleus is essential for the transcriptional activation of a nuclear stress response to mitochondrial depolarization and for supporting basal mitochondrial biogenesis in differentiating adipocytes and brown adipose tissue (BAT) from mice. In the nucleus, GPS2 recruitment to target gene promoters regulates histone H3K9 demethylation and RNA POL2 activation through inhibition of Ubc13-mediated ubiquitination. These findings, together, reveal an additional layer of regulation of mitochondrial gene transcription, uncover a direct mitochondria-nuclear communication pathway, and indicate that GPS2 retrograde signaling is a key component of the mitochondrial stress response in mammals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hematopoietic stem cells (HSCs) maintain a quiescent state during homeostasis, but with acute infection, they exit the quiescent state to increase the output of immune cells, the so-called "emergency hematopoiesis." However, HSCs' response to severe infection during septic shock and the pathological impact remain poorly elucidated. Here, we report that the histone demethylase KDM1A/LSD1, serving as a critical regulator of mammalian hematopoiesis, is a negative regulator of the response to inflammation in HSCs during endotoxic shock typically observed during acute bacterial or viral infection. Inflammation-induced LSD1 deficiency results in an acute expansion of a pathological population of hyperproliferative and hyperinflammatory myeloid progenitors, resulting in a septic shock phenotype and acute death. Unexpectedly, in vivo administration of bacterial lipopolysaccharide (LPS) to wild-type mice results in acute suppression of LSD1 in HSCs with a septic shock phenotype that resembles that observed following induced deletion of LSD1 The suppression of LSD1 in HSCs is caused, at least in large part, by a cohort of inflammation-induced microRNAs. Significantly, reconstitution of mice with bone marrow progenitor cells expressing inhibitors of these inflammation-induced microRNAs blocked the suppression of LSD1 in vivo following acute LPS administration and prevented mortality from endotoxic shock. Our results indicate that LSD1 activators or miRNA antagonists could serve as a therapeutic approach for life-threatening septic shock characterized by dysfunction of HSCs., Competing Interests: The authors declare no conflict of interest.

Long-range enhancer interactions critically regulate gene expression, yet little is known about how their coordinated activities contribute to CNS development or how this may, in turn, relate to disease states. By examining the regulation of the transcription factor NFIA in the developing spinal cord, we identified long-range enhancers that recapitulate NFIA expression across glial and neuronal lineages in vivo. Complementary genetic studies found that Sox9-Brn2 and Isl1-Lhx3 regulate enhancer activity and NFIA expression in glial and neuronal populations. Chromatin conformation analysis revealed that these enhancers and transcription factors form distinct architectures within these lineages in the spinal cord. In glioma models, the glia-specific architecture is present in tumors, and these enhancers are required for NFIA expression and contribute to glioma formation. By delineating three-dimensional mechanisms of gene expression regulation, our studies identify lineage-specific chromatin architectures and associated enhancers that regulate cell fate and tumorigenesis in the CNS.

An effect of thyroid hormone (TH) on erythropoiesis has been known for more than a century but the molecular mechanism(s) by which TH affects red cell formation is still elusive. Here we demonstrate an essential role of TH during terminal human erythroid cell differentiation; specific depletion of TH from the culture medium completely blocked terminal erythroid differentiation and enucleation. Treatment with TRβ agonists stimulated premature erythroblast differentiation in vivo and alleviated anemic symptoms in a chronic anemia mouse model by regulating erythroid gene expression. To identify factors that cooperate with TRβ during human erythroid terminal differentiation, we conducted RNA-seq in human reticulocytes and identified nuclear receptor coactivator 4 (NCOA4) as a critical regulator of terminal differentiation. Furthermore, Ncoa4 -/- mice are anemic in perinatal periods and fail to respond to TH by enhanced erythropoiesis. Genome-wide analysis suggests that TH promotes NCOA4 recruitment to chromatin regions that are in proximity to Pol II and are highly associated with transcripts abundant during terminal differentiation. Collectively, our results reveal the molecular mechanism by which TH functions during red blood cell formation, results that are potentially useful to treat certain anemias., Competing Interests: The authors declare no conflict of interest.

The idea that signal-dependent transcription might involve the generation of transient DNA nicks or even breaks in the regulatory regions of genes, accompanied by activation of DNA damage repair pathways, would seem to be counterintuitive, as DNA damage is usually considered harmful to cellular integrity. However, recent studies have generated a substantial body of evidence that now argues that programmed DNA single- or double-strand breaks can, at least in specific cases, have a role in transcription regulation. Here, we discuss the emerging functions of DNA breaks in the relief of DNA torsional stress and in promoter and enhancer activation.