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KMT2D, a H3K4me1 methyltransferase primarily regulating enhancers, is a leading cause of KABUKI syndrome. This multisystem disorder leads to craniofacial and cognitive abnormalities, possibly through neural crest and neuronal lineages. However, the impacted cell-of-origin and molecular mechanism of KMT2D during the development of KABUKI disease remains unknown. Here we have optimized a brain organoid model to investigate neural crest and neuronal differentiation. To pinpoint KMT2D's enhancer target, we developed a genome-wide cis-regulatory element explorer (GREE) based on single-cell multiomic integration. Single cell RNA-seq revealed that KMT2D-knockout (KO) and patient-derived organoids exhibited neural crest deformities and GABAergic overproduction. Mechanistically, GREE identified that KMT2D targets a roof-plate-like niche cell and activates the niche cell-specific WNT3A enhancer, providing the microenvironment for neural crest and neuronal development. Interestingly, KMT2D-mutated mice displayed decreased WNT3A expression in the diencephalon roof plate, indicating impaired niche cell function. Deleting the WNT3A enhancer in the organoids presented phenotypic similarities to KMT2D-depletion, emphasizing the WNT3A enhancer as the predominant target of KMT2D. Conversely, reactivating WNT signaling in KMT2D-KO rescued the lineage defects by restoring the microenvironment. Overall, our discovery of KMT2D's primary target provides insights for reconciling complex phenotypes of KABUKI syndrome and establishes a new paradigm for dissecting the mechanisms of genetic disorders from genotype to phenotype., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Enhancers and promoters are classically considered to be bound by a small set of transcription factors (TFs) in a sequence-specific manner. This assumption has come under increasing skepticism as the datasets of ChIP-seq assays of TFs have expanded. In particular, high-occupancy target (HOT) loci attract hundreds of TFs with often no detectable correlation between ChIP-seq peaks and DNA-binding motif presence. Here, we used a set of 1003 TF ChIP-seq datasets (HepG2, K562, H1) to analyze the patterns of ChIP-seq peak co-occurrence in combination with functional genomics datasets. We identified 43,891 HOT loci forming at the promoter (53%) and enhancer (47%) regions. HOT promoters regulate housekeeping genes, whereas HOT enhancers are involved in tissue-specific process regulation. HOT loci form the foundation of human super-enhancers and evolve under strong negative selection, with some of these loci being located in ultraconserved regions. Sequence-based classification analysis of HOT loci suggested that their formation is driven by the sequence features, and the density of mapped ChIP-seq peaks across TF-bound loci correlates with sequence features and the expression level of flanking genes. Based on the affinities to bind to promoters and enhancers we detected five distinct clusters of TFs that form the core of the HOT loci. We report an abundance of HOT loci in the human genome and a commitment of 51% of all TF ChIP-seq binding events to HOT locus formation thus challenging the classical model of enhancer activity and propose a model of HOT locus formation based on the existence of large transcriptional condensates., Competing Interests: SH, IO No competing interests declared

Enhancers are cis-regulatory DNA sequences that are widely distributed throughout the genome. They can precisely regulate the expression of target genes. Since the features of enhancer segments are difficult to detect, we propose DeepDualEnhancer, a DNABert-based method using a multi-scale convolutional neural network, BiLSTM, for enhancer identification. We first designed the DeepDualEnhancer method based only on the DNA sequence input. It mainly consists of a multi-scale Convolutional Neural Network, and BiLSTM to extract features by DNABert and embedding, respectively. Meanwhile, we collected new datasets from the enhancer-promoter interaction field and designed the method DeepDualEnhancer-genomic for inputting DNA sequences and genomic signals, which consists of the transformer sequence attention. Extensive comparisons of our method with 20 other excellent methods through 5-fold cross validation, ablation experiments, and an independent test demonstrated that DeepDualEnhancer achieves the best performance. It is also found that the inclusion of genomic signals helps the enhancer recognition task to be performed better.

Extrachromosomal DNA (ecDNA) is a major contributor to treatment resistance and poor outcome for patients with cancer 1,2 . Here we examine the diversity of ecDNA elements across cancer, revealing the associated tissue, genetic and mutational contexts. By analysing data from 14,778 patients with 39 tumour types from the 100,000 Genomes Project, we demonstrate that 17.1% of tumour samples contain ecDNA. We reveal a pattern highly indicative of tissue-context-based selection for ecDNAs, linking their genomic content to their tissue of origin. We show that not only is ecDNA a mechanism for amplification of driver oncogenes, but it also a mechanism that frequently amplifies immunomodulatory and inflammatory genes, such as those that modulate lymphocyte-mediated immunity and immune effector processes. Moreover, ecDNAs carrying immunomodulatory genes are associated with reduced tumour T cell infiltration. We identify ecDNAs bearing only enhancers, promoters and lncRNA elements, suggesting the combinatorial power of interactions between ecDNAs in trans. We also identify intrinsic and environmental mutational processes linked to ecDNA, including those linked to its formation, such as tobacco exposure, and progression, such as homologous recombination repair deficiency. Clinically, ecDNA detection was associated with tumour stage, more prevalent after targeted therapy and cytotoxic treatments, and associated with metastases and shorter overall survival. These results shed light on why ecDNA is a substantial clinical problem that can cooperatively drive tumour growth signals, alter transcriptional landscapes and suppress the immune system., (© 2024. The Author(s).)

Chronic inflammation and tissue fibrosis are common responses that worsen organ function, yet the molecular mechanisms governing their cross-talk are poorly understood. In diseased organs, stress-induced gene expression changes fuel maladaptive cell state transitions 1 and pathological interaction between cellular compartments. Although chronic fibroblast activation worsens dysfunction in the lungs, liver, kidneys and heart, and exacerbates many cancers 2 , the stress-sensing mechanisms initiating transcriptional activation of fibroblasts are poorly understood. Here we show that conditional deletion of the transcriptional co-activator Brd4 in infiltrating Cx3cr1 + macrophages ameliorates heart failure in mice and significantly reduces fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1 + cells identified a large enhancer proximal to interleukin-1β (IL-1β, encoded by Il1b), and a series of CRISPR-based deletions revealed the precise stress-dependent regulatory element that controls Il1b expression. Secreted IL-1β activated a fibroblast RELA-dependent (also known as p65) enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL-1β neutralization improved cardiac function and tissue fibrosis in heart failure. Systemic IL-1β inhibition or targeted Il1b deletion in Cx3cr1 + cells prevented stress-induced Meox1 expression and fibroblast activation. The elucidation of BRD4-dependent cross-talk between a specific immune cell subset and fibroblasts through IL-1β reveals how inflammation drives profibrotic cell states and supports strategies that modulate this process in heart disease and other chronic inflammatory disorders featuring tissue remodelling., Competing Interests: Competing interests D.S. is scientific co-founder, shareholder and director of Tenaya Therapeutics. S.M.H. is an executive, officer and shareholder of Amgen and is a scientific co-founder and shareholder of Tenaya Therapeutics. T.A.M. received funding from Italfarmaco for an unrelated project. K.S.P. is a shareholder of Tenaya Therapeutics., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Autoactivation of lineage-determining transcription factors mediates bistable expression, generating distinct cell phenotypes essential for complex body plans. Classical type 1 dendritic cell (cDC1) and type 2 dendritic cell (cDC2) subsets provide nonredundant functions for defense against distinct immune challenges. Interferon regulatory factor 8 (IRF8), the cDC1 lineage-determining transcription factor, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32-kb enhancer, responsible for IRF8 autoactivation, is naturally suboptimized with low-affinity IRF8 binding sites. Introducing multiple high-affinity IRF8 sites into the Irf8 +32-kb enhancer causes a gain-of-function effect, leading to erroneous IRF8 autoactivation in specified cDC2 progenitors, redirecting them toward cDC1 and a novel hybrid DC subset with mixed-lineage phenotypes. Further, this also causes a loss-of-function effect, reducing Irf8 expression in cDC1s. These developmental alterations critically impair both cDC1-dependent and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of cDCs required for normal immune function., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In this study, we characterize a novel lncRNA-producing gene locus that we name Syntenic Cardiovascular Conserved Region-Associated lncRNA-6 (scar-6) and functionally validate its role in coagulation and cardiovascular function. A 12-bp deletion of the scar-6 locus in zebrafish (scar-6 gib007Δ12/Δ12 ) results in cranial hemorrhage and vascular permeability. Overexpression, knockdown and rescue with the scar-6 lncRNA modulates hemostasis in zebrafish. Molecular investigation reveals that the scar-6 lncRNA acts as an enhancer lncRNA (elncRNA), and controls the expression of prozb, an inhibitor of factor Xa, through an enhancer element in the scar-6 locus. The scar-6 locus suppresses loop formation between prozb and scar-6 sequences, which might be facilitated by the methylation of CpG islands via the prdm14-PRC2 complex whose binding to the locus might be stabilized by the scar-6 elncRNA transcript. Binding of prdm14 to the scar-6 locus is impaired in scar-6 gib007Δ12/Δ12 zebrafish. Finally, activation of the PAR2 receptor in scar-6 gib007Δ12/Δ12 zebrafish triggers NF-κB-mediated endothelial cell activation, leading to vascular dysfunction and hemorrhage. We present evidence that the scar-6 locus plays a role in regulating the expression of the coagulation cascade gene prozb and maintains vascular homeostasis., (© 2024. The Author(s).)

Cis-regulatory elements (CREs) are integral to the spatiotemporal and quantitative expression dynamics of target genes, thus directly influencing phenotypic variation and evolution. However, many of these CREs become highly susceptible to transcriptional silencing when in a transgenic state, particularly when organised as tandem repeats. We investigated the mechanism of this phenomenon and found that three of the six selected flower-specific CREs were prone to transcriptional silencing when in a transgenic context. We determined that this silencing was caused by the ectopic expression of non-coding RNAs (ncRNAs), which were processed into 24-nt small interfering RNAs (siRNAs) that drove RNA-directed DNA methylation (RdDM). Detailed analyses revealed that aberrant ncRNA transcription within the AGAMOUS enhancer (AGe) in a transgenic context was significantly enhanced by an adjacent CaMV35S enhancer (35Se). This particular enhancer is known to mis-activate the regulatory activities of various CREs, including the AGe. Furthermore, an insertion of 35Se approximately 3.5 kb upstream of the AGe in its genomic locus also resulted in the ectopic induction of ncRNA/siRNA production and de novo methylation specifically in the AGe, but not other regions, as well as the production of mutant flowers. This confirmed that interactions between the 35Se and AGe can induce RdDM activity in both genomic and transgenic states. These findings highlight a novel epigenetic role for CRE-CRE interactions in plants, shedding light on the underlying forces driving hypermethylation in transgenes, duplicate genes/enhancers, and repetitive transposons, in which interactions between CREs are inevitable., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity., (© 2024 Wiley Periodicals LLC.)

The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Epithelial-to-mesenchymal transitions (EMTs) and extracellular matrix (ECM) remodeling are distinct yet important processes during carcinoma invasion and metastasis. Transforming growth factor β (TGF-β) and RAS, signaling through SMAD and RAS-responsive element-binding protein 1 (RREB1), jointly trigger expression of EMT and fibrogenic factors as two discrete arms of a common transcriptional response in carcinoma cells. Here, we demonstrate that both arms come together to form a program for lung adenocarcinoma metastasis and identify chromatin determinants tying the expression of the constituent genes to TGF-β and RAS inputs. RREB1 localizes to H4K16acK20ac marks in histone H2A.Z-loaded nucleosomes at enhancers in the fibrogenic genes interleukin-11 (IL11), platelet-derived growth factor-B (PDGFB), and hyaluronan synthase 2 (HAS2), as well as the EMT transcription factor SNAI1, priming these enhancers for activation by a SMAD4-INO80 nucleosome remodeling complex in response to TGF-β. These regulatory properties segregate the fibrogenic EMT program from RAS-independent TGF-β gene responses and illuminate the operation and vulnerabilities of a bifunctional program that promotes metastatic outgrowth., Competing Interests: Declaration of interests J.M. owns company stock in Scholar Rock. C.M.R. has consulted regarding oncology drug development with Amgen, Astra Zeneca, Chugai, Daiichi Sankyo, Hoffman-La Roche, and Jazz Pharmaceuticals; C.M.R. serves on the scientific advisory boards of Auron, Bridge Medicines, DISCO, Earli, and Harpoon Therapeutics. S.W.L. is a consultant and holds equity in Blueprint Medicines, ORIC Pharmaceuticals, Mirimus, PMV Pharmaceuticals, Faeth Therapeutics, and Senecea Therapeutics, and is a consultant for Fate Therapeutics. T.T. is a consultant and holds equity in Lime Therapeutics; the Tammela Lab receives research support from Ono Pharmaceuticals Co., Ltd. (unrelated to this work). T.T.’s spouse is an employee of Recursion Pharmaceuticals. R.P.K. is a co-founder of and consultant for Econic Biosciences., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Obesity-induced β cell dysfunction contributes to the onset of type 2 diabetes. Nevertheless, elucidating epigenetic mechanisms underlying islet dysfunction at single cell level remains challenging. Here we profile single-nuclei RNA along with enhancer marks H3K4me1 or H3K27ac in islets from lean or obese mice. Our study identifies distinct gene signatures and enhancer states correlating with β cell dysfunction trajectory. Intriguingly, while many metabolic stress-induced genes exhibit concordant changes in both H3K4me1 and H3K27ac at their enhancers, expression changes of specific subsets are solely attributable to either H3K4me1 or H3K27ac dynamics. Remarkably, a subset of H3K4me1 + H3K27ac - primed enhancers prevalent in lean β cells and occupied by FoxA2 are largely absent after metabolic stress. Lastly, cell-cell communication analysis identified the nerve growth factor (NGF) as protective paracrine signaling for β cells through repressing ER stress. In summary, our findings define the heterogeneous enhancer responses to metabolic challenges in individual β cells., (© 2024. The Author(s).)

Genes are often regulated by multiple enhancers. It is poorly understood how the individual enhancer activities are combined to control promoter activity. Anecdotal evidence has shown that enhancers can combine sub-additively, additively, synergistically, or redundantly. However, it is not clear which of these modes are more frequent in mammalian genomes. Here, we systematically tested how pairs of enhancers activate promoters using a three-way combinatorial reporter assay in mouse embryonic stem cells. By assaying about 69,000 enhancer-enhancer-promoter combinations we found that enhancer pairs generally combine near-additively. This behaviour was conserved across seven developmental promoters tested. Surprisingly, these promoters scale the enhancer signals in a non-linear manner that depends on promoter strength. A housekeeping promoter showed an overall different response to enhancer pairs, and a smaller dynamic range. Thus, our data indicate that enhancers mostly act additively, but promoters transform their collective effect non-linearly., Competing Interests: MM, Bv No competing interests declared, FC co-founder of enGene Statistics GmbH, (© 2023, Martinez-Ara et al.)

Transcriptional regulation, orchestrated by the interplay between transcription factors (TFs) and enhancers, governs gene expression dynamics crucial for cellular processes. While gross qualitative fluctuations in transcription factor-dependent gene expression patterning have a long history of characterization, the roles of these factors in the nuclei retaining expression in the presence or absence of these factors are now observable using modern techniques. Our study investigates the impact of Suppressor of Hairless (Su(H)), a broadly expressed transcription factor, on enhancer-driven transcriptional modulation using Drosophila early embryos as a model system. Building upon previous findings, we employ super-resolution microscopy to dissect Su(H)'s influence on sog-Distal ( sogD ) enhancer activity specifically in nuclei with preserved sogD -driven expression in the absence of Su(H) binding. We demonstrate that Su(H) occupancy perturbations alter expression levels and bursting dynamics. Notably, Su(H) absence during embryonic development exhibits region-specific effects, inhibiting expression dorsally and stabilizing expression ventrally, implying a nuanced role in enhancer regulation. Our findings shed light on the intricate mechanisms that govern transcriptional dynamics and suggest a critical patterning role for Notch/Hairless signaling in sog expression as embryos transition to gastrulation.

CD4 + T cells play a crucial role in adaptive immune responses and have been implicated in the pathogenesis of autoimmune diseases (ADs). Despite numerous studies, the molecular mechanisms underlying T cell dysregulation in ADs remain incompletely understood. Here, we used chromatin immunoprecipitation (ChIP)-sequencing of active chromatin and transcriptomic data from CD4 + T cells of healthy donors and patients with systemic lupus erythematosus (SLE), psoriasis, juvenile idiopathic arthritis (JIA), and Graves' disease to investigate the role of enhancers in AD pathogenesis. By generating enhancer-based gene regulatory networks (eGRNs), we identified disease-specific dysregulated pathways and potential downstream target genes of enhancers harboring AD-associated single-nucleotide polymorphisms (SNPs), which we also validated using chromatin-capture (HiC) data and CRISPR interference (CRISPRi) in primary CD4 + T cells. Our results suggest that alterations in the regulatory landscapes of CD4 + T cells, including enhancers, contribute to the development of ADs and provide a basis for developing new therapeutic approaches., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Currently, routine diagnostics for spinocerebellar ataxia (SCA) look for polyQ repeat expansions and conventional variations affecting the proteins encoded by known SCA genes. However, ~40% of the patients still remain without a genetic diagnosis after routine tests. Increasing evidence suggests that variations in the enhancer regions of genes involved in neurodegenerative disorders can also cause disease. Since the enhancers of SCA genes are not yet known, it remains to be determined whether variations in these regions are a cause of SCA. In this pilot project, we aimed to identify the enhancers of the SCA genes ATXN1 , ATXN3 , TBP and ITPR1 in the human cerebellum using 4C-seq, publicly available datasets, reciprocal 4C-seq, and luciferase assays. We then screened these enhancers for copy number variants (CNVs) in a cohort of genetically undiagnosed SCA patients. We identified two active enhancers for each of the four SCA genes. CNV analysis did not reveal any CNVs in the enhancers of the four SCA genes in the genetically undiagnosed SCA patients. However, in one patient, we noted a CNV deletion with an unknown clinical significance near one of the ITPR1 enhancers. These results not only reveal elements involved in SCA gene regulation but can also lead to the discovery of novel SCA-causing genetic variants. As enhancer variations are being increasingly recognized as a cause of brain disorders, screening the enhancers of ATXN1 , ATXN3 , TBP and ITPR1 for variations other than CNVs and identifying and screening enhancers of other SCA genes might elucidate the genetic cause in undiagnosed patients.

Enhancers are central for the regulation of metazoan transcription but have proven difficult to study, primarily due to a myriad of interdependent variables shaping their activity. Consequently, synthetic biology has emerged as the main approach for dissecting mechanisms of enhancer function. We start by reviewing simple but highly parallel reporter assays, which have been successful in quantifying the complexity of the activator/coactivator mechanisms at enhancers. We then describe studies that examine how enhancers function in the genomic context and in combination with other enhancers, revealing that they activate genes through a variety of different mechanisms, working together as a system. Here, we primarily focus on synthetic reporter genes that can quantify the dynamics of enhancer biology through time. We end by considering the consequences of having many genes and enhancers within a 'local environment', which we believe leads to correlated gene expression and likely reports on the general principles of enhancer biology.

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically up-regulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally acting chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Williams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Growing evidence supports the transcription of enhancer RNAs (eRNAs) and their important roles in gene regulation. However, their interactions with other biomolecules and their corresponding functionality remain poorly understood. In an attempt to facilitate mechanistic research, this study presents eRNA-IDO, the first integrative computational platform for the identification, interactome discovery, and functional annotation of human eRNAs. eRNA-IDO comprises two modules: eRNA-ID and eRNA-Anno. Functionally, eRNA-ID can identify eRNAs from de novo assembled transcriptomes. eRNA-ID includes eight kinds of enhancer makers, enabling users to customize enhancer regions flexibly and conveniently. In addition, eRNA-Anno provides cell-/tissue-specific functional annotation for both new and known eRNAs by analyzing the eRNA interactome from prebuilt or user-defined networks between eRNAs and protein-coding genes. The prebuilt networks include the Genotype-Tissue Expression (GTEx)-based co-expression networks in normal tissues, The Cancer Genome Atlas (TCGA)-based co-expression networks in cancer tissues, and omics-based eRNA-centric regulatory networks. eRNA-IDO can facilitate research on the biogenesis and functions of eRNAs. The eRNA-IDO server is freely available at http://bioinfo.szbl.ac.cn/eRNA&#95;IDO/., (© The Author(s) 2024. Published by Oxford University Press and Science Press on behalf of the Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation and Genetics Society of China.)

Annotation of newly sequenced genomes frequently includes genes, but rarely covers important non-coding genomic features such as the cis -regulatory modules-e.g., enhancers and silencers-that regulate gene expression. Here, we begin to remedy this situation by developing a workflow for rapid initial annotation of insect regulatory sequences, and provide a searchable database resource with enhancer predictions for 33 genomes. Using our previously developed SCRMshaw computational enhancer prediction method, we predict over 2.8 million regulatory sequences along with the tissues where they are expected to be active, in a set of insect species ranging over 360 million years of evolution. Extensive analysis and validation of the data provides several lines of evidence suggesting that we achieve a high true-positive rate for enhancer prediction. One, we show that our predictions target specific loci, rather than random genomic locations. Two, we predict enhancers in orthologous loci across a diverged set of species to a significantly higher degree than random expectation would allow. Three, we demonstrate that our predictions are highly enriched for regions of accessible chromatin. Four, we achieve a validation rate in excess of 70% using in vivo reporter gene assays. As we continue to annotate both new tissues and new species, our regulatory annotation resource will provide a rich source of data for the research community and will have utility for both small-scale (single gene, single species) and large-scale (many genes, many species) studies of gene regulation. In particular, the ability to search for functionally related regulatory elements in orthologous loci should greatly facilitate studies of enhancer evolution even among distantly related species., Competing Interests: HA, ET, KD, JR, TD, XH, YT, MH No competing interests declared, (© 2024, Asma et al.)

Well-differentiated low-grade lung neuroendocrine tumors (lung carcinoids or LNETs) are histopathologically classified as typical and atypical LNETs, but each subtype is still heterogeneous at both the molecular level and its clinical manifestation. Here, we report genome-wide profiles of primary LNETs' cis-regulatory elements by H3K27ac ChIP-seq with matching RNA-seq profiles. Analysis of these regulatory landscapes revealed three regulatory subtypes, independent of the typical/atypical classification. We identified unique differentiation signals that delineate each subtype. The "proneuronal" subtype emerges under the influence of ASCL1, SOX4, and TCF4 transcription factors, embodying a pronounced proneuronal signature. The "luminal-like" subtype is characterized by gain of acetylation at markers of luminal cells and GATA2 activation and loss of LRP5 and OTP. The "HNF+" subtype is characterized by a robust enhancer landscape driven by HNF1A, HNF4A, and FOXA3, with notable acetylation and expression of FGF signaling genes, especially FGFR3 and FGFR4, pivotal components of the FGF pathway. Our findings not only deepen the understanding of LNETs' regulatory and developmental diversity but also spotlight the HNF+ subtype's reliance on FGFR signaling. We demonstrate that targeting this pathway with FGF inhibitors curtails tumor growth both in vitro and in xenograft models, unveiling a potential vulnerability and paving the way for targeted therapies. Overall, our work provides an important resource for studying LNETs to reveal regulatory networks, differentiation signals, and therapeutically relevant dependencies., Competing Interests: Competing interests statement:The authors declare no competing interest.

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3' untranslated region (3'UTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management., Competing Interests: XW, JL, JC, WC, WX, KW, LL, JH, JC, HZ No competing interests declared, (© 2024, Wei, Liu et al.)

The BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS), a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A +/- induced pluripotent stem cells to model CNCC specification, we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: In lung squamous cell carcinoma (LUSC), Black patients show significantly higher incidence and lower overall survival than White patients. Although socioeconomic factors likely contribute to this survival disparity, genomic factors have yet to be elucidated in LUSC., Methods: Using 416 LUSC tumor samples in the Cancer Genome Atlas (TCGA), we assessed genomic and transcriptomic profiles by ancestry. We replicated our analyses in pan-cancer data from TCGA, the American Association of Cancer Research (AACR) Genomics Evidence Neoplasia Information Exchange (GENIE), and Columbia University Medical Center., Results: We found increased MYC amplification, LUSC-specific MYC enhancer amplification, and chromosome arm 8q (chr8q) gain to be significantly associated with genetic AFR (African) ancestry in LUSC in TCGA. Furthermore, expression of MYC target genes was significantly enriched in AFR samples. Local ancestry analysis identified correlation of chr8q gain with AFR ancestry at the MYC locus in TCGA. We also found a significant correlation between chr8q and AFR ancestry in multiple cancer types and pan-cancer in TCGA. Similarly, in a pan-cancer subset of AACR GENIE data, we found a significant correlation between chr8q gain and race., Conclusion: Together, our data suggest that ancestry may influence amplification of not only MYC but also its enhancer in LUSC. They also suggest a role for genetic ancestry in chr8q aneuploidy in cancer. These studies further define and expand patients who may benefit from future anti- MYC therapeutic approaches.

Understanding how cell identity is encoded by the genome and acquired during differentiation is a central challenge in cell biology. I have developed a theoretical framework called EnhancerNet, which models the regulation of cell identity through the lens of transcription factor-enhancer interactions. I demonstrate that autoregulation in these interactions imposes a constraint on the model, resulting in simplified dynamics that can be parameterized from observed cell identities. Despite its simplicity, EnhancerNet recapitulates a broad range of experimental observations on cell identity dynamics, including enhancer selection, cell fate induction, hierarchical differentiation through multipotent progenitor states and direct reprogramming by transcription factor overexpression. The model makes specific quantitative predictions, reproducing known reprogramming recipes and the complex haematopoietic differentiation hierarchy without fitting unobserved parameters. EnhancerNet provides insights into how new cell types could evolve and highlights the functional importance of distal regulatory elements with dynamic chromatin in multicellular evolution., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

PD-1 is a key negative regulator of CD8 + T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8 + T cells in chronic infection, creating a 'sweet spot' of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1-knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8 + T cell dysfunction while avoiding excess immunopathology., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Enhancers are noncoding regulatory elements that instruct spatial and temporal specificity of gene transcription in response to a variety of intrinsic and extrinsic signals during development. Although it has long been postulated that enhancers physically interact with target promoters through the formation of stable loops, recent studies have changed this static view: sequence-specific transcription factors (TFs) and coactivators are dynamically recruited to enhancers and assemble so-called transcription hubs. Dynamic assembly of transcription hubs appears to serve as a key scaffold to integrate regulatory information encoded by surrounding genome and biophysical properties of transcription machineries. In this review, we outline emerging new models of transcriptional regulation by enhancers and discuss future perspectives., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Transposable elements (TEs) have emerged as important factors in establishing the cell type-specific gene regulatory networks and evolutionary novelty of embryonic and placental development. Recently, studies on the role of TEs and their dysregulation in cancers have shed light on the transcriptional, transpositional, and regulatory activity of TEs, revealing that the activation of developmental transcriptional programs by TEs may have a role in the dedifferentiation of cancer cells to the progenitor-like cell states. This essay reviews the recent evidence of the cis-regulatory TEs (henceforth crTE) in normal development and malignancy as well as the key transcription factors and regulatory pathways that are implicated in both cell states, and presents existing gaps remaining to be studied, limitations of current technologies, and therapeutic possibilities., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer-promoter (E-P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward- and outward-oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD-boundary-crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long-distance E-P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA-mediated TAD boundary function via R-loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E-P interactions is developmentally regulated., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

The genomes of living lungfishes can inform on the molecular-developmental basis of the Devonian sarcopterygian fish-tetrapod transition. We de novo sequenced the genomes of the African (Protopterus annectens) and South American lungfishes (Lepidosiren paradoxa). The Lepidosiren genome (about 91 Gb, roughly 30 times the human genome) is the largest animal genome sequenced so far and more than twice the size of the Australian (Neoceratodus forsteri) 1 and African 2 lungfishes owing to enlarged intergenic regions and introns with high repeat content (about 90%). All lungfish genomes continue to expand as some transposable elements (TEs) are still active today. In particular, Lepidosiren's genome grew extremely fast during the past 100 million years (Myr), adding the equivalent of one human genome every 10 Myr. This massive genome expansion seems to be related to a reduction of PIWI-interacting RNAs and C2H2 zinc-finger and Krüppel-associated box (KRAB)-domain protein genes that suppress TE expansions. Although TE abundance facilitates chromosomal rearrangements, lungfish chromosomes still conservatively reflect the ur-tetrapod karyotype. Neoceratodus' limb-like fins still resemble those of their extinct relatives and remained phenotypically static for about 100 Myr. We show that the secondary loss of limb-like appendages in the Lepidosiren-Protopterus ancestor was probably due to loss of sonic hedgehog limb-specific enhancers., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The migration is the key step for thymic T cells to enter circulation and then lymph nodes (LNs), essential for future immune surveillance. Although promoter-based transcriptional regulation through Foxo1, Klf2, Ccr7, and Sell regulates T-cell migration, it remains largely unexplored whether and how enhancers are involved in this process. Here we found that the conditional deletion of Med1, a component of the mediator complex and a mediator between enhancers and RNA polymerase II, caused a reduction of both CD4 + and CD8 + T cells in LNs, as well as a decrease of CD8 + T cells in the spleen. Importantly, Med1 deletion hindered the migration of thymic αβT cells into the circulation and then into LNs, accompanied by the downregulation of KLF2, CCR7, and CD62L. Mechanistically, Med1 promotes Klf2 transcription by facilitating Foxo1 binding to the Klf2 enhancer. Furthermore, forced expression of Klf2 rescued Ccr7 and Sell expression, as well as αβT-cell migration into LNs. Collectively, our study unveils a crucial role for Med1 in regulating the enhancer-based Foxo1-Klf2 transcriptional program and the migration of αβT cells into LNs, providing valuable insights into the molecular mechanisms underlying T-cell migration., (© 2024 Wiley‐VCH GmbH.)

A key question in regulatory genomics is whether cis-regulatory elements (CREs) are modular elements that can function anywhere in the genome, or whether they are adapted to certain genomic locations. To distinguish between these possibilities we develop MPIRE (Massively Parallel Integrated Regulatory Elements), a technology for recurrently assaying CREs at thousands of defined locations across the genome in parallel. MPIRE allows us to separate the intrinsic activity of CREs from the effects of their genomic environments. We apply MPIRE to assay three insulator sequences at thousands of genomic locations and find that each insulator functions in locations with distinguishable properties. All three insulators can block enhancers, but each insulator blocks specific enhancers at specific locations. However, only ALOXE3 appears to block heterochromatin silencing. We conclude that insulator function is highly context dependent and that MPIRE is a robust method for revealing the context dependencies of CREs., (© 2024. The Author(s).)

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders., (© 2024. The Author(s).)

The β-catenin-dependent canonical Wnt signaling is pivotal in organ development, tissue homeostasis, and cancer. Here, we identified an upstream enhancer of Ctnnb1 - the coding gene for β-catenin, named ieCtnnb1 ( i ntestinal e nhancer of Ctnnb1 ), which is crucial for intestinal homeostasis. ieCtnnb1 is predominantly active in the base of small intestinal crypts and throughout the epithelia of large intestine. Knockout of ieCtnnb1 led to a reduction in Ctnnb1 transcription, compromising the canonical Wnt signaling in intestinal crypts. Single-cell sequencing revealed that ieCtnnb1 knockout altered epithelial compositions and potentially compromised functions of small intestinal crypts. While deletion of ieCtnnb1 hampered epithelial turnovers in physiologic conditions, it prevented occurrence and progression of Wnt/β-catenin-driven colorectal cancers. Human ieCTNNB1 drove reporter gene expression in a pattern highly similar to mouse ieCtnnb1. ieCTNNB1 contains a single-nucleotide polymorphism associated with CTNNB1 expression levels in human gastrointestinal epithelia. The enhancer activity of ieCTNNB1 in colorectal cancer tissues was stronger than that in adjacent normal tissues. HNF4α and phosphorylated CREB1 were identified as key trans-factors binding to ieCTNNB1 and regulating CTNNB1 transcription. Together, these findings unveil an enhancer-dependent mechanism controlling the dosage of Wnt signaling and homeostasis in intestinal epithelia., Competing Interests: XH, CZ, JT, JW, XM, GZ, MW, MY, YL, YZ No competing interests declared, (© 2024, Hua et al.)

Neuronal activity-regulated gene expression plays a crucial role in sculpting neural circuits that underpin adaptive brain function. Transcriptional enhancers are now recognized as key components of gene regulation that orchestrate spatiotemporally precise patterns of gene transcription. We propose that the dynamics of enhancer activation uniquely position these genomic elements to finely tune activity-dependent cellular plasticity. Enhancer specificity and modularity can be exploited to gain selective genetic access to specific cell states, and the precise modulation of target gene expression within restricted cellular contexts enabled by targeted enhancer manipulation allows for fine-grained evaluation of gene function. Mounting evidence also suggests that enduring stimulus-induced changes in enhancer states can modify target gene activation upon restimulation, thereby contributing to a form of cell-wide metaplasticity. We advocate for focused exploration of activity-dependent enhancer function to gain new insight into the mechanisms underlying brain plasticity and cognitive dysfunction., Competing Interests: Declaration of interests M.E.G. serves as a member of the Neuron advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Large-scale sequencing efforts have been undertaken to understand the mutational landscape of the coding genome. However, the vast majority of variants occur within non-coding genomic regions. We designed an integrative computational and experimental framework to identify recurrently mutated non-coding regulatory regions that drive tumor progression. Applying this framework to sequencing data from a large prostate cancer patient cohort revealed a large set of candidate drivers. We used (1) in silico analyses, (2) massively parallel reporter assays, and (3) in vivo CRISPR interference screens to systematically validate metastatic castration-resistant prostate cancer (mCRPC) drivers. One identified enhancer region, GH22I030351, acts on a bidirectional promoter to simultaneously modulate expression of the U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. SF3A1 and CCDC157 promote tumor growth in vivo. We nominated a number of transcription factors, notably SOX6, to regulate expression of SF3A1 and CCDC157. Our integrative approach enables the systematic detection of non-coding regulatory regions that drive human cancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWAS) have identified hundreds of genetic signals associated with autoimmune disease. The majority of these signals are located in non-coding regions and likely impact cis -regulatory elements (cRE). Because cRE function is dynamic across cell types and states, profiling the epigenetic status of cRE across physiological processes is necessary to characterize the molecular mechanisms by which autoimmune variants contribute to disease risk. We localized risk variants from 15 autoimmune GWAS to cRE active during TCR-CD28 co-stimulation of naïve human CD4+ T cells. To characterize how dynamic changes in gene expression correlate with cRE activity, we measured transcript levels, chromatin accessibility, and promoter-cRE contacts across three phases of naive CD4+ T cell activation using RNA-seq, ATAC-seq, and HiC. We identified ~1200 protein-coding genes physically connected to accessible disease-associated variants at 423 GWAS signals, at least one-third of which are dynamically regulated by activation. From these maps, we functionally validated a novel stretch of evolutionarily conserved intergenic enhancers whose activity is required for activation-induced IL2 gene expression in human and mouse, and is influenced by autoimmune-associated genetic variation. The set of genes implicated by this approach are enriched for genes controlling CD4+ T cell function and genes involved in human inborn errors of immunity, and we pharmacologically validated eight implicated genes as novel regulators of T cell activation. These studies directly show how autoimmune variants and the genes they regulate influence processes involved in CD4+ T cell proliferation and activation., Competing Interests: MP, PS, RT, ZT, ZM, JP, PM, PS, CS, DC, SG, AW No competing interests declared, (© 2024, Pahl, Sharma, Thomas et al.)

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner., (© 2024. The Author(s).)

The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here, we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression program at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C, promoter-capture Hi-C, CUT&Tag for H3K27ac and RNA-seq. We find that genes involved in axonal regeneration form long-range, complex chromatin loops, and that cohesin is required for the full induction of the regenerative transcriptional program. Importantly, loss of cohesin results in disruption of chromatin architecture and severely impaired nerve regeneration. Complex enhancer-promoter loops are also enriched in the human fetal cortical plate, where the axonal growth potential is highest, and are lost in mature adult neurons. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent long-range promoter interactions in nerve regeneration., Competing Interests: Competing interests statement:The authors declare no competing interest.

Recent advances in high-resolution mapping of spatial interactions among regulatory elements support the existence of complex topological assemblies of enhancers and promoters known as enhancer-promoter hubs or cliques. Yet, organization principles of these multi-interacting enhancer-promoter hubs and their potential role in regulating gene expression in cancer remain unclear. Here, we systematically identify enhancer-promoter hubs in breast cancer, lymphoma, and leukemia. We find that highly interacting enhancer-promoter hubs form at key oncogenes and lineage-associated transcription factors potentially promoting oncogenesis of these diverse cancer types. Genomic and optical mapping of interactions among enhancer and promoter elements further show that topological alterations in hubs coincide with transcriptional changes underlying acquired resistance to targeted therapy in T cell leukemia and B cell lymphoma. Together, our findings suggest that enhancer-promoter hubs are dynamic and heterogeneous topological assemblies with the potential to control gene expression circuits promoting oncogenesis and drug resistance., (© 2024. The Author(s).)

Transitions between the fed and fasted state are common in mammals. The liver orchestrates adaptive responses to feeding/fasting by transcriptionally regulating metabolic pathways of energy usage and storage. Transcriptional and enhancer dynamics following cessation of fasting (refeeding) have not been explored. We examined the transcriptional and chromatin events occurring upon refeeding in mice, including kinetic behavior and molecular drivers. We found that the refeeding response is temporally organized with the early response focused on ramping up protein translation while the later stages of refeeding drive a bifurcated lipid synthesis program. While both the cholesterol biosynthesis and lipogenesis pathways were inhibited during fasting, most cholesterol biosynthesis genes returned to their basal levels upon refeeding while most lipogenesis genes markedly overshoot above pre-fasting levels. Gene knockout, enhancer dynamics, and ChIP-seq analyses revealed that lipogenic gene overshoot is dictated by LXRα. These findings from unbiased analyses unravel the mechanism behind the long-known phenomenon of refeeding fat overshoot., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Korenfeld et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although the study of leukemogenesis has traditionally focused on protein-coding genes, the role of enhancer dysregulation is becoming increasingly recognized. The advent of high-throughput sequencing, together with a better understanding of enhancer biology, has revealed how various genetic and epigenetic lesions produce oncogenic enhancers that drive transformation. These aberrations include translocations that lead to enhancer hijacking, point mutations that modulate enhancer activity, and copy number alterations that modify enhancer dosage. In this review, we describe these mechanisms in the context of leukemia and discuss potential therapeutic avenues to target these regulatory elements. Significance: Large-scale sequencing projects have uncovered recurrent gene mutations in leukemia, but the picture remains incomplete: some patients harbor no such aberrations, whereas others carry only a few that are insufficient to bring about transformation on their own. One of the missing pieces is enhancer dysfunction, which only recently has emerged as a critical driver of leukemogenesis. Knowledge of the various mechanisms of enhancer dysregulation is thus key for a complete understanding of leukemia and its causes, as well as the development of targeted therapies in the era of precision medicine., (©2024 American Association for Cancer Research.)

Gene upregulation through genome editing is important for plant research and breeding. Targeted insertion of short transcriptional enhancers (STEs) into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis. Here, we introduce an "in locus activation" technique in rice that leverages well-characterized STEs for refined, heritable, and multiplexed gene upregulation. To address the scarcity of potent enhancers, we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts. The in locus integration of these STEs into eight rice genes resulted in substantial transcriptional upregulation in the edited plants, with up to 869.1-fold increases in their transcript levels. Employing a variety of STEs, we achieved delicate control of gene expression, enabling the fine-tuning of key phenotypic traits such as plant height. Our approach also enabled efficient multiplexed gene upregulation, with up to four genes activated simultaneously, significantly enhancing the nicotinamide mononucleotide metabolic pathway. Importantly, heritability studies from the T0 to T3 generations confirmed the stable and heritable nature of STE-driven gene activation. Collectively, our work demonstrates that coupled with STE mining, leveraging genome editing for in locus activation and gene upregulation holds great promise to be widely adopted in fundamental plant research and crop breeding., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Our study highlights the discovery of recurrent copy number alterations in noncoding regions, specifically blood enhancer cluster (BENC-CNA), in B-precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. We demonstrate that BENC-CNA acts as a super-enhancer, driving MYC expression and possibly contributing to the immortalization and proliferative advantage of BCP-ALL cells in vitro., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

While understanding the genetic underpinnings of osteogenesis has far-reaching implications for skeletal diseases and evolution, a comprehensive characterization of the osteoblastic regulatory landscape in non-mammalian vertebrates is still lacking. Here, we compared the ATAC-Seq profile of Xenopus tropicalis (Xt) osteoblasts to a variety of non mineralizing control tissues, and identified osteoblast-specific nucleosome free regions (NFRs) at 527 promoters and 6747 distal regions. Sequence analyses, Gene Ontology, RNA-Seq and ChIP-Seq against four key histone marks confirmed that the distal regions correspond to bona fide osteogenic transcriptional enhancers exhibiting a shared regulatory logic with mammals. We report 425 regulatory regions conserved with human and globally associated to skeletogenic genes. Of these, 35 regions have been shown to impact human skeletal phenotypes by GWAS, including one trps1 enhancer and the runx2 promoter, two genes which are respectively involved in trichorhinophalangeal syndrome type I and cleidocranial dysplasia. Intriguingly, 60 osteoblastic NFRs also align to the genome of the elephant shark, a species lacking osteoblasts and bone tissue. To tackle this paradox, we chose to focus on dlx5 because its conserved promoter, known to integrate regulatory inputs during mammalian osteogenesis, harbours an osteoblast-specific NFR in both frog and human. Hence, we show that dlx5 is expressed in Xt and elephant shark odontoblasts, supporting a common cellular and genetic origin of bone and dentine. Taken together, our work (i) unravels the Xt osteogenic regulatory landscape, (ii) illustrates how cross-species comparisons harvest data relevant to human biology and (iii) reveals that a set of genes including bnc2, dlx5, ebf3, mir199a, nfia, runx2 and zfhx4 drove the development of a primitive form of mineralized skeletal tissue deep in the vertebrate lineage., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutate histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleash cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupts the deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal levels of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs., (© 2024. The Author(s).)

The senescent microenvironment and aged cells per se contribute to tissue remodeling, chronic inflammation, and age-associated dysfunction. However, the metabolic and epigenomic bases of the senescence-associated secretory phenotype (SASP) remain largely unknown. Here, we show that ATP-citrate lyase (ACLY), a key enzyme in acetyl-coenzyme A (CoA) synthesis, is essential for the pro-inflammatory SASP, independent of persistent growth arrest in senescent cells. Citrate-derived acetyl-CoA facilitates the action of SASP gene enhancers. ACLY-dependent de novo enhancers augment the recruitment of the chromatin reader BRD4, which causes SASP activation. Consistently, specific inhibitions of the ACLY-BRD4 axis suppress the STAT1-mediated interferon response, creating the pro-inflammatory microenvironment in senescent cells and tissues. Our results demonstrate that ACLY-dependent citrate metabolism represents a selective target for controlling SASP designed to promote healthy aging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, ∼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

During the progression of proliferative vitreoretinopathy (PVR) following ocular trauma, previously quiescent retinal pigment epithelial (RPE) cells transition into a state of rapid proliferation, migration, and secretion. The elusive molecular mechanisms behind these changes have hindered the development of effective pharmacological treatments, presenting a pressing clinical challenge. In this study, by monitoring the dynamic changes in chromatin accessibility and various histone modifications, we chart the comprehensive epigenetic landscape of RPE cells in male mice subjected to traumatic PVR. Coupled with transcriptomic analysis, we reveal a robust correlation between enhancer activation and the upregulation of the PVR-associated gene programs. Furthermore, by constructing transcription factor regulatory networks, we identify the aberrant activation of enhancer-driven RANK-NFATc1 pathway as PVR advanced. Importantly, we demonstrate that intraocular interventions, including nanomedicines inhibiting enhancer activity, gene therapies targeting NFATc1 and antibody therapeutics against RANK pathway, effectively mitigate PVR progression. Together, our findings elucidate the epigenetic basis underlying the activation of PVR-associated genes during RPE cell fate transitions and offer promising therapeutic avenues targeting epigenetic modulation and the RANK-NFATc1 axis for PVR management., (© 2024. The Author(s).)

The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow some physical crosstalk across regulatory domains, which is at odds with the specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites cooperatively foster the robust insulation of regulatory domains. By genetically dissecting a couple of representative loci in mouse embryonic stem cells, we show that CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), while developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the insulation of regulatory domains, which in turn might help interpreting the pathological consequences of certain structural variants., (© 2024. The Author(s).)