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The recognition of core promoter sequences by TFIID is the first step in RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is a trilobular complex, composed of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs). Why and how TAFs are necessary for the formation of TFIID domains and how they contribute to transcription initiation remain unclear. Inducible TAF7 or TAF10 depletion, followed by comprehensive analysis of TFIID subcomplex formation, chromatin binding, and nascent transcription in mouse embryonic stem cells, result in the formation of a TAF7-lacking TFIID or a minimal core-TFIID complex, respectively. These partial complexes support TBP recruitment at promoters and nascent Pol II transcription at most genes early after depletion, but importantly, TAF10 is necessary for efficient Pol II pausing. We show that partially assembled TFIID complexes can sustain Pol II transcription initiation but cannot replace holo-TFIID over several cell divisions and/or development., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aging is the main risk factor of cognitive neurodegenerative diseases such as Alzheimer's disease, with epigenome alterations as a contributing factor. Here, we compared transcriptomic/epigenomic changes in the hippocampus, modified by aging and by tauopathy, an AD-related feature. We show that the cholesterol biosynthesis pathway is severely impaired in hippocampal neurons of tauopathic but not of aged mice pointing to vulnerability of these neurons in the disease. At the epigenomic level, histone hyperacetylation was observed at neuronal enhancers associated with glutamatergic regulations only in the tauopathy. Lastly, a treatment of tau mice with the CSP-TTK21 epi-drug that restored expression of key cholesterol biosynthesis genes counteracted hyperacetylation at neuronal enhancers and restored object memory. As acetyl-CoA is the primary substrate of both pathways, these data suggest that the rate of the cholesterol biosynthesis in hippocampal neurons may trigger epigenetic-driven changes, that may compromise the functions of hippocampal neurons in pathological conditions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Super-enhancers (SEs) are stretches of enhancers ensuring a high level of expression of key genes associated with cell function. The identification of cancer-specific SE-driven genes is a powerful means for the development of innovative therapeutic strategies. Here, we identify a MITF/SOX10/TFIIH-dependent SE promoting the expression of BAHCC1 in a broad panel of melanoma cells. BAHCC1 is highly expressed in metastatic melanoma and is required for tumor engraftment, growth, and dissemination. Integrative genomics analyses reveal that BAHCC1 is a transcriptional regulator controlling expression of E2F/KLF-dependent cell-cycle and DNA-repair genes. BAHCC1 associates with BRG1-containing remodeling complexes at the promoters of these genes. BAHCC1 silencing leads to decreased cell proliferation and delayed DNA repair. Consequently, BAHCC1 deficiency cooperates with PARP inhibition to induce melanoma cell death. Our study identifies BAHCC1 as an SE-driven gene expressed in melanoma and demonstrates how its inhibition can be exploited as a therapeutic target., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5'-methylcytosine., Methods: Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments., Results: Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity., Conclusions: Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions., (© 2023. The Author(s).)

Early-life exposure to high-fat diets (HF) can program metabolic and cognitive alterations in adult offspring. Although the hippocampus plays a crucial role in memory and metabolic homeostasis, few studies have reported the impact of maternal HF on this structure. We assessed the effects of maternal HF during lactation on physiological, metabolic, and cognitive parameters in young adult offspring mice. To identify early-programming mechanisms in the hippocampus, we developed a multi-omics strategy in male and female offspring. Maternal HF induced a transient increased body weight at weaning, and a mild glucose intolerance only in 3-month-old male mice with no change in plasma metabolic parameters in adult male and female offspring. Behavioral alterations revealed by a Barnes maze test were observed both in 6-month-old male and female mice. The multi-omics strategy unveiled sex-specific transcriptomic and proteomic modifications in the hippocampus of adult offspring. These studies that were confirmed by regulon analysis show that, although genes whose expression was modified by maternal HF were different between sexes, the main pathways affected were similar with mitochondria and synapses as main hippocampal targets of maternal HF. The effects of maternal HF reported here may help to better characterize sex-dependent molecular pathways involved in cognitive disorders and neurodegenerative diseases.

Cytoplasmic mislocalization of the nuclear Fused in Sarcoma (FUS) protein is associated to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS accumulation is recapitulated in the frontal cortex and spinal cord of heterozygous Fus ∆NLS/+ mice. Yet, the mechanisms linking FUS mislocalization to hippocampal function and memory formation are still not characterized. Herein, we show that in these mice, the hippocampus paradoxically displays nuclear FUS accumulation. Multi-omic analyses showed that FUS binds to a set of genes characterized by the presence of an ETS/ELK-binding motifs, and involved in RNA metabolism, transcription, ribosome/mitochondria and chromatin organization. Importantly, hippocampal nuclei showed a decompaction of the neuronal chromatin at highly expressed genes and an inappropriate transcriptomic response was observed after spatial training of Fus ∆NLS/+ mice. Furthermore, these mice lacked precision in a hippocampal-dependent spatial memory task and displayed decreased dendritic spine density. These studies shows that mutated FUS affects epigenetic regulation of the chromatin landscape in hippocampal neurons, which could participate in FTD/ALS pathogenic events. These data call for further investigation in the neurological phenotype of FUS-related diseases and open therapeutic strategies towards epigenetic drugs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Mitotic entry correlates with the condensation of the chromosomes, changes in histone modifications, exclusion of transcription factors from DNA, and the broad downregulation of transcription. However, whether mitotic condensation influences transcription in the subsequent interphase is unknown. Here, we show that preventing one chromosome to condense during mitosis causes it to fail resetting of transcription. Rather, in the following interphase, the affected chromosome contains unusually high levels of the transcription machinery, resulting in abnormally high expression levels of genes in cis , including various transcription factors. This subsequently causes the activation of inducible transcriptional programs in trans , such as the GAL genes, even in the absence of the relevant stimuli. Thus, mitotic chromosome condensation exerts stringent control on interphase gene expression to ensure the maintenance of basic cellular functions and cell identity across cell divisions. Together, our study identifies the maintenance of transcriptional homeostasis during interphase as an unexpected function of mitosis and mitotic chromosome condensation.

Background: Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder that primarily affects the cerebellum and retina. SCA7 is caused by a polyglutamine expansion in the ATXN7 protein, a subunit of the transcriptional coactivator SAGA that acetylates histone H3 to deposit narrow H3K9ac mark at DNA regulatory elements of active genes. Defective histone acetylation has been presented as a possible cause for gene deregulation in SCA7 mouse models. However, the topography of acetylation defects at the whole genome level and its relationship to changes in gene expression remain to be determined., Methods: We performed deep RNA-sequencing and chromatin immunoprecipitation coupled to high-throughput sequencing to examine the genome-wide correlation between gene deregulation and alteration of the active transcription marks, e.g. SAGA-related H3K9ac, CBP-related H3K27ac and RNA polymerase II (RNAPII), in a SCA7 mouse retinopathy model., Results: Our analyses revealed that active transcription marks are reduced at most gene promoters in SCA7 retina, while a limited number of genes show changes in expression. We found that SCA7 retinopathy is caused by preferential downregulation of hundreds of highly expressed genes that define morphological and physiological identities of mature photoreceptors. We further uncovered that these photoreceptor genes harbor unusually broad H3K9ac profiles spanning the entire gene bodies and have a low RNAPII pausing. This broad H3K9ac signature co-occurs with other features that delineate superenhancers, including broad H3K27ac, binding sites for photoreceptor specific transcription factors and expression of enhancer-related non-coding RNAs (eRNAs). In SCA7 retina, downregulated photoreceptor genes show decreased H3K9 and H3K27 acetylation and eRNA expression as well as increased RNAPII pausing, suggesting that superenhancer-related features are altered., Conclusions: Our study thus provides evidence that distinctive epigenetic configurations underlying high expression of cell-type specific genes are preferentially impaired in SCA7, resulting in a defect in the maintenance of identity features of mature photoreceptors. Our results also suggest that continuous SAGA-driven acetylation plays a role in preserving post-mitotic neuronal identity., (© 2022. The Author(s).)

Cisplatin is a potent chemotherapeutic drug that is widely used in the treatment of various solid cancers. However, its clinical effectiveness is strongly limited by frequent severe adverse effects, in particular nephrotoxicity and chemotherapy-induced peripheral neuropathy. Thus, there is an urgent medical need to identify novel strategies that limit cisplatin-induced toxicity. In the present study, we show that the FDA-approved adenosine A2A receptor antagonist istradefylline (KW6002) protected from cisplatin-induced nephrotoxicity and neuropathic pain in mice with or without tumors. Moreover, we also demonstrate that the antitumoral properties of cisplatin were not altered by istradefylline in tumor-bearing mice and could even be potentiated. Altogether, our results support the use of istradefylline as a valuable preventive approach for the clinical management of patients undergoing cisplatin treatment.

Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. In the mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying their protective effect is unknown. Here, we show that TRIM28 is required to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of Trim28, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type, different from gonadal embryonic progenitors. TRIM28 is recruited on chromatin in the proximity of FOXL2 to maintain the ovarian pathway and to repress testicular-specific genes. The role of TRIM28 in ovarian maintenance depends on its E3-SUMO ligase activity that regulates the sex-specific SUMOylation profile of ovarian-specific genes. Our study identifies TRIM28 as a key factor in protecting the adult ovary from the testicular pathway., (© 2022. The Author(s).)

The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Caffeine is the most widely consumed psychoactive substance in the world. Strikingly, the molecular pathways engaged by its regular consumption remain unclear. We herein addressed the mechanisms associated with habitual (chronic) caffeine consumption in the mouse hippocampus using untargeted orthogonal omics techniques. Our results revealed that chronic caffeine exerts concerted pleiotropic effects in the hippocampus at the epigenomic, proteomic, and metabolomic levels. Caffeine lowered metabolism-related processes (e.g., at the level of metabolomics and gene expression) in bulk tissue, while it induced neuron-specific epigenetic changes at synaptic transmission/plasticity-related genes and increased experience-driven transcriptional activity. Altogether, these findings suggest that regular caffeine intake improves the signal-to-noise ratio during information encoding, in part through fine-tuning of metabolic genes, while boosting the salience of information processing during learning in neuronal circuits.

Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, the mechanisms that orchestrate the global and dramatic transcriptional changes leading to adipocyte differentiation remain to be fully unraveled. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing endogenous sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like PPARγ/RXR and their co-factors are associated with the transcription of adipogenic genes. Finally, using RXR as a model, we found that sumoylation may regulate adipogenic transcription by supporting the chromatin occurrence of transcription factors. Our data demonstrate that the sumoylation pathway supports the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides the scientists in the field of cellular differentiation and development with an in-depth resource of the dynamics of the SUMO-chromatin landscape, SUMO-regulated transcription and endogenous sumoylation sites during adipocyte differentiation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Cova et al.)

EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations., (© 2021. The Author(s).)

The Rett syndrome protein MeCP2 was described as a methyl-CpG-binding protein, but its exact function remains unknown. Here we show that mouse MeCP2 is a microsatellite binding protein that specifically recognizes hydroxymethylated CA repeats. Depletion of MeCP2 alters chromatin organization of CA repeats and lamina-associated domains and results in nucleosome accumulation on CA repeats and genome-wide transcriptional dysregulation. The structure of MeCP2 in complex with a hydroxymethylated CA repeat reveals a characteristic DNA shape, with considerably modified geometry at the 5-hydroxymethylcytosine, which is recognized specifically by Arg 133 , a key residue whose mutation causes Rett syndrome. Our work identifies MeCP2 as a microsatellite DNA binding protein that targets the 5hmC-modified CA-rich strand and maintains genome regions nucleosome-free, suggesting a role for MeCP2 dysfunction in Rett syndrome., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Epigenetic modifications and nucleosome positioning play an important role in modulating gene expression. However, how the patterns of epigenetic modifications and nucleosome positioning are established around promoters is not well understood. Here, we have addressed these questions in a series of genome-wide experiments coupled to a novel bioinformatic analysis approach. Our data reveal a clear correlation between CpG density, promoter activity and accumulation of active or repressive histone marks. CGI boundaries define the chromatin promoter regions that will be epigenetically modified. CpG-rich promoters are targeted by histone modifications and histone variants, while CpG-poor promoters are regulated by DNA methylation. CGIs boundaries, but not transcriptional activity, are essential determinants of H2A.Z positioning in vicinity of the promoters, suggesting that the presence of H2A.Z is not related to transcriptional control. Accordingly, H2A.Z depletion has no impact on gene expression of arrested mouse embryonic fibroblasts. Therefore, the underlying DNA sequence, the promoter CpG density and, to a lesser extent, transcriptional activity, are key factors implicated in promoter chromatin architecture., Competing Interests: Declarations of Interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Histone post-translational modifications (PTMs) are key players in chromatin regulation. The identification of novel histone acylations raises important questions regarding their role in transcription. In this study, we characterize the role of an acylation on the lateral surface of the histone octamer, H3K122 succinylation (H3K122succ), in chromatin function and transcription. Using chromatin succinylated at H3K122 in in vitro transcription assays, we show that the presence of H3K122succ is sufficient to stimulate transcription. In line with this, we found in our ChIP assays H3K122succ enriched on promoters of active genes and H3K122succ enrichment scaling with gene expression levels. Furthermore, we show that the co-activators p300/CBP can succinylate H3K122 and identify sirtuin 5 (SIRT5) as a new desuccinylase. By applying single molecule FRET assays, we demonstrate a direct effect of H3K122succ on nucleosome stability, indicating an important role for histone succinylation in modulating chromatin dynamics. Together, these data provide the first insights into the mechanisms underlying transcriptional regulation by H3K122succ., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Polycystic ovary syndrome (PCOS) is the most common reproductive and metabolic disorder affecting women of reproductive age. PCOS has a strong heritable component, but its pathogenesis has been unclear. Here, we performed RNA sequencing and genome-wide DNA methylation profiling of ovarian tissue from control and third-generation PCOS-like mice. We found that DNA hypomethylation regulates key genes associated with PCOS and that several of the differentially methylated genes are also altered in blood samples from women with PCOS compared with healthy controls. Based on this insight, we treated the PCOS mouse model with the methyl group donor S-adenosylmethionine and found that it corrected their transcriptomic, neuroendocrine, and metabolic defects. These findings show that the transmission of PCOS traits to future generations occurs via an altered landscape of DNA methylation and propose methylome markers as a possible diagnostic landmark for the condition, while also identifying potential candidates for epigenetic-based therapy., Competing Interests: Declaration of interests P.G., N.E.H.M., I.P., A.-L.B., and V.P. disclose that they are inventors of a submitted patent application by the INSERM (Institut National de la Santé et de la Recherche Médicale) covering methods and kits for diagnostic and treatment of PCOS. All other authors do not have competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The IKZF1 gene, which encodes the Ikaros transcription factor, is frequently deleted or mutated in patients with B-cell precursor acute lymphoblastic leukemias that express oncogenes, like BCR-ABL, which activate the JAK-STAT5 pathway. Ikaros functionally antagonizes the transcriptional programs downstream of IL-7/STAT5 during B cell development, as well as STAT5 activity in leukemic cells. However, the mechanisms by which Ikaros interferes with STAT5 function is unknown. We studied the genomic distribution of Ikaros and STAT5 on chromatin in a murine pre-B cell line, and found that both proteins colocalize on >60% of STAT5 target regions. Strikingly, Ikaros activity leads to widespread loss of STAT5 binding at most of its genomic targets within two hours of Ikaros induction, suggesting a direct mechanism. Ikaros did not alter the level of total or phosphorylated STAT5 proteins, nor did it associate with STAT5. Using sequences from the Cish, Socs2 and Bcl6 genes that Ikaros and STAT5 target, we show that both proteins bind overlapping sequences at GGAA motifs. Our results demonstrate that Ikaros antagonizes STAT5 DNA binding, in part by competing for common target sequences. Our study has implications for understanding the functions of Ikaros and STAT5 in B cell development and transformation., Competing Interests: The authors have declared that no competing interests exist.

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.

The role of brain cell-type-specific functions and profiles in pathological and non-pathological contexts is still poorly defined. Such cell-type-specific gene expression profiles in solid, adult tissues would benefit from approaches that avoid cellular stress during isolation. Here, we developed such an approach and identified highly selective transcriptomic signatures in adult mouse striatal direct and indirect spiny projection neurons, astrocytes, and microglia. Integrating transcriptomic and epigenetic data, we obtained a comprehensive model for cell-type-specific regulation of gene expression in the mouse striatum. A cross-analysis with transcriptomic and epigenomic data generated from mouse and human Huntington's disease (HD) brains shows that opposite epigenetic mechanisms govern the transcriptional regulation of striatal neurons and glial cells and may contribute to pathogenic and compensatory mechanisms. Overall, these data validate this less stressful method for the investigation of cellular specificity in the adult mouse brain and demonstrate the potential of integrative studies using multiple databases., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chromatin acetylation, a critical regulator of synaptic plasticity and memory processes, is thought to be altered in neurodegenerative diseases. Here, we demonstrate that spatial memory and plasticity (LTD, dendritic spine formation) deficits can be restored in a mouse model of tauopathy following treatment with CSP-TTK21, a small-molecule activator of CBP/p300 histone acetyltransferases (HAT). At the transcriptional level, CSP-TTK21 re-established half of the hippocampal transcriptome in learning mice, likely through increased expression of neuronal activity genes and memory enhancers. At the epigenomic level, the hippocampus of tauopathic mice showed a significant decrease in H2B but not H3K27 acetylation levels, both marks co-localizing at TSS and CBP enhancers. Importantly, CSP-TTK21 treatment increased H2B acetylation levels at decreased peaks, CBP enhancers, and TSS, including genes associated with plasticity and neuronal functions, overall providing a 95% rescue of the H2B acetylome in tauopathic mice. This study is the first to provide in vivo proof-of-concept evidence that CBP/p300 HAT activation efficiently reverses epigenetic, transcriptional, synaptic plasticity, and behavioral deficits associated with Alzheimer's disease lesions in mice., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Prenatal exposure to androgens during brain development in male individuals may participate to increase their susceptibility to develop neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability. However, little is known about the action of androgens in human neural cells., Methods: We used human neural stem cells differentiated from embryonic stem cells to investigate targets of androgens., Results: RNA sequencing revealed that treatment with dihydrotestosterone (DHT) leads to subtle but significant changes in the expression of about 200 genes, encoding proteins of extracellular matrix or involved in signal transduction of growth factors (e.g., insulin/insulin growth factor 1). We showed that the most differentially expressed genes (DEGs), RGCC, RNF144B, NRCAM, TRIM22, FAM107A, IGFBP5, and LAMA2, are reproducibly regulated by different androgens in different genetic backgrounds. We showed, by overexpressing the androgen receptor in neuroblastoma cells SH-SY5Y or knocking it down in human neural stem cells, that this regulation involves the androgen receptor. A chromatin immunoprecipitation combined with direct sequencing analysis identified androgen receptor-bound sequences in nearly half of the DHT-DEGs and in numerous other genes. DHT-DEGs appear enriched in genes involved in ASD (ASXL3, NLGN4X, etc.), associated with ASD (NRCAM), or differentially expressed in patients with ASD (FAM107A, IGFBP5). Androgens increase human neural stem cell proliferation and survival in nutrient-deprived culture conditions, with no detectable effect on regulation of neurite outgrowth., Conclusions: We characterized androgen action in neural progenitor cells, identifying DHT-DEGs that appear to be enriched in genes related to ASD. We also showed that androgens increase proliferation of neuronal precursors and protect them from death during their differentiation in nutrient-deprived conditions., (Copyright © 2018 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Histones are highly covalently modified, but the functions of many of these modifications remain unknown. In particular, it is unclear how histone marks are coupled to cellular metabolism and how this coupling affects chromatin architecture. We identified histone H3 Lys14 (H3K14) as a site of propionylation and butyrylation in vivo and carried out the first systematic characterization of histone propionylation. We found that H3K14pr and H3K14bu are deposited by histone acetyltransferases, are preferentially enriched at promoters of active genes and are recognized by acylation-state-specific reader proteins. In agreement with these findings, propionyl-CoA was able to stimulate transcription in an in vitro transcription system. Notably, genome-wide H3 acylation profiles were redefined following changes to the metabolic state, and deletion of the metabolic enzyme propionyl-CoA carboxylase altered global histone propionylation levels. We propose that histone propionylation, acetylation and butyrylation may act in combination to promote high transcriptional output and to couple cellular metabolism with chromatin structure and function.

Fragile-X syndrome (FXS) is a frequent genetic form of intellectual disability (ID). The main recurrent mutagenic mechanism causing FXS is the expansion of a CGG repeat sequence in the 5'-UTR of the FMR1 gene, therefore, routinely tested in ID patients. We report here three FMR1 intragenic pathogenic variants not affecting this sequence, identified using high-throughput sequencing (HTS): a previously reported hemizygous deletion encompassing the last exon of FMR1, too small to be detected by array-CGH and inducing decreased expression of a truncated form of FMRP protein, in three brothers with ID (family 1) and two splice variants in boys with sporadic ID: a de novo variant c.990+1G>A (family 2) and a maternally inherited c.420-8A>G variant (family 3). After clinical reevaluation, the five patients presented features consistent with FXS (mean Hagerman's scores=15). We conducted a systematic review of all rare non-synonymous variants previously reported in FMR1 in ID patients and showed that six of them are convincing pathogenic variants. This study suggests that intragenic FMR1 variants, although much less frequent than CGG expansions, are a significant mutational mechanism leading to FXS and demonstrates the interest of HTS approaches to detect them in ID patients with a negative standard work-up.

Epigenetic and transcriptional alterations are both implicated in Huntington's disease (HD), a progressive neurodegenerative disease resulting in degeneration of striatal neurons in the brain. However, how impaired epigenetic regulation leads to transcriptional dysregulation in HD is unclear. Here, we investigated enhancer RNAs (eRNAs), a class of long non-coding RNAs transcribed from active enhancers. We found that eRNAs are expressed from many enhancers of mouse striatum and showed that a subset of those eRNAs are deregulated in HD vs control mouse striatum. Enhancer regions producing eRNAs decreased in HD mouse striatum were associated with genes involved in striatal neuron identity. Consistently, they were enriched in striatal super-enhancers. Moreover, decreased eRNA expression in HD mouse striatum correlated with down-regulation of associated genes. Additionally, a significant number of RNA Polymerase II (RNAPII) binding sites were lost within enhancers associated with decreased eRNAs in HD vs control mouse striatum. Together, this indicates that loss of RNAPII at HD mouse enhancers contributes to reduced transcription of eRNAs, resulting in down-regulation of target genes. Thus, our data support the view that eRNA dysregulation in HD striatum is a key mechanism leading to altered transcription of striatal neuron identity genes, through reduced recruitment of RNAPII at super-enhancers.

The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation.

The tripartite motif (TRIM) family of proteins plays important roles in innate immunity and antimicrobial infection. None of these proteins has been shown to directly regulate transcription of genes in monocyte/macrophage except TRIM33 that we have recently shown to be a macrophage specific transcriptional inhibitor of Ifnb1. Using ChIP-seq analyses, we now report that TRIM33 is bound to two fold more genes in immature than in mature myeloid cell lines. When located near the same genes, TRIM33 is bound to different sequences in the two cell lines suggesting a role of TRIM33 in both immature and mature myeloid cells. Accordingly, expression of TRIM33 in immature myeloid cells is necessary for efficient production of small peritoneal macrophages, monocytes and bone marrow derived macrophage (BMDM) and TRIM33 targets a subset of genes involved in the inflammatory response only in mature myeloid cells. Functionally, this targeting is associated with impaired repression of pathways regulating the late phases of lipopolysaccharide (LPS) activation of BMDM and a high sensitivity to LPS in vivo when the trim33 gene is inactivated in mature myeloid cells. These findings pinpoint TRIM33 as an important transcriptional actor of monocyte/macrophage mediated inflammation.

Background: Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders., Methods: We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption., Results: We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases., Conclusions: We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease., Trial Registration Numbers: NCT01746121 and NCT02397824., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

T-cell development is accompanied by epigenetic changes that ensure the silencing of stem cell-related genes and the activation of lymphocyte-specific programmes. How transcription factors influence these changes remains unclear. We show that the Ikaros transcription factor forms a complex with Polycomb repressive complex 2 (PRC2) in CD4(-)CD8(-) thymocytes and allows its binding to more than 500 developmentally regulated loci, including those normally activated in haematopoietic stem cells and others induced by the Notch pathway. Loss of Ikaros in CD4(-)CD8(-) cells leads to reduced histone H3 lysine 27 trimethylation and ectopic gene expression. Furthermore, Ikaros binding triggers PRC2 recruitment and Ikaros interacts with PRC2 independently of the nucleosome remodelling and deacetylation complex. Our results identify Ikaros as a fundamental regulator of PRC2 function in developing T cells.

Collier, the single Drosophila COE (Collier/EBF/Olf-1) transcription factor, is required in several developmental processes, including head patterning and specification of muscle and neuron identity during embryogenesis. To identify direct Collier (Col) targets in different cell types, we used ChIP-seq to map Col binding sites throughout the genome, at mid-embryogenesis. In vivo Col binding peaks were associated to 415 potential direct target genes. Gene Ontology analysis revealed a strong enrichment in proteins with DNA binding and/or transcription-regulatory properties. Characterization of a selection of candidates, using transgenic CRM-reporter assays, identified direct Col targets in dorso-lateral somatic muscles and specific neuron types in the central nervous system. These data brought new evidence that Col direct control of the expression of the transcription regulators apterous and eyes-absent (eya) is critical to specifying neuronal identities. They also showed that cross-regulation between col and eya in muscle progenitor cells is required for specification of muscle identity, revealing a new parallel between the myogenic regulatory networks operating in Drosophila and vertebrates. Col regulation of eya, both in specific muscle and neuronal lineages, may illustrate one mechanism behind the evolutionary diversification of Col biological roles.

Mouse embryonic stem cells (ESCs) treated with all-trans retinoic acid differentiate into a homogenous population of glutamatergic neurons. Although differentiation is initiated through activation of target genes by the retinoic acid receptors, the downstream transcription factors specifying neuronal fate are less well characterised. Here, we show that the transcription factor Brn2 (also known as Pou3f2) is essential for the neuronal differentiation programme. By integrating results from RNA-seq following Brn2 silencing with results from Brn2 ChIP-seq, we identify a set of Brn2 target genes required for the neurogenic programme. Further integration of Brn2 ChIP-seq data from retinoic-acid-treated ESCs and P19 cells with data from ESCs differentiated into neuronal precursors by Fgf2 treatment and that from fibroblasts trans-differentiated into neurons by ectopic Brn2 expression showed that Brn2 occupied a distinct but overlapping set of genomic loci in these differing conditions. However, a set of common binding sites and target genes defined the core of the Brn2-regulated neuronal programme, among which was that encoding the transcription factor Zic1. Small hairpin RNA (shRNA)-mediated silencing of Zic1 prevented ESCs from differentiating into neuronal precursors, thus defining a hierarchical Brn2-Zic1 axis that is essential to specify neural fate in retinoic-acid-treated ESCs., (© 2015. Published by The Company of Biologists Ltd.)

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

Background: Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation., Methods: We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases., Results: We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients' clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders., Conclusions: With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

The functions of the TAF subunits of mammalian TFIID in physiological processes remain poorly characterised. In this study, we describe a novel function of TAFs in directing genomic occupancy of a transcriptional activator. Using liver-specific inactivation in mice, we show that the TAF4 subunit of TFIID is required for post-natal hepatocyte maturation. TAF4 promotes pre-initiation complex (PIC) formation at post-natal expressed liver function genes and down-regulates a subset of embryonic expressed genes by increased RNA polymerase II pausing. The TAF4-TAF12 heterodimer interacts directly with HNF4A and in vivo TAF4 is necessary to maintain HNF4A-directed embryonic gene expression at post-natal stages and promotes HNF4A occupancy of functional cis-regulatory elements adjacent to the transcription start sites of post-natal expressed genes. Stable HNF4A occupancy of these regulatory elements requires TAF4-dependent PIC formation highlighting that these are mutually dependent events. Local promoter-proximal HNF4A-TFIID interactions therefore act as instructive signals for post-natal hepatocyte differentiation.

Intellectual disability (ID) is characterized by an extraordinary genetic heterogeneity, with >250 genes that have been implicated in monogenic forms of ID. Because this complexity precluded systematic testing for mutations and because clinical features are often non-specific, for some of these genes only few cases or families have been unambiguously documented. It is the case of the X-linked gene encoding monoamine oxidase A (MAOA), for which only one nonsense mutation has been identified in Brunner syndrome, characterized in a single family by mild non-dysmorphic ID and impulsive, violent and aggressive behaviors. We have performed targeted high-throughput sequencing of 220 genes, including MAOA, in patients with undiagnosed ID. We identified a c.797_798delinsTT (p.C266F) missense mutation in MAOA in a boy with autism spectrum disorder, attention deficit and autoaggressive behavior. Two maternal uncles carry the mutation and have severe ID, with a history of maltreatment in early childhood. This novel missense mutation decreases MAOA enzymatic activity, leading to abnormal levels of urinary monoamines. The identification of this new point mutation confirms, for the first time since 1993, the monogenic implication of the MAOA gene in ID of various degrees, autism and behavioral disturbances. The variable expressivity of the mutation observed in male patients of this family may involve gene-environment interactions, and the identification of a perturbation in monoamine metabolism should be taken into account when prescribing psychoactive drugs in such patients.

The Notch signaling pathway is activated in many cell types, but its effects are cell type- and stage-specific. In the immune system, Notch activity is required for the differentiation of T cell progenitors, but it is reduced in more mature thymocytes, in which Notch is oncogenic. Studies based on single-gene models have suggested that the tumor suppressor protein Ikaros plays an important role in repressing the transcription of Notch target genes. We used genome-wide analyses, including chromatin immunoprecipitation sequencing, to identify genes controlled by Notch and Ikaros in gain- and loss-of-function experiments. We found that Ikaros bound to and directly repressed the expression of most genes that are activated by Notch. Specific deletion of Ikaros in thymocytes led to the persistent expression of Notch target genes that are essential for T cell maturation, as well as the rapid development of T cell leukemias in mice. Expression of Notch target genes that are normally silent in T cells, but are activated by Notch in other cell types, occurred in T cells of mice genetically deficient in Ikaros. We propose that Ikaros shapes the timing and repertoire of the Notch transcriptional response in T cells through widespread targeting of elements adjacent to Notch regulatory sequences. These results provide a molecular framework for understanding the regulation of tissue-specific and tumor-related Notch responses.

Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.

Congenital myopathies are severe muscle disorders affecting adults as well as children in all populations. The diagnosis of congenital myopathies is constrained by strong clinical and genetic heterogeneity. Moreover, the majority of patients present with unspecific histological features, precluding purposive molecular diagnosis and demonstrating the need for an alternative and more efficient diagnostic approach. We used exome sequencing complemented by histological and ultrastructural analysis of muscle biopsies to identify the causative mutations in eight patients with clinically different skeletal muscle pathologies, ranging from a fatal neonatal myopathy to a mild and slowly progressive myopathy with adult onset. We identified RYR1 (ryanodine receptor) mutations in six patients and NEB (nebulin) mutations in two patients. We found novel missense and nonsense mutations, unraveled small insertions/deletions and confirmed their impact on splicing and mRNA/protein stability. Histological and ultrastructural findings of the muscle biopsies of the patients validated the exome sequencing results. We provide the evidence that an integrated strategy combining exome sequencing with clinical and histopathological investigations overcomes the limitations of the individual approaches to allow a fast and efficient diagnosis, accelerating the patient's access to a better healthcare and disease management. This is of particular interest for the diagnosis of congenital myopathies, which involve very large genes like RYR1 and NEB as well as genetic and phenotypic heterogeneity.

How a more plastic chromatin state is maintained and reversed during development is unknown. Heterochromatin-mediated silencing of repetitive elements occurs in differentiated cells. Here, we used repetitive elements, including retrotransposons, as model loci to address how and when heterochromatin forms during development. RNA sequencing throughout early mouse embryogenesis revealed that repetitive-element expression is dynamic and stage specific, with most repetitive elements becoming repressed before implantation. We show that LINE-1 and IAP retrotransposons become reactivated from both parental genomes after fertilization. Chromatin immunoprecipitation for H3K4me3 and H3K9me3 in 2- and 8-cell embryos indicates that their developmental silencing follows loss of activating marks rather than acquisition of conventional heterochromatic marks. Furthermore, short LINE-1 RNAs regulate LINE-1 transcription in vivo. Our data indicate that reprogramming after mammalian fertilization comprises a robust transcriptional activation of retrotransposons and that repetitive elements are initially regulated through RNA.