Search

Background Cellular senescence is a permanent state of replicative arrest defined by a specific pattern of gene expression. The epigenome in senescent cells is sculptured in order to sustain the new transcriptional requirements, particularly at enhancers and super-enhancers. How these distal regulatory elements are dynamically modulated is not completely defined. Results Enhancer regions are defined by the presence of H3K27 acetylation marks, which can be modulated by class IIa HDACs, as part of multi-protein complexes. Here, we explore the regulation of class IIa HDACs in different models of senescence. We find that HDAC4 is polyubiquitylated and degraded during all types of senescence and it selectively binds and monitors H3K27ac levels at specific enhancers and super-enhancers that supervise the senescent transcriptome. Frequently, these HDAC4-modulated elements are also monitored by AP-1/p300. The deletion of HDAC4 in transformed cells which have bypassed oncogene-induced senescence is coupled to the re-appearance of senescence and the execution of the AP-1/p300 epigenetic program. Conclusions Overall, our manuscript highlights a role of HDAC4 as an epigenetic reader and controller of enhancers and super-enhancers that supervise the senescence program. More generally, we unveil an epigenetic checkpoint that has important consequences in aging and cancer.

The lysine acetyltransferases type 3 (KAT3) family members CBP and p300 are important transcriptional co-activators, but their specific functions in adult post-mitotic neurons remain unclear. Here, we show that the combined elimination of both proteins in forebrain excitatory neurons of adult mice resulted in a rapidly progressing neurological phenotype associated with severe ataxia, dendritic retraction and reduced electrical activity. At the molecular level, we observed the downregulation of neuronal genes, as well as decreased H3K27 acetylation and pro-neural transcription factor binding at the promoters and enhancers of canonical neuronal genes. The combined deletion of CBP and p300 in hippocampal neurons resulted in the rapid loss of neuronal molecular identity without de- or transdifferentiation. Restoring CBP expression or lysine acetylation rescued neuronal-specific transcription in cultured neurons. Together, these experiments show that KAT3 proteins maintain the excitatory neuron identity through the regulation of histone acetylation at cell type-specific promoter and enhancer regions., M.L. is recipient of a Santiago Grisolia fellowship given by the Generalitat Valenciana, J.M.C. is recipient of a fellowship from the Spanish Ministry of Education, Culture and Sport (MECD), J.F.-A. and C.M.N. are recipients of fellowships from the Spanish Ministry of Science and Innovation (MICINN). The ultrastructure research was supported by the Polish National Science Center Grant UMO-2014/15/N/NZ3/04468 and by the European Regional Development Fund POIG 01.01.02-00-008/08. J.P.L.-A. research is supported by Grants RYC-2015-18056 and RTI2018-102260-B-I00 from MICINN co-financed by ERDF. A.B. research is supported by Grants SAF2017-87928-R, PCIN-2015-192-C02-01, and SEV-2017-0723 from MICINN co-financed by ERDF, PROMETEO/2016/026 from the Generalitat Valenciana, and RGP0039/2017 from the Human Frontiers Science Program Organization (HFSPO). The Instituto de Neurociencias is a “Centre of Excellence Severo Ochoa”.

Regulation of RNA polymerase II (Pol2) elongation in the promoter-proximal region is an important and ubiquitous control point for gene expression in metazoans. We report that transcription of the adenovirus 5 E4 region is regulated during the release of paused Pol2 into productive elongation by recruitment of the super-elongation complex, dependent on promoter H3K18/27 acetylation by CBP/p300. We also establish that this is a general transcriptional regulatory mechanism that applies to ~7% of expressed protein-coding genes in primary human airway epithelial cells. We observed that a homeostatic mechanism maintains promoter, but not enhancer, H3K18/27ac in response to extensive inhibition of CBP/p300 acetyl transferase activity by the highly specific small molecule inhibitor A-485. Further, our results suggest a function for BRD4 association at enhancers in regulating paused Pol2 release at nearby promoters. Taken together, our results uncover the processes regulating transcriptional elongation by promoter region histone H3 acetylation and homeostatic maintenance of promoter, but not enhancer, H3K18/27ac in response to inhibition of CBP/p300 acetyl transferase activity.

Cardiovascular disease (CVD) is the biggest cause of mortality and morbidity worldwide, with 80% of the deaths from CVD due to myocardial infarction. Extensive cellular, tissue, and organ remodeling occurs through CVD initiation and progression, which is mediated by stable alterations in gene expression. Covalent modifications on histone proteins regulate chromatin structure and change the accessibility of gene promoter and enhancer regions to transcriptional factors. In this manner, histone modifications and modifiers act to dynamically tune transcription. Histone methylation is generally repressive, whereas acetylation promotes gene expression but the effect of each histone modification is highly context-specific. Dynamic changes in histone modification profiles have been described in cardi0myocyte hypertrophy, heart failure, and vascular disease, with experimental studies revealing these changes as necessary for the initiation or development of CVD. Moreover, preventing these disease-associated changes to histone marks, by modulating histone modifiers, can attenuate or aggravate the CVD phenotype.

Gene expression programs determine cell fate in embryonic development and their dysregulation results in disease. Transcription factors (TFs) control gene expression by binding to enhancers, but how TFs select and activate their target enhancers is still unclear. HOX TFs share conserved homeodomains with highly similar sequence recognition properties, yet they impart the identity of different animal body parts. To understand how HOX TFs control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. HOXA2 and HOXA3 directly cooperate with TALE TFs and selectively target different subsets of a broad TALE chromatin platform. Binding of HOX and tissue-specific TFs convert low affinity TALE binding into high confidence, tissue-specific binding events, which bear the mark of active enhancers. We propose that HOX paralogs, alone and in combination with tissue-specific TFs, generate tissue-specific transcriptional outputs by modulating the activity of TALE TFs at selected enhancers., Author summary The different anatomical features that develop along the animal body axis are determined by HOX transcription factors. Each member of the HOX family appears in a well-defined territory of the developing embryo, and instruct a specific gene expression program. In stark contrast to their specificity in vivo, which is central to developmental programs, HOX transcription factors display highly similar sequence recognition properties in vitro. To understand how HOX transcription factors control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. We find that HOXA2 and HOXA3 occupy a large set of high-confidence, non-overlapping genomic regions, that are also bound by three aminoacid loop extension (TALE) transcription factors. We identify three main determinants of HOX paralog-selective binding: recognition of unique variants of the HOX-PBX motif, differential affinity at shared HOX-PBX motifs and, additionally, contribution of tissue-specific transcription factors. These mechanisms result in high-confidence, cooperative HOX-TALE binding at different genomic locations, which bear the mark of active enhancers. We propose that HOX paralogs operate, alone and in concert with tissue-specific transcription factors, to switch on TALE function at selected enhancers.

SummaryRegulation of RNA Polymerase II (Pol2) elongation in the promoter proximal region is an important and ubiquitous control point for gene expression in metazoan cells. We report that transcription of the adenovirus 5 E4 region is regulated during the release of paused Pol2 into productive elongation by recruitment of the super elongation complex (SEC), dependent on promoter H3K18/27 acetylation by CBP/p300. We also establish that this is a general transcriptional regulatory mechanism for ∼6% of genes expressed with FPKM>1 in primary human airway epithelial cells. We observed that a homeostatic mechanism maintains promoter, but not enhancer H3K18/27ac in response to extensive inhibition of CBP/p300 acetyl transferase activity by the highly specific small molecule inhibitor A-485. Further, our results suggest a function for BRD4 association at enhancers in regulating paused Pol2 release at nearby promoters. Taken together, our results uncover processes regulating transcriptional elongation by promoter region histone H3 acetylation and homeostatic maintenance of promoter, but not enhancer, H3K18/27ac in response to inhibition of CBP/p300 acetyl transferase activity.

Cooperation between DNA, RNA and protein regulates gene expression and controls differentiation through interactions that connect regions of nucleic acids and protein domains and through the assembly of biomolecular condensates. Here, we report that endoderm differentiation is regulated by the interaction between the long non-coding RNA (lncRNA) DIGIT and the bromodomain and extraterminal domain protein BRD3. BRD3 forms phase-separated condensates of which the formation is promoted by DIGIT, occupies enhancers of endoderm transcription factors and is required for endoderm differentiation. BRD3 binds to histone H3 acetylated at lysine 18 (H3K18ac) in vitro and co-occupies the genome with H3K18ac. DIGIT is also enriched in regions of H3K18ac, and the depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings show that cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest that protein-lncRNA phase-separated condensates have a broader role as regulators of transcription.

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements., Super-enhancer are usually defined by high levels of chromatin modification and associate with cell-specific gene expression. Here, the authors define hyperacetylated chromatin domains (HCDs) by using histone hyperacetylation peak breadth information and show that HCDs associated more closely with cell identity than super-enhancers.

The closely related acetyltransferases CBP and p300 are key regulators of gene expression in metazoans. CBP/p300 acetylate several specific lysine residues within nucleosomes, including histone H3 lysine 27 (H3K27), a hallmark of active enhancers and promoters. However, it has remained largely unclear how specificity of CBP/p300 towards H3K27 is achieved. Here we show that the TAZ2 domain of CBP is required for efficient acetylation of H3K27, while curbing activity towards other lysine residues within nucleosomes. We find that TAZ2 is a sequence-independent DNA binding module, promoting interaction between CBP and nucleosomes, thereby enhancing enzymatic activity and regulating substrate specificity of CBP. TAZ2 is further required to stabilize CBP binding to chromatin in mouse embryonic stem cells, facilitating specificity towards H3K27 and modulating gene expression. These findings reveal a crucial role of TAZ2 in regulating H3K27ac, while highlighting the importance of correct site-specific acetylation for proper regulation of gene expression.

The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers, which are approximately 5,000 basepairs apart, exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of the observed enhancer cooperativity. We generated genetic deletions of individual estrogen receptor α (ER) bound enhancers and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that a cluster of two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences between enhancers, we found that the genomic location in which a full ERE resides strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing active chromatin environment within an enhancer cluster is also needed in order for estrogen-driven gene regulation to occur.

Hormone-dependent activation of enhancers includes histone hyperacetylation and mediator recruitment. Histone hyperacetylation is mostly explained by a bimodal switch model, where histone deacetylases (HDACs) disassociate from chromatin, and histone acetyl transferases (HATs) are recruited. This model builds on decades of research on steroid receptor regulation of transcription. Yet, the general concept of the bimodal switch model has not been rigorously tested genome wide. We have used a genomics approach to study enhancer hyperacetylation by the thyroid hormone receptor (TR), described to operate as a bimodal switch. H3 acetylation, HAT and HDAC ChIP-seq analyses of livers from hypo- and hyperthyroid wildtype, TR deficient and NCOR1 disrupted mice reveal three types of thyroid hormone (T3)-regulated enhancers. One subset of enhancers is bound by HDAC3-NCOR1 in the absence of hormone and constitutively occupy TR and HATs irrespective of T3 levels, suggesting a poised enhancer state in absence of hormone. In presence of T3, HDAC3-NCOR1 dissociates from these enhancers leading to histone hyperacetylation, suggesting a histone acetylation rheostat function of HDACs at poised enhancers. Another subset of enhancers, not occupied by HDACs, is hyperacetylated in a T3-dependent manner, where TR is recruited to chromatin together with HATs. Lastly, a subset of enhancers, is not occupied directly by TR yet requires TR for histone hyperacetylation. This indirect enhancer activation involves co-association with TR bound enhancers within super-enhancers or topological associated domains. Collectively, this demonstrates various mechanisms controlling hormone-dependent transcription and adds significant details to the otherwise simple bimodal switch model., Author summary Thyroid hormone (T3) is a central regulator of growth, thermogenesis, heart rate and metabolism. In the liver T3 binds thyroid hormone receptor beta (TRβ) controlling expression of genes involved in processes such as lipid and cholesterol metabolism. The molecular mechanisms controlling TR-dependent gene regulation are centred on a bimodal switch model. In the absence of T3 co-repressors bind TR reducing gene expression. When hormone binds TR, co-repressors dissociate, and co-activators are recruited inducing gene expression. This model predominates the current understanding of T3-regulated gene expression. However, only a few studies have tested this model by genome-wide approaches. We have quantified histone3 acetylation genome-wide in the liver of hypo- and hyperthyroid mice and identified gene regulatory regions regulated by T3. Probing TR and co-regulators at these regulatory regions, and analysing histone3 acetylation in mouse models for disrupted co-repressor and TR activity, reveal additional insights to the mechanisms regulating T3-dependent gene expression. We suggest a revision of the prevailing bimodal switch model which helps understanding T3-regulated gene expression in tissues such as liver. We hope that this study, together with future studies, will add new perspectives on nuclear receptor-mediated transcriptional regulation to reveal general principles.

Skeletal muscles consist of fibers of differing metabolic activities and contractility, which become remodeled in response to chronic exercise, but the epigenomic basis for muscle identity and adaptation remains poorly understood. Here, we used chromatin immunoprecipitation sequencing of dimethylated histone 3 lysine 4 and acetylated histone 3 lysine 27 as well as transposase-accessible chromatin profiling to dissect cis-regulatory networks across muscle groups. We demonstrate that in vivo enhancers specify muscles in accordance with myofiber composition, show little resemblance to cultured myotube enhancers, and identify glycolytic and oxidative muscle-specific regulators. Moreover, we find that voluntary wheel running and muscle-specific peroxisome proliferator–activated receptor gamma coactivator-1 alpha (Pgc1a) transgenic (mTg) overexpression, which stimulate endurance performance in mice, result in markedly different changes to the epigenome. Exercise predominantly leads to enhancer hypoacetylation, whereas mTg causes hyperacetylation at different sites. Integrative analysis of regulatory regions and gene expression revealed that exercise and mTg are each associated with myocyte enhancer factor (MEF) 2 and estrogen-related receptor (ERR) signaling and transcription of genes promoting oxidative metabolism. However, exercise was additionally associated with regulation by retinoid X receptor (RXR), jun proto-oncogene (JUN), sine oculis homeobox factor (SIX), and other factors. Overall, our work defines the unique enhancer repertoires of skeletal muscles in vivo and reveals that divergent exercise-induced or PGC1α-driven epigenomic programs direct partially convergent transcriptional networks., The epigenomic basis for the identity and adaptation of different muscle groups is poorly understood. This study reveals chromatin landscapes across muscle groups and their dynamic response to either exercise or overexpression of PPAR gamma coactivator 1 alpha.

Chromosome dynamics and cellular clocks Many genes undergo daily or circadian changes in their rate of transcription. Kim et al. explored the mechanism by which the circadian clock is linked to chromosome dynamics (see the Perspective by Diettrich Mallet de Lima and Göndör). They used a chromosome conformation capture technique (Hi-C) to identify the interactions of adjacent DNA fragments and determine how DNA looping that altered such interactions changed over daily cycles. The repressive transcription factor Rev-erbα, which functions as part of the mammalian clock mechanism, appears to bind to chromatin and recruit a protein complex that evicts other proteins that enhance looping, thus favoring enhancer-promoter interactions. Science , this issue p. 1274 ; see also p. 1212

Histone deacetylase inhibitors (HDACIs) are known to alter gene expression by both up- and down-regulation of protein-coding genes in normal and cancer cells. However, the exact regulatory mechanisms of action remain uncharacterized. Here we investigated genome wide dose-dependent epigenetic and transcriptome changes in response to HDACI largazole in a transformed and a non-transformed cell line. Exposure to low nanomolar largazole concentrations (

Parkinson disease (PD) is a complex neurodegenerative disorder of largely unknown etiology. While several genetic risk factors have been identified, the involvement of epigenetics in the pathophysiology of PD is mostly unaccounted for. We conducted a histone acetylome-wide association study in PD, using brain tissue from two independent cohorts of cases and controls. Immunoblotting revealed increased acetylation at several histone sites in PD, with the most prominent change observed for H3K27, a marker of active promoters and enhancers. Chromatin immunoprecipitation sequencing (ChIP-seq) further indicated that H3K27 hyperacetylation in the PD brain is a genome-wide phenomenon, with a strong predilection for genes implicated in the disease, includingSNCA, PARK7, PRKNandMAPT. Integration of the ChIP-seq with transcriptomic data revealed that the correlation between promoter H3K27 acetylation and gene expression is attenuated in PD patients, suggesting that H3K27 acetylation may be decoupled from transcription in the PD brain. Our findings strongly suggest that dysregulation of histone acetylation plays an important role in the pathophysiology of PD and identify novel epigenetic signatures associated with the disease.

Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.

CREB mediates effects of cyclic AMP on cellular gene expression. Ubiquitous CREB target genes are induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell-specific genes by targeting CBP/p300 to promoter-distal enhancer regions. Subsequent increases in histone acetylation facilitate recruitment of the coactivators CRTC2 and BRD4, leading to release of RNA polymerase II over the target gene body. Indeed, CREB-induced hyperacetylation of chromatin over superenhancers promoted beta cell-restricted gene expression, which is sensitive to inhibitors of CBP/p300 and BRD4 activity. Neurod1 appears critical in establishing nucleosome-free regions for recruitment of CREB to beta cell-specific enhancers. Deletion of a CREB-Neurod1-bound enhancer within the Lrrc10b-Syt7 superenhancer disrupted the expression of both genes and decreased beta cell function. Our results demonstrate how cross talk between signal-dependent and lineage-determining factors promotes the expression of cell-type-specific gene programs in response to extracellular cues.

The PLZF transcription factor is essential for osteogenic differentiation of hMSCs; however, its regulation and molecular function during this process is not fully understood. Here, we revealed that the ZBTB16 locus encoding PLZF, is repressed by Polycomb (PcG) and H3K27me3 in naive hMSCs. At the pre-osteoblast stage of differentiation, the locus lost PcG binding and H3K27me3, gained JMJD3 recruitment, and H3K27ac resulting in high expression of PLZF. Subsequently, PLZF was recruited to osteogenic enhancers, influencing H3K27 acetylation and expression of nearby genes important for osteogenic function. Furthermore, we identified a latent enhancer within the ZBTB16/PLZF locus itself that became active, gained PLZF, p300 and Mediator binding and looped to the promoter of the nicotinamide N-methyltransferase (NNMT) gene. The increased expression of NNMT correlated with a decline in SAM levels, which is dependent on PLZF and is required for osteogenic differentiation.

Enhancer profiling is a powerful approach for discovering cis-regulatory elements that define the core transcriptional regulatory circuits of normal and malignant cells. Gene control through enhancer activity is often dominated by a subset of lineage-specific transcription factors. By integrating measures of chromatin accessibility and enrichment for H3K27 acetylation, we have generated regulatory landscapes of chronic lymphocytic leukemia (CLL) samples and representative cell lines. With super enhancer-based modeling of regulatory circuits and assessments of transcription factor dependencies, we discover that the essential super enhancer factor PAX5 dominates CLL regulatory nodes and is essential for CLL cell survival. Targeting enhancer signaling via BET bromodomain inhibition disrupts super enhancer-dependent gene expression with selective effects on CLL core regulatory circuitry, conferring potent anti-tumor activity.

SUMMARY Neuronal activity-inducible gene transcription correlates with rapid and transient increases in histone acetylation at promoters and enhancers of activity-regulated genes. Exactly how histone acetylation modulates transcription of these genes has remained unknown. We used single-cell in situ transcriptional analysis to show that Fos and Npas4 are transcribed in stochastic bursts in mouse neurons and that membrane depolarization increases mRNA expression by increasing burst frequency. We then expressed dCas9-p300 or dCas9-HDAC8 fusion proteins to mimic or block activity-induced histone acetylation locally at enhancers. Adding histone acetylation increased Fos transcription by prolonging burst duration and resulted in higher Fos protein levels and an elevation of resting membrane potential. Inhibiting histone acetylation reduced Fos transcription by reducing burst frequency and impaired experience-dependent Fos protein induction in the hippocampus in vivo. Thus, activity-inducible histone acetylation tunes the transcriptional dynamics of experience-regulated genes to affect selective changes in neuronal gene expression and cellular function., In Brief Using CRISPR-mediated epigenome editing, Chen et al. show that enhancer histone acetylation fine-tunes the activity-dependent transcription of Fos in neurons., Graphical Abstract

Summary Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6 ) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes.

The sex chromosomes are enriched with germline genes that are activated during the late stages of spermatogenesis. Due to meiotic sex chromosome inactivation (MSCI), these sex chromosome-linked genes must escape silencing for activation in spermatids, thereby ensuring their functions for male reproduction. RNF8, a DNA damage response protein, and SCML2, a germline-specific Polycomb protein, are two major, known regulators of this process. Here, we show that RNF8 and SCML2 cooperate to regulate ubiquitination during meiosis, an early step to establish active histone modifications for subsequent gene activation. Double mutants of Rnf8 and Scml2 revealed that RNF8-dependent monoubiquitination of histone H2A at Lysine 119 (H2AK119ub) is deubiquitinated by SCML2, demonstrating interplay between RNF8 and SCML2 in ubiquitin regulation. Additionally, we identify distinct functions of RNF8 and SCML2 in the regulation of ubiquitination: SCML2 deubiquitinates RNF8-independent H2AK119ub but does not deubiquitinate RNF8-dependent polyubiquitination. RNF8-dependent polyubiquitination is required for the establishment of H3K27 acetylation, a marker of active enhancers, while persistent H2AK119ub inhibits establishment of H3K27 acetylation. Following the deposition of H3K27 acetylation, H3K4 dimethylation is established as an active mark on poised promoters. Together, we propose a model whereby regulation of ubiquitin leads to the organization of poised enhancers and promoters during meiosis, which induce subsequent gene activation from the otherwise silent sex chromosomes in postmeiotic spermatids., Author summary To produce unimpaired sperm, precise activation of germline-specific genes is an essential step during the late stages of spermatogenesis. However, sex chromosomes carrying these genes become silenced in a chromosome-wide manner during meiosis in a process called meiotic sex chromosome inactivation. Sex chromosome inactivation is maintained from meiosis into postmeiotic spermatids. Thus, to ensure the function of sex chromosome-linked (sex-linked) genes required for male reproduction, these genes must escape silencing for activation in spermatids. Here, we unravel the epigenetic mechanisms that underlie the activation of sex-linked genes from otherwise inactive sex chromosomes in the male germline. We determine the mechanism by which two factors regulate gene activation: one is RNF8, a DNA damage response protein, and the other is SCML2, a germline-specific Polycomb protein. Our data suggest that, during meiosis, RNF8 and SCML2 cooperate to regulate ubiquitination, which establishes active epigenetic modifications on enhancers and promoters for subsequent gene activation; these memories are maintained through meiotic divisions to induce gene activation in spermatids. Importantly, this study uncovers novel epigenetic mechanisms that underlie specific gene activation in spermatids and illuminates potential causes of male infertility.

In eukaryotic chromatin, islands of histone tail acetylation are found near transcription start sites and enhancers, whereas linker histones (LHs) are localized in intergenic regions with wild-type (WT) histone tails. However, the structural mechanisms by which acetylation, in combination with LH binding, modulates chromatin compaction and hence transcription regulation are unknown. To explore the folding propensity by which these features may govern gene expression, we analyze 20 kb fibers that contain regularly spaced acetylation islands of two sizes (2 or 5 kb) with various LH levels by mesoscale modeling. Specifically, we investigate the effect of acetylating each histone tail (H3, H4, H2A, and H2B) individually, in combination (H3 and H4, or all tails), and adding LH to WT regions. We find that fibers with acetylated H4 tails lose local contacts (

The transcriptional co-activator p300 is a histone acetyltransferase (HAT) that is typically recruited to transcriptional enhancers and regulates gene expression by acetylating chromatin. Here we show that the activation of p300 directly depends on the activation and oligomerization status of transcription factor ligands. Using two model transcription factors, IRF3 and STAT1, we demonstrate that transcription factor dimerization enables the trans-autoacetylation of p300 in a highly conserved and intrinsically disordered autoinhibitory lysine-rich loop, resulting in p300 activation. We describe a crystal structure of p300 in which the autoinhibitory loop invades the active site of a neighbouring HAT domain, revealing a snapshot of a trans-autoacetylation reaction intermediate. Substrate access to the active site involves the rearrangement of an autoinhibitory RING domain. Our data explain how cellular signalling and the activation and dimerization of transcription factors control the activation of p300, and therefore explain why gene transcription is associated with chromatin acetylation.

In vitro studies have demonstrated an increase in GnRH gene expression associated with an elevated secretory response to kisspeptin administration, suggesting that kisspeptin mediates GnRH expression at both the secretory and pretranslational levels. However, the kisspeptin-mediated intracellular mechanisms associated with the dynamic chromatin modifications modulating GnRH gene expression are unclear. The studies in this manuscript describe specific histone modifications on the enhancer and promoter of the mouse GnRH (mGnRH) gene induced by kisspeptin in GnRH neuronal cell lines (GT1-7 cells). ChIP assays followed by quantitative real-time PCR (qPCR) demonstrate that 15 and 45 min of 10(-9) M kisspeptin significantly increased histone 3 acetylation (H3Ac) at the kisspeptin response element (KsRE) contained between -3446 and -2806 bp of the mGnRH enhancer (GnRHen) in GT1-7 cells, while no changes were observed in the downstream neuron-specific element (NSE). Moreover, kisspeptin specifically induced acetylation of H3AcK14 and K27 and trimethylation of H3 lysine 4 at the KsRE (markers of active chromatin) and no changes in dimethylation of H3K9 (a marker associated with gene repression). Occupancy of RNA Pol II (RNAPII) and a differential carboxyl-terminal domain (CTD) phosphorylation pattern was observed. An interaction between the NSE and the KsRE via a chromatin loop in the mGnRH gene by kisspeptin was detected by the chromosome conformation capture assay (3C). In conclusion, these results demonstrate that kisspeptin induces histone acetylation/methylation and consequently enhances the formation of a chromatin loop in the mGnRH gene which results in known increase in kisspeptin-dependent mGnRH expression.

Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases.

Summary Chromatin remodeling complexes play essential roles in metazoan development through widespread control of gene expression, but the precise molecular mechanisms by which they do this in vivo remain ill defined. Using an inducible system with fine temporal resolution, we show that the nucleosome remodeling and deacetylation (NuRD) complex controls chromatin architecture and the protein binding repertoire at regulatory regions during cell state transitions. This is primarily exerted through its nucleosome remodeling activity while deacetylation at H3K27 follows changes in gene expression. Additionally, NuRD activity influences association of RNA polymerase II at transcription start sites and subsequent nascent transcript production, thereby guiding the establishment of lineage-appropriate transcriptional programs. These findings provide a detailed molecular picture of genome-wide modulation of lineage-specific transcription by an essential chromatin remodeling complex as well as insight into the orchestration of molecular events involved in transcriptional transitions in vivo. Video Abstract, Graphical Abstract, Highlights • NuRD increases nucleosome density, expelling TFs or inhibiting recruitment • NuRD displaces RNA Pol II from TSSs, reducing nascent transcription • Local gains in TF and Mediator occupancy can be indirect effects of NuRD activity • Resetting protein binding at regulatory elements can promote or suppress transcription, Bornelöv et al. define how NuRD, an abundant chromatin remodeling complex, fine-tunes gene expression. NuRD controls nucleosome positioning across regulatory elements genome-wide, controlling access of DNA-binding proteins to enhancers and promoters. This resetting of the transcription factor repertoire at regulatory elements restricts expression from some loci and induces transcription at others.

Steady-state gene expression across the cell cycle has been studied extensively. However, transcriptional gene regulation and the dynamics of histone modification at different cell-cycle stages are largely unknown. By applying a combination of global nuclear run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and histone-modification Chip sequencing (ChIP-seq), we depicted a comprehensive transcriptional landscape at the G0/G1, G1/S, and M phases of breast cancer MCF-7 cells. Importantly, GRO-seq and RNA-seq analysis identified different cell-cycle-regulated genes, suggesting a lag between transcription and steady-state expression during the cell cycle. Interestingly, we identified genes actively transcribed at early M phase that are longer in length and have low expression and are accompanied by a global increase in active histone 3 lysine 4 methylation (H3K4me2) and histone 3 lysine 27 acetylation (H3K27ac) modifications. In addition, we identified 2,440 cell-cycle-regulated enhancer RNAs (eRNAs) that are strongly associated with differential active transcription but not with stable expression levels across the cell cycle. Motif analysis of dynamic eRNAs predicted Kruppel-like factor 4 (KLF4) as a key regulator of G1/S transition, and this identification was validated experimentally. Taken together, our combined analysis characterized the transcriptional and histone-modification profile of the human cell cycle and identified dynamic transcriptional signatures across the cell cycle.

The innate immune response to pathogenic challenge is a complex, multi-staged process involving thousands of genes. While numerous transcription factors that act as master regulators of this response have been identified, the temporal complexity of gene expression changes in response to pathogen-associated molecular pattern receptor stimulation strongly suggest that additional layers of regulation remain to be uncovered. The evolved pathogen response program in mammalian innate immune cells is understood to reflect a compromise between the probability of clearing the infection and the extent of tissue damage and inflammatory sequelae it causes. Because of that, a key challenge to delineating the regulators that control the temporal inflammatory response is that an innate immune regulator that may confer a selective advantage in the wild may be dispensable in the lab setting. In order to better understand the complete transcriptional response of primary macrophages to the bacterial endotoxin lipopolysaccharide (LPS), we designed a method that integrates temporally resolved gene expression and chromatin-accessibility measurements from mouse macrophages. By correlating changes in transcription factor binding site motif enrichment scores, calculated within regions of accessible chromatin, with the average temporal expression profile of a gene cluster, we screened for transcriptional factors that regulate the cluster. We have validated our predictions of LPS-stimulated transcriptional regulators using ChIP-seq data for three transcription factors with experimentally confirmed functions in innate immunity. In addition, we predict a role in the macrophage LPS response for several novel transcription factors that have not previously been implicated in immune responses. This method is applicable to any experimental situation where temporal gene expression and chromatin-accessibility data are available.

BRD4 governs pathological cardiac gene expression by binding acetylated chromatin, resulting in enhanced RNA polymerase II (Pol II) phosphorylation and transcription elongation. Here, we describe a signal-dependent mechanism for regulation of BRD4 in cardiomyocytes. BRD4 expression is suppressed by microRNA-9 (miR-9), which targets the 3′ untranslated region of the Brd4 transcript. In response to stress stimuli, miR-9 is downregulated, leading to derepression of BRD4 and enrichment of BRD4 at long-range super-enhancers (SEs) associated with pathological cardiac genes. A miR-9 mimic represses stimulus-dependent targeting of BRD4 to SEs and blunts Pol II phosphorylation at proximal transcription start sites, without affecting BRD4 binding to SEs that control constitutively expressed cardiac genes. These findings suggest that dynamic enrichment of BRD4 at SEs genome-wide serves a crucial role in the control of stress-induced cardiac gene expression, and define a miR-dependent signaling mechanism for the regulation of chromatin state and Pol II phosphorylation.

How tissue regeneration programs are triggered by injury has received limited research attention. Here we investigate the existence of enhancer regulatory elements that are activated in regenerating tissue. Transcriptomic analyses reveal that leptin b (lepb) is highly induced in regenerating hearts and fins of zebrafish. Epigenetic profiling identified a short DNA sequence element upstream and distal to lepb that acquires open chromatin marks during regeneration and enables injury-dependent expression from minimal promoters. This element could activate expression in injured neonatal mouse tissues and was divisible into tissue-specific modules sufficient for expression in regenerating zebrafish fins or hearts. Simple enhancer-effector transgenes employing lepb-linked sequences upstream of pro- or anti-regenerative factors controlled the efficacy of regeneration in zebrafish. Our findings provide evidence for ‘tissue regeneration enhancer elements’ (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs. An injury-dependent enhancer element is identified that activates gene expression in regenerating zebrafish tissues and can be engineered into DNA constructs that increase tissue regenerative capacity; the element is also active in injured mouse tissue. Ken Poss and colleagues identify an injury-dependent enhancer element that activates gene expression in regenerating zebrafish tissues. They find that the element, which they term a 'tissue regeneration enhancer element' (TREE), is divisible into tissue-specific modules that can each direct expression in zebrafish hearts or fins. The identified element can be used to direct the expression of pro- or anti-regenerative factors in zebrafish tissues and thus control the efficiency of regeneration. Finally, by engineering TREEs upstream of mitogenic factor genes, the authors demonstrate their ability to boost tissue repair in injured mouse tissue.

Nucleosome positioning at transcription start sites is known to regulate gene expression by altering DNA accessibility to transcription factors; however, its role at enhancers is poorly understood. We investigated nucleosome positioning at the androgen receptor (AR) enhancers of TMPRSS2, KLK2, and KLK3/PSA in prostate cancer cells. Surprisingly, a population of enhancer modules in androgen-deprived cultures showed nucleosome-depleted regions (NDRs) in all three loci. Under androgen-deprived conditions, NDRs at the TMPRSS2 enhancer were maintained by the pioneer AR transcriptional collaborator GATA-2. Androgen treatment resulted in AR occupancy, an increased number of enhancer modules with NDRs without changes in footprint width, increased levels of histone H3 acetylation (AcH3), and dimethylation (H3K4me2) at nucleosomes flanking the NDRs. Our data suggest that, in the absence of ligand, AR enhancers exist in an equilibrium in which a percentage of modules are occupied by nucleosomes while others display NDRs. We propose that androgen treatment leads to the disruption of the equilibrium toward a nucleosome-depleted state, rather than to enhancer de novo “remodeling.” This allows the recruitment of histone modifiers, chromatin remodelers, and ultimately gene activation. The “receptive” state described here could help explain AR signaling activation under very low ligand concentrations.

The Spt–Ada–Gcn5–acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5′ end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression.

In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian β-globin gene loci, and determined that within the murine locus, neither the β-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic β-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ϵ-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine β-globin locus results in significant decrease in the expression of the embryonic β-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for β-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure.

The intestinal expression of genes involved in carbohydrate digestion and absorption, such as sucrase-isomaltase (SI) and sodium-dependent glucose cotransporter (SGLT1), is higher in rodents fed a high-starch/low-fat (HS) diet than in those fed a low-starch/high-fat (LS) diet. In the present study, we investigated whether the HS diet-induced induction of SI and SGLT1 in the rat jejunum is coordinately regulated by nuclear transcription factors, histone acetylation, or histone acetyltransferases. HS diet intake induced jejunal expression of a histone acetyltransferase, general control of amino acid synthesis (GCN5), concurrently with the SI and SGLT1 genes; however, gene expression of nuclear transcription factors such as hepatocyte nuclear factor-1, caudal type homeobox-2, and GATA-binding protein-4 was unaffected by the HS diet. Acetylation of histones H3/H4 and binding of acetyltransferase GCN5 on the promoter/enhancer and transcribed regions of SI and SGLT1 genes were significantly higher in HS diet-fed rats than in LS diet-fed rats, but transcription factor binding was not affected by the HS diet. Our results suggest that the concomitant induction of SI and SGLT1 genes in the jejunum by the HS diet is closely associated with the binding of GCN5 and acetylation of histones H3/H4 on these genes.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3′ enhancer (E3′), and a further downstream enhancer (Ed). Previous studies on B lymphocytes derived from mutant embryonic stem cells have shown that deletion of either Ei or E3′ significantly reduces Igκ gene rearrangement, whereas the combined deletion of both Ei and E3′ eliminates such recombination. Furthermore, deletion of either E3′ or Ed significantly reduces rearranged Igκ gene transcription. To determine whether the combined presence of both E3′ and Ed are essential for Igκ gene expression, we generated homozygous double knockout (DKO) mice with targeted deletions in both elements. Significantly, homozygous DKO mice were unable to generate κ+ B cells both in bone marrow and the periphery and exhibited surface expression almost exclusively of Igλ-chains, despite the fact that they possessed potentially functional rearranged Igκ genes. Compared with their single-enhancer–deleted counterparts, Igκ loci in homozygous DKO mice exhibited dramatically reduced germline and rearranged gene transcription, lower levels of gene rearrangement and histone H3 acetylation, and markedly increased DNA methylation. This contributed to a partial developmental block at the pre-B cell stage of development. We conclude that the two downstream enhancers are essential in Igκ gene expression and that Ei in homozygous DKO mice is incapable of triggering Igκ gene transcription. Furthermore, these results reveal unexpected compensatory roles for Ed in E3′ knockout mice in triggering germline transcription and Vκ gene rearrangements to both Jκ and RS elements.

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites, as well as a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (

The transcriptional mechanisms by which temporary exposure to developmental signals instigates adipocyte differentiation are unknown. During early adipogenesis, we find transient enrichment of the glucocorticoid receptor (GR), CCAAT/enhancer-binding protein beta (CEBPbeta), p300, mediator subunit 1, and histone H3 acetylation near genes involved in cell proliferation, development, and differentiation, including the gene encoding the master regulator of adipocyte differentiation, peroxisome proliferator-activated receptor gamma2 (PPARgamma2). Occupancy and enhancer function are triggered by adipogenic signals, and diminish upon their removal. GR, which is important for adipogenesis but need not be active in the mature adipocyte, functions transiently with other enhancer proteins to propagate a new program of gene expression that includes induction of PPARgamma2, thereby providing a memory of the earlier adipogenic signal. Thus, the conversion of preadipocyte to adipocyte involves the formation of an epigenomic transition state that is not observed in cells at the beginning or end of the differentiation process.

Maltase and glucoamylase are derived from the same mRNA and are responsible for digestion of starch in the small intestine. Their jejunal activities in rodents are induced by a high-starch/low-fat (HS)-diet. However, it is unknown whether jejunal expression of the maltase-glucoamylase (Mgam) gene is enhanced by the HS-diet. In this study, we found that jejunal Mgam mRNA was increased by a HS-diet in mice. We showed that the HS-diet increased acetylation of histones, bindings of a coactivator, Creb binding protein (CREBBP), and the transcriptional factors caudal type homeobox 2 (CDX2) and HNF1 homeobox (HNF1) in the promoter/enhancer and transcriptional regions of Mgam gene. This suggests that the increase in the jejunal activity of maltase and glucoamylase caused by a HS-diet in mice is regulated at the mRNA level through histone acetylation and binding of CREBBP, CDX2 and HNF1 in the promoter/enhancer and transcriptional regions of Mgam gene.

We have previously reported that dietary fructose rapidly induces jejunal sucrase–isomaltase (SI) gene expression in rats. In this study, we confirmed in mice that SI mRNA was induced 6 h after force-feeding fructose, but not glucose. Using the chromatin immunoprecipitation assay, we revealed that histones H3 and H4 on the promoter/enhancer regions of the SI gene in mice given fructose were highly acetylated, compared with those given glucose or water. These results suggest that acute induction of SI gene expression by dietary fructose is associated with acetylation of histones H3 and H4 on the SI gene.

Mitogen-activated protein kinase (MAPK) signaling pathways are key mediators of eukaryotic transcriptional responses to extracellular signals. These pathways control gene expression in a number of ways including the phosphorylation and regulation of transcription factors, co-regulatory proteins and chromatin proteins. MAPK pathways therefore target multiple components of transcriptional complexes at gene promoters and can regulate DNA binding, protein stability, cellular localization, transactivation or repression, and nucleosome structure. Recent work has uncovered further complexities in the mechanisms by which MAPKs control gene expression including their roles as integral components of transcription factor complexes and their interplay with other post-translational modification pathways. In this review I discuss these advances with particular focus on how MAPK signals are integrated by transcription factor complexes to provide specific transcriptional responses and how this relates to cellular function.

The biological actions of 1,25-(OH)2D3 are mediated by the vitamin D receptor (VDR), a protein that binds to target genes and alters their expression. 1,25-(OH)2D3 is also capable of inducing transcription of the VDR gene itself. In the present study, we explored both the capacity of 1,25-(OH)2D3 to induce VDR gene expression in bone cells and the mechanism instrumental to this up-regulation. After establishing the ability of 1,25-(OH)2D3 to stimulate VDR mRNA up-regulation both in bone in vivo and in osteoblastic cells, we screened the mouse VDR gene locus from 20 kb upstream of the gene’s transcriptional start site (TSS) to 10 kb downstream of the final exon to identify VDR binding sites using chromatin immunoprecipitation-DNA microarray (ChIP-chip) analysis. Three conserved regions were identified 20, 27, and 29 kb downstream of the TSS. VDR binding to these sites in response to 1,25-(OH)2D3 was confirmed by ChIP analysis and was accompanied by differential localization of retinoid X receptor, histone acetylation, and RNA polymerase II recruitment. One of these regions was able to confer 1,25-(OH)2D3 regulation to downstream promoters, thereby permitting identification and characterization of the regulatory element located within. Importantly, a highly conserved region within the human VDR gene analogous to that discovered in the mouse was also capable of mediating 1,25-(OH)2D3 response. Our results demonstrate that 1,25-(OH)2D3 and its receptor autoregulate the expression of the VDR gene. The location of these regulatory regions and their apparent distances from the TSS are consistent with new findings suggesting the emerging relevance of distant enhancers. (Molecular Endocrinology 20: 1231–1247, 2006)

INTRODUCTION Brain activity drives both behavior and regulated gene expression in neurons. Although past studies have identified activity-induced signaling and gene regulation cascades in cultured neurons, much less is known about how activity- dependent transcriptional networks are affected by the variations in cell-type composition, network interconnections, and firing patterns that comprise behaviorally active brain circuits in vivo. Dual mechanism model for behaviorally regulated gene expression diversity. ( Left ) Song brain circuit and zebra finch song motif (transcribed using https://my.scorecloud.com). ( Middle ) Song nucleus–specific (RA, red; Area X, blue) singing- regulated genes (gene A and gene B) in response to neural f ring (yellow). ( Right ) Region-general EATF and region-specific TF only bind to genomic DNA (lines) with region-specific acetylated histone 3 (H3K27ac peaks) and then transcribe their mRNAs (green arrow). RATIONALE We tested the hypothesis that behaviorally regulated gene expression is anatomically and temporally diverse and that the key determinants of this diversity are networks of transcription factors, their genomic binding sites, and epigenetic chromatin states. We analyzed genome-wide, singing-regulated gene expression across time in the four major forebrain regions of the song control system in songbirds, a model of speech production in humans. We then performed a transcription factor motif analysis to identify gene regulatory networks enriched in each song nucleus and measured acetylation of histone 3 at lysine 27 (H3K27ac) to identify chromatin regions that were transcriptionally active in the genomes of song nuclei before and after singing. RESULTS We found that singing was associated with differential regulation of about 10% of all genes in the avian genome that came in several waves across time. Less than 1% of these genes were comparably regulated in all song nuclei tested, and these comprised a core set dominated by immediate-early gene (IEG) transcription factors. By contrast, the vast majority of singing-regulated genes were regulated in only one or a subset of song nuclei, such that each song nucleus had its own dominant subset of genes regulated with defined temporal profiles, controlling a variety of functions. The promoters of many of the singing-regulated genes contained binding motifs for known early-activated transcription factors (EATFs) that become active in response to neural firing, some of which were expressed differentially between song nuclei at baseline. One EATF, calcium-response factor ( CaRF ), was tested with RNA interference knockdown in cultured neurons and found to regulate the predicted genes in response to neural activity, but was also found to modulate their expression even at baseline. More strikingly, we found with H3K27ac analysis that many song nucleus–specific singing-regulated genes did not show increased chromatin regulatory element activity after singing but rather already had primed region-specific regulatory activity before singing began. CONCLUSIONS We propose a dual mechanism for the diversity of behaviorally regulated genes across different brain regions in vivo (see the figure). First, the neural activity associated with singing activates EATFs, and some TFs differentially expressed in brain regions at baseline, to trigger region-specific expression of their target genes. Second, brain region–specific enhancers near activity- regulated genes are waiting in an epigenetically primed state, ready to modulate transcription of general and song nucleus–specific genes at a moment’s notice when the neurons fire. The combination of these two mechanisms underlies a great diversity of behaviorally regulated gene expression, permitting each nucleus to perform its particular function in this complex behavior.

One of the central issues facing current biology concerns the molecular mechanisms whereby stem cells give rise to differentiated progeny. Hematopoiesis is the best-characterized stem cell system, and experiments with mice and lower vertebrates have identified a small number of transcription factors, including SCL, LMO-2, and RUNX-1, which are central to the formation and/or behavior of hematopoietic stem cells (HSCs). Tight transcriptional control of these key regulatory genes is frequently critical for their biological functions, but the mechanisms responsible are poorly understood. The SCL gene encodes a basic helix-loop-helix protein and is expressed in blood, in endothelium, and within specific regions of the central nervous system, a pattern of expression that is highly conserved across vertebrate species from mammals to teleost fish (reviewed in reference 4). Within the hematopoietic system, SCL is expressed in hemangioblasts, HSCs, and a subset of hematopoietic lineages, including both primitive and definitive erythroblasts. Although not required for self-renewal of adult HSCs (9, 46), targeted mutation of the SCL gene has shown that it is essential for the development of all hematopoietic lineages in mice (57, 61) and during murine embryonic stem (ES) cell differentiation (13, 14, 62). However, SCL−/− mouse embryos and ES cells both generate endothelial cells (62, 73), suggesting that SCL is required for lineage commitment to blood cell formation. Consistent with this concept, ectopic expression of SCL during zebra fish development results in excessive formation of hemangioblasts (19). Maintenance of SCL expression is required for normal differentiation along erythroid and megakaryocytic lineages (32, 46), whereas failure to downregulate SCL transcription during T-cell differentiation is associated with T-cell acute lymphoblastic leukemia (T-ALL) (reviewed in reference 4). Current evidence therefore demonstrates that appropriate transcriptional regulation is essential for the biological functions of SCL, and this focuses attention on the mechanisms whereby transcription of SCL itself is initiated and maintained. Several lines of work have helped define the size of the transcriptional domain necessary for the normal pattern of SCL transcription. A 130-kb human yeast artificial chromosome containing both flanking genes was able to completely rescue the lethal SCL-/- phenotype in mice (66). Consistent with this result, the pufferfish SCL genomic locus gave rise to appropriate expression in transgenic zebra fish (3), and comparisons of SCL flanking genes during vertebrate evolution revealed a limited region of conserved synteny likely to contain regulatory elements responsible for the conserved pattern of SCL expression (21, 24). Analysis of chromatin structure, together with large-scale comparative genomic sequence analysis, has led to identification of SCL enhancers with activity in transfection assays (16, 25) or in transgenic mice (6, 24, 26, 64, 67). These approaches have so far revealed a panel of five enhancers, each of which targets expression to a specific subdomain of the normal pattern of SCL expression and two of which are active in blood and/or endothelial cells. The +18/19 enhancer is sufficient to direct reporter gene expression to hematopoietic progenitors and endothelial cells during development (64), to the vast majority of long-term-repopulating HSCs from adult bone marrow and fetal liver (65), and to putative hemangioblasts within frog dorsolateral plate mesoderm (26). Expression of an SCL cDNA under the control of this enhancer in transgenic mice selectively rescued the formation of early hematopoietic progenitors in SCL−/− embryos (65), and transgenic mice in which β-geo is driven by this enhancer have been used to identify endoglin as a novel HSC marker (7). Fine mapping demonstrated that the +19 component was sufficient for enhancer activity in transgenic mouse embryos, and biochemical characterization has shown that the +19 element is activated by a novel multiprotein complex containing Fli-1, Elf-1, and GATA-2 (26). These data suggest that this enhancer functions as a nodal point for the integration of signals responsible for establishing the transcriptional program for blood cell development. Interestingly, ES cells in which the +18/19 element was deleted from the endogenous SCL locus were still capable of forming blood cells in vitro and in vivo (23). This observation led to the characterization of a second enhancer (−4 element), which targets expression to endothelial cells and hematopoietic progenitors and which is also bound by Fli-1 and Elf-1 (23). However, several questions remain. In particular, little is known about the hematopoietic expression of other genes within the SCL locus, and it is unclear whether individual SCL enhancers also regulate the transcription of neighboring genes. Furthermore, the +18/19 and −4 elements target hematopoietic progenitors but not erythroid cells, and yet SCL itself is normally expressed in the primitive and definitive erythroid lineages. As a consequence, we have previously postulated the existence of a separate erythroid enhancer necessary for maintaining SCL expression following erythroid commitment (65). In this paper, we describe the pattern of transcription of SCL and neighboring genes in a panel of hematopoietic cell lines representing multiple lineages. We show that SCL exhibits unexpected coexpression with its downstream neighbor MAP17, suggesting that they share regulatory elements. A systematic survey of histone acetylation throughout the SCL locus resulted in identification of a novel erythroid enhancer 40 kb downstream of SCL exon 1a. This element functions as an erythroid-restricted enhancer in vitro; directs expression to primitive, but not definitive, erythroid cells in vivo; and provides a powerful tool for studying the poorly understood primitive erythroid lineage.

Transcriptional activation is associated commonly with recruitment of histone acetylases, while repression involves histone deacetylases (HDACs). Here, we provide evidence to suggest that STAT5 activates gene expression by recruiting HDAC. The interleukin-3 (IL-3)-dependent expression of the Id-1 gene, encoding a helix-loop-helix (HLH) transcriptional inhibitor, is activated by both C/EBPbeta and STAT5 transcription factors bound to its pro-B-cell enhancer (PBE), but is inhibited by HDAC inhibitors in Ba/F3 cells. STAT5 interacts with HDAC1 in the PBE region, resulting in deacetylation of histones, as well as C/EBPbeta, whose acetylation diminishes its DNA-binding activity. Consistently, expression of an acetylation-resistant mutant of C/EBPbeta results in IL-3-independent expression of the Id-1 gene. Thus, we propose a novel mechanism by which STAT5 mediates the deacetylation of C/EBPbeta, allowing transcriptional activation.

Objective Although the expression of hepatic lipogenic genes is enhanced in insulin resistance, the underlying mechanism is unclear. To reveal the details, the aim of this study was to investigate whether the expression of hepatic lipogenic genes are mediated by epigenetic regulation and specific transcription factors in an insulin resistance model of rats. Methods Using a rat model of insulin resistance (SHR/NDmc-cp), we investigated the relationship between hepatic expression of the lipogenic gene fatty-acid synthase ( Fasn ), binding of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to the Fasn gene, and histone modifications in the region of the Fasn gene by real-time reverse transcriptase polymerase chain reaction, immunoblotting, and chromatin immunoprecipitation assay . Results Compared with control rats, Fasn mRNA expression and protein levels were higher in the livers of SHR/NDmc-cp rats, as were protein expression levels and Fasn binding of ChREBP and RNA polymerase II. Moreover, compared with the livers of control rats, levels of mono-methylated histone H3 lysine (K) 4 and acetylated histone H4 were higher in the promoter/enhancer region of the Fasn gene in the livers of SHR/NDmc-cp rats. Levels of trimethylated histone H3K4 and acetylated histone H3 were higher in the transcribed region. Conclusion The results of this study indicate that expression of the Fasn gene in the livers of insulin-resistant rats is associated with increased H3K4 methylation, increased histone H3 acetylation, and increased H4 acetylation, and also, binding levels of ChREBP to promoter/enhancer region of Fasn gene is involved in the Fasn gene expression caused by hyperglycemia.