RESUMO
The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.
Assuntos
Cromatina/metabolismo , Protaminas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/fisiologia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Fertilização/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/metabolismo , Oócitos/fisiologia , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Protaminas/genética , Proteínas Serina-Treonina Quinases/fisiologia , Splicing de RNA/genética , Splicing de RNA/fisiologia , Espermatozoides/metabolismo , Fatores de Transcrição/metabolismo , Zigoto/metabolismoRESUMO
Growth factor receptors rank among the most important oncogenic pathways, but pharmacologic inhibitors often demonstrate limited benefit as monotherapy. Here, we show that epidermal growth factor receptor (EGFR) signaling repressed N6-methyladenosine (m6A) levels in glioblastoma stem cells (GSCs), whereas genetic or pharmacologic EGFR targeting elevated m6A levels. Activated EGFR induced non-receptor tyrosine kinase SRC to phosphorylate the m6A demethylase, AlkB homolog 5 (ALKBH5), thereby inhibiting chromosomal maintenance 1 (CRM1)-mediated nuclear export of ALKBH5 to permit sustained mRNA m6A demethylation in the nucleus. ALKBH5 critically regulated ferroptosis through m6A modulation and YTH N6-methyladenosine RNA binding protein (YTHDF2)-mediated decay of the glutamate-cysteine ligase modifier subunit (GCLM). Pharmacologic targeting of ALKBH5 augmented the anti-tumor efficacy of EGFR and GCLM inhibitors, supporting an EGFR-ALKBH5-GCLM oncogenic axis. Collectively, EGFR reprograms the epitranscriptomic landscape through nuclear retention of the ALKBH5 demethylase to protect against ferroptosis, offering therapeutic paradigms for the treatment of lethal cancers.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Receptores ErbB , Ferroptose , Glioblastoma , Humanos , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Receptores ErbB/genética , Ferroptose/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , RNA Mensageiro/genéticaRESUMO
The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1ß with the NCoR/HDAC3 complex, resulting in the activation of PGC1ß and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.
Assuntos
Osteoclastos , RNA , Humanos , Camundongos , Animais , Proteínas Correpressoras/genética , Osteoclastos/metabolismo , Ligante RANK/genética , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Expressão GênicaRESUMO
The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking-a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Simples , Reparo do DNA , DNA Topoisomerases Tipo I/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína Homóloga a MRE11 , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The idea that signal-dependent transcription might involve the generation of transient DNA nicks or even breaks in the regulatory regions of genes, accompanied by activation of DNA damage repair pathways, would seem to be counterintuitive, as DNA damage is usually considered harmful to cellular integrity. However, recent studies have generated a substantial body of evidence that now argues that programmed DNA single- or double-strand breaks can, at least in specific cases, have a role in transcription regulation. Here, we discuss the emerging functions of DNA breaks in the relief of DNA torsional stress and in promoter and enhancer activation.
Assuntos
Dano ao DNA/genética , DNA/química , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Regulação da Expressão Gênica , HumanosRESUMO
Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.
Assuntos
Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Estrogênios/metabolismo , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Complexos Multiproteicos/metabolismoRESUMO
Distal enhancers characterized by the H3K4me(1) mark play critical roles in developmental and transcriptional programs. However, potential roles of specific distal regulatory elements in regulating RNA polymerase II (Pol II) promoter-proximal pause release remain poorly investigated. Here, we report that a unique cohort of jumonji C-domain-containing protein 6 (JMJD6) and bromodomain-containing protein 4 (Brd4) cobound distal enhancers, termed anti-pause enhancers (A-PEs), regulate promoter-proximal pause release of a large subset of transcription units via long-range interactions. Brd4-dependent JMJD6 recruitment on A-PEs mediates erasure of H4R3me(2(s)), which is directly read by 7SK snRNA, and decapping/demethylation of 7SK snRNA, ensuring the dismissal of the 7SK snRNA/HEXIM inhibitory complex. The interactions of both JMJD6 and Brd4 with the P-TEFb complex permit its activation and pause release of regulated coding genes. The functions of JMJD6/ Brd4-associated dual histone and RNA demethylase activity on anti-pause enhancers have intriguing implications for these proteins in development, homeostasis, and disease.
Assuntos
Elementos Facilitadores Genéticos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Ciclo Celular , Células HEK293 , Humanos , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/metabolismoRESUMO
The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay-referred to as chromatin isolation by RNA-Cas13a complex-to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17ß-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.
Assuntos
Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , RNA Antissenso/química , RNA Antissenso/genética , Ativação Transcricional/genética , Linhagem Celular , Homólogo 5 da Proteína Cromobox/metabolismo , Proteína Substrato Associada a Crk , Receptor alfa de Estrogênio/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Ligantes , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/metabolismo , Estabilidade de RNA , Proteína 28 com Motivo Tripartido/metabolismoRESUMO
The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).
Assuntos
Fator de Ligação a CCCTC/genética , Elementos Facilitadores Genéticos , Predisposição Genética para Doença , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cromatina , Proteínas Cromossômicas não Histona/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Células MCF-7 , Neoplasias/genética , Células-Tronco Neurais , Oncogenes , Doença de Parkinson/genética , CoesinasRESUMO
In diseased organs, stress-activated signalling cascades alter chromatin, thereby triggering maladaptive cell state transitions. Fibroblast activation is a common stress response in tissues that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains unclear1,2. Pharmacological inhibition of bromodomain and extra-terminal domain (BET) proteins alleviates cardiac dysfunction3-7, providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here we use single-cell epigenomic analyses of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch that underlies the activation of fibroblasts. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed previously undescribed DNA elements, the accessibility of which dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer that regulated the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program and was required for TGFß-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFß-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction and demonstrate its upregulation after activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide previously unknown trans- and cis-targets for treating fibrotic disease.
Assuntos
Elementos Facilitadores Genéticos , Fibroblastos/citologia , Cardiopatias/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Epigenômica , Regulação da Expressão Gênica , Humanos , Camundongos , Proteínas/antagonistas & inibidores , Análise de Célula Única , Transcriptoma , Fator de Crescimento Transformador beta/metabolismoRESUMO
The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1ß complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1ß coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1ß complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.
Assuntos
Histonas , Fator 6 Associado a Receptor de TNF , Ativação Transcricional , Proteínas Correpressoras/genética , Histonas/genética , Histonas/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição AP-1/metabolismo , Receptor 4 Toll-Like/metabolismo , Transdução de Sinais , NF-kappa B/genética , NF-kappa B/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Although eukaryotic nuclei contain distinct architectural structures associated with noncoding RNAs (ncRNAs), their potential relationship to regulated transcriptional programs remains poorly understood. Here, we report that methylation/demethylation of Polycomb 2 protein (Pc2) controls relocation of growth-control genes between Polycomb bodies (PcGs) and interchromatin granules (ICGs) in response to growth signals. This movement is the consequence of binding of methylated and unmethylated Pc2 to the ncRNAs TUG1 and MALAT1/NEAT2, located in PcGs and ICGs, respectively. These ncRNAs mediate assembly of multiple corepressors/coactivators and can serve to switch mark recognition by "readers" of the histone code. Additionally, binding of NEAT2 to unmethylated Pc2 promotes E2F1 SUMOylation, leading to activation of the growth-control gene program. These observations delineate a molecular pathway linking the actions of subnuclear structure-specific ncRNAs and nonhistone protein methylation to relocation of transcription units in the three-dimensional space of the nucleus, thus achieving coordinated gene expression programs.
Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica , RNA não Traduzido/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cromatina/metabolismo , Fator de Transcrição E2F1/metabolismo , Células HeLa , Humanos , Ligases , Metilação , Metiltransferases/metabolismo , Dados de Sequência Molecular , Proteínas do Grupo Polycomb , RNA Longo não Codificante , Proteínas Repressoras/química , Sumoilação , Transcrição Gênica , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
As most of the mitochondrial proteome is encoded in the nucleus, mitochondrial functions critically depend on nuclear gene expression and bidirectional mito-nuclear communication. However, mitochondria-to-nucleus communication pathways in mammals are incompletely understood. Here, we identify G-Protein Pathway Suppressor 2 (GPS2) as a mediator of mitochondrial retrograde signaling and a transcriptional activator of nuclear-encoded mitochondrial genes. GPS2-regulated translocation from mitochondria to nucleus is essential for the transcriptional activation of a nuclear stress response to mitochondrial depolarization and for supporting basal mitochondrial biogenesis in differentiating adipocytes and brown adipose tissue (BAT) from mice. In the nucleus, GPS2 recruitment to target gene promoters regulates histone H3K9 demethylation and RNA POL2 activation through inhibition of Ubc13-mediated ubiquitination. These findings, together, reveal an additional layer of regulation of mitochondrial gene transcription, uncover a direct mitochondria-nuclear communication pathway, and indicate that GPS2 retrograde signaling is a key component of the mitochondrial stress response in mammals.
Assuntos
Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Transdução de Sinais/fisiologia , Células 3T3-L1 , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/genética , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metilação , Camundongos , Mitocôndrias/genética , Regiões Promotoras Genéticas/fisiologia , Ativação Transcricional/fisiologiaRESUMO
Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.
Assuntos
Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/genética , Proteínas F-Box/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Histona Desmetilases com o Domínio Jumonji/genética , Ubiquitina-Proteína Ligases Nedd4/genética , RNA Polimerase II/genética , Sequência de Bases , Sítios de Ligação , Sistemas CRISPR-Cas , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Proteínas F-Box/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células MCF-7 , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ligação Proteica , RNA/genética , RNA/metabolismo , RNA Polimerase II/metabolismo , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
Whereas the actions of enhancers in gene transcriptional regulation are well established, roles of JmjC-domain-containing proteins in mediating enhancer activation remain poorly understood. Here, we report that recruitment of the JmjC-domain-containing protein 6 (JMJD6) to estrogen receptor alpha (ERα)-bound active enhancers is required for RNA polymerase II recruitment and enhancer RNA production on enhancers, resulting in transcriptional pause release of cognate estrogen target genes. JMJD6 is found to interact with MED12 in the mediator complex to regulate its recruitment. Unexpectedly, JMJD6 is necessary for MED12 to interact with CARM1, which methylates MED12 at multiple arginine sites and regulates its chromatin binding. Consistent with its role in transcriptional activation, JMJD6 is required for estrogen/ERα-induced breast cancer cell growth and tumorigenesis. Our data have uncovered a critical regulator of estrogen/ERα-induced enhancer coding gene activation and breast cancer cell potency, providing a potential therapeutic target of ER-positive breast cancers.
Assuntos
Neoplasias da Mama/enzimologia , Proliferação de Células , Receptor alfa de Estrogênio/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Complexo Mediador/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Ativação Transcricional , Animais , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Células MCF-7 , Complexo Mediador/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Transporte Proteico , Proteína-Arginina N-Metiltransferases/genética , Transdução de Sinais , Ativação Transcricional/efeitos dos fármacosRESUMO
The resolution limit of chromatin conformation capture methodologies (3Cs) has restrained their application in detection of fine-level chromatin structure mediated by cis-regulatory elements (CREs). Here, we report two 3C-derived methods, Tri-4C and Tri-HiC, which utilize multirestriction enzyme digestions for ultrafine mapping of targeted and genome-wide chromatin interaction, respectively, at up to one hundred basepair resolution. Tri-4C identified CRE loop interaction networks and quantitatively revealed their alterations underlying dynamic gene control. Tri-HiC uncovered global fine-gauge regulatory interaction networks, identifying >20-fold more enhancer:promoter (E:P) loops than in situ Hi-C. In addition to vastly improved identification of subkilobase-sized E:P loops, Tri-HiC also uncovered interaction stripes and contact domain insulation from promoters and enhancers, revealing their loop extrusion behaviors resembling the topologically associating domain boundaries. Tri-4C and Tri-HiC provide robust approaches to achieve the high-resolution interactome maps required for characterizing fine-gauge regulatory chromatin interactions in analysis of development, homeostasis, and disease.
Assuntos
Cromossomos , Genoma , Mapeamento Cromossômico/métodos , Genoma/genética , Cromatina/genética , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Gene regulation by transcriptional enhancers is the dominant mechanism driving cell type- and signal-specific transcriptional diversity in metazoans. However, over four decades since the original discovery, how enhancers operate in the nuclear space remains largely enigmatic. Recent multidisciplinary efforts combining real-time imaging, genome sequencing, and biophysical strategies provide insightful but conflicting models of enhancer-mediated gene control. Here, we review the discovery and progress in enhancer biology, emphasizing the recent findings that acutely activated enhancers assemble regulatory machinery as mesoscale architectural structures with distinct physical properties. These findings help formulate novel models that explain several mysterious features of the assembly of transcriptional enhancers and the mechanisms of spatial control of gene expression.
Assuntos
DNA Viral , Elementos Facilitadores Genéticos , Sequência de Bases , Núcleo Celular/genética , Regulação da Expressão Gênica/genéticaRESUMO
The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E2 program appears to depend on GR SUMOylation, which leads to stable trans-recruitment of the GR-N-CoR/SMRT-HDAC3 corepressor complex on these enhancers. Together, these results uncover a mechanism by which competitive recruitment of DNA-binding nuclear receptors/transcription factors in trans to hot spot enhancers serves as an effective biological strategy for trans-repression, with clear implications for breast cancer and other diseases.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptor Cross-Talk , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Dexametasona/farmacologia , Regulação para Baixo , Elementos Facilitadores Genéticos , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Células MCF-7 , Complexos Multiproteicos , Mutação , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Ligação Proteica , Interferência de RNA , Receptor Cross-Talk/efeitos dos fármacos , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Transdução de Sinais , Sumoilação , Transcrição Gênica/efeitos dos fármacos , Transcriptoma , TransfecçãoRESUMO
The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (â¼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.
Assuntos
Compartimento Celular , Núcleo Celular , Eucariotos , Ribonucleoproteínas , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromossomos/genética , Eucariotos/citologia , Eucariotos/genética , Genes Essenciais , Genoma/genética , Salpicos Nucleares/genética , Ribonucleoproteínas/metabolismo , Transcrição GênicaRESUMO
Nurr1, an orphan nuclear receptor, plays an essential role in the generation and maintenance of dopaminergic neurons in the brain. Rare mutations in Nurr1 are associated with familial Parkinson's disease, but the underlying basis for this relationship has not been established. Here, we demonstrate that Nurr1 unexpectedly functions to inhibit expression of pro-inflammatory neurotoxic mediators in both microglia and astrocytes. Reduced Nurr1 expression results in exaggerated inflammatory responses in microglia that are further amplified by astrocytes, leading to the production of factors that cause death of tyrosine hydroxylase-expressing neurons. Nurr1 exerts anti-inflammatory effects by docking to NF-kappaB-p65 on target inflammatory gene promoters in a signal-dependent manner. Subsequently, Nurr1 recruits the CoREST corepressor complex, resulting in clearance of NF-kappaB-p65 and transcriptional repression. These studies suggest that Nurr1 protects against loss of dopaminergic neurons in Parkinson's disease in part by limiting the production of neurotoxic mediators by microglia and astrocytes.