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1.
Nucleic Acids Res ; 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36464162

RESUMO

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. We identify four distinct homodimerization interfaces on the GR-LBD surface, which can associate into 20 topologically different homodimers. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution, quantitative fluorescence microscopy in living cells, and transcriptomic analyses. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work illustrates the unique flexibility of GR's LBD and suggests different dimeric conformations within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design.

2.
Proc Natl Acad Sci U S A ; 119(15): e2119429119, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35377791

RESUMO

Charge density waves (CDWs) have been observed in nearly all families of copper-oxide superconductors. But the behavior of these phases across different families has been perplexing. In La-based cuprates, the CDW wavevector is an increasing function of doping, exhibiting the so-called Yamada behavior, while in Y- and Bi-based materials the behavior is the opposite. Here, we report a combined resonant soft X-ray scattering (RSXS) and neutron scattering study of charge and spin density waves in isotopically enriched La1.8−xEu0.2SrxCuO4 over a range of doping 0.07≤x≤0.20. We find that the CDW amplitude is temperature independent and develops well above experimentally accessible temperatures. Further, the CDW wavevector shows a nonmonotonic temperature dependence, exhibiting Yamada behavior at low temperature with a sudden change occurring near the spin ordering temperature. We describe these observations using a Landau­Ginzburg theory for an incommensurate CDW in a metallic system with a finite charge compressibility and spin-CDW coupling. Extrapolating to high temperature, where the CDW amplitude is small and spin order is absent, our analysis predicts a decreasing wavevector with doping, similar to Y and Bi cuprates. Our study suggests that CDW order in all families of cuprates forms by a common mechanism.

3.
Sci Adv ; 8(13): eabj8360, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35353576

RESUMO

The cohesin complex is central to chromatin looping, but mechanisms by which these long-range chromatin interactions are formed and persist remain unclear. We demonstrate that interactions between a transcription factor (TF) and the cohesin loader NIPBL regulate enhancer-dependent gene activity. Using mass spectrometry, genome mapping, and single-molecule tracking methods, we demonstrate that the glucocorticoid (GC) receptor (GR) interacts with NIPBL and the cohesin complex at the chromatin level, promoting loop extrusion and long-range gene regulation. Real-time single-molecule experiments show that loss of cohesin markedly diminishes the concentration of TF molecules at specific nuclear confinement sites, increasing TF local concentration and promoting gene regulation. Last, patient-derived acute myeloid leukemia cells harboring cohesin mutations exhibit a reduced response to GCs, suggesting that the GR-NIPBL-cohesin interaction is defective in these patients, resulting in poor response to GC treatment.


Assuntos
Proteínas Cromossômicas não Histona , Receptores de Glucocorticoides , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Humanos , Receptores de Glucocorticoides/genética
4.
Nat Commun ; 12(1): 1987, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790284

RESUMO

A widely regarded model for glucocorticoid receptor (GR) action postulates that dimeric binding to DNA regulates unfavorable metabolic pathways while monomeric receptor binding promotes repressive gene responses related to its anti-inflammatory effects. This model has been built upon the characterization of the GRdim mutant, reported to be incapable of DNA binding and dimerization. Although quantitative live-cell imaging data shows GRdim as mostly dimeric, genomic studies based on recovery of enriched half-site response elements suggest monomeric engagement on DNA. Here, we perform genome-wide studies on GRdim and a constitutively monomeric mutant. Our results show that impairing dimerization affects binding even to open chromatin. We also find that GRdim does not exclusively bind half-response elements. Our results do not support a physiological role for monomeric GR and are consistent with a common mode of receptor binding via higher order structures that drives both the activating and repressive actions of glucocorticoids.


Assuntos
DNA/metabolismo , Estudo de Associação Genômica Ampla/métodos , Multimerização Proteica , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , DNA/genética , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , Humanos , Camundongos , Mutação , Ligação Proteica , Receptores de Glucocorticoides/genética , Elementos de Resposta/genética , Transdução de Sinais/genética
5.
Mol Cell ; 81(7): 1484-1498.e6, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33561389

RESUMO

Transcription factors (TFs) regulate gene expression by binding to specific consensus motifs within the local chromatin context. The mechanisms by which TFs navigate the nuclear environment as they search for binding sites remain unclear. Here, we used single-molecule tracking and machine-learning-based classification to directly measure the nuclear mobility of the glucocorticoid receptor (GR) in live cells. We revealed two distinct and dynamic low-mobility populations. One accounts for specific binding to chromatin, while the other represents a confinement state that requires an intrinsically disordered region (IDR), implicated in liquid-liquid condensate subdomains. Further analysis showed that the dwell times of both subpopulations follow a power-law distribution, consistent with a broad distribution of affinities on the GR cistrome and interactome. Together, our data link IDRs with a confinement state that is functionally distinct from specific chromatin binding and modulates the transcriptional output by increasing the local concentration of TFs at specific sites.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Receptores de Glucocorticoides/química , Fatores de Transcrição/química , Animais , Feminino , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Ratos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Mol Cell ; 75(6): 1161-1177.e11, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31421980

RESUMO

Genes are transcribed in a discontinuous pattern referred to as RNA bursting, but the mechanisms regulating this process are unclear. Although many physiological signals, including glucocorticoid hormones, are pulsatile, the effects of transient stimulation on bursting are unknown. Here we characterize RNA synthesis from single-copy glucocorticoid receptor (GR)-regulated transcription sites (TSs) under pulsed (ultradian) and constant hormone stimulation. In contrast to constant stimulation, pulsed stimulation induces restricted bursting centered around the hormonal pulse. Moreover, we demonstrate that transcription factor (TF) nuclear mobility determines burst duration, whereas its bound fraction determines burst frequency. Using 3D tracking of TSs, we directly correlate TF binding and RNA synthesis at a specific promoter. Finally, we uncover a striking co-bursting pattern between TSs located at proximal and distal positions in the nucleus. Together, our data reveal a dynamic interplay between TF mobility and RNA bursting that is responsive to stimuli strength, type, modality, and duration.


Assuntos
Glucocorticoides/farmacologia , Regiões Promotoras Genéticas , RNA/biossíntese , Receptores de Glucocorticoides/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética/efeitos dos fármacos , Animais , Camundongos , RNA/genética
7.
Genome Res ; 29(8): 1223-1234, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31337711

RESUMO

Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher-order structure that drives both the activating and repressive actions of glucocorticoids.


Assuntos
Cromatina/ultraestrutura , Células Epiteliais/efeitos dos fármacos , Genoma , Glucocorticoides/farmacologia , RNA Mensageiro/genética , Receptores de Glucocorticoides/química , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Cromatina/química , DNA/genética , DNA/metabolismo , Elementos Facilitadores Genéticos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Edição de Genes/métodos , Glucocorticoides/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , Ligação Proteica , Estrutura Quaternária de Proteína , RNA Mensageiro/metabolismo , Ratos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional
8.
Nucleic Acids Res ; 46(1): 203-214, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29126175

RESUMO

Glucocorticoid hormone plays a major role in metabolism and disease. The hormone-bound glucocorticoid receptor (GR) binds to a specific set of enhancers in different cell types, resulting in unique patterns of gene expression. We have addressed the role of chromatin structure in GR binding by mapping nucleosome positions in mouse adenocarcinoma cells. Before hormone treatment, GR-enhancers exist in one of three chromatin states: (i) Nucleosome-depleted enhancers that are DNase I-hypersensitive, associated with the Brg1 chromatin remodeler and flanked by nucleosomes incorporating histone H2A.Z. (ii) Nucleosomal enhancers that are DNase I-hypersensitive, marked by H2A.Z and associated with Brg1. (iii) Nucleosomal enhancers that are inaccessible to DNase I, incorporate little or no H2A.Z and lack Brg1. Hormone-induced GR binding results in nucleosome shifts at all types of GR-enhancer, coinciding with increased recruitment of Brg1. We propose that nucleosome-depleted GR-enhancers are formed and maintained by other transcription factors which recruit Brg1 whereas, at nucleosomal enhancers, GR behaves like a pioneer factor, interacting with nucleosomal sites and recruiting Brg1 to remodel the chromatin.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos , Nucleossomos/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Dexametasona/metabolismo , Dexametasona/farmacologia , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Histonas/genética , Histonas/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/efeitos dos fármacos , Nucleossomos/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Glucocorticoides/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos
9.
Nat Commun ; 8: 15896, 2017 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-28635963

RESUMO

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other's dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level.


Assuntos
Imagem Molecular/métodos , Receptores de Esteroides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cromatina/metabolismo , Corticosterona/farmacologia , DNA Helicases/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Ratos , Receptores de Glucocorticoides/análise , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/análise , Receptores de Esteroides/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Célula Única/métodos , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo
10.
Proc Natl Acad Sci U S A ; 113(29): 8236-41, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27382178

RESUMO

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand-regulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR's oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors.


Assuntos
DNA/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Fibroblastos/metabolismo , Camundongos Knockout , Multimerização Proteica , Receptores de Glucocorticoides/genética
11.
PLoS Biol ; 12(3): e1001813, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24642507

RESUMO

Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.


Assuntos
Receptores de Glucocorticoides/química , Animais , Células Cultivadas , DNA/metabolismo , Camundongos , Multimerização Proteica , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/metabolismo
12.
Nat Struct Mol Biol ; 21(1): 73-81, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24317492

RESUMO

ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes.


Assuntos
Montagem e Desmontagem da Cromatina , Genoma , Animais , Sítios de Ligação , Camundongos
13.
Immunity ; 35(6): 919-31, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-22195747

RESUMO

Follicular helper T (Tfh) cells comprise an important subset of helper T cells; however, their relationship with other helper lineages is incompletely understood. Herein, we showed interleukin-12 acting via the transcription factor STAT4 induced both Il21 and Bcl6 genes, generating cells with features of both Tfh and Th1 cells. However, STAT4 also induced the transcription factor T-bet. With ChIP-seq, we defined the genome-wide targets of T-bet and found that it repressed Bcl6 and other markers of Tfh cells, thereby attenuating the nascent Tfh cell-like phenotype in the late phase of Th1 cell specification. Tfh-like cells were rapidly generated after Toxoplasma gondii infection in mice, but T-bet constrained Tfh cell expansion and consequent germinal center formation and antibody production. Our data argue that Tfh and Th1 cells share a transitional stage through the signal mediated by STAT4, which promotes both phenotypes. However, T-bet represses Tfh cell functionalities, promoting full Th1 cell differentiation.


Assuntos
Diferenciação Celular , Células Th1/citologia , Células Th1/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/parasitologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Imunofenotipagem , Interferon gama/metabolismo , Interleucina-12/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-6 , Fator de Transcrição STAT4/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th1/metabolismo , Toxoplasma
14.
Cell ; 146(4): 544-54, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21835447

RESUMO

The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.


Assuntos
Montagem e Desmontagem da Cromatina , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Vírus do Tumor Mamário do Camundongo , Camundongos , Modelos Biológicos , Método de Monte Carlo , Nucleossomos/metabolismo , Receptores de Estrogênio/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo
15.
Mol Cell ; 43(1): 145-55, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21726817

RESUMO

Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome.


Assuntos
Cromatina/metabolismo , Modelos Genéticos , Receptores de Glucocorticoides/metabolismo , Fator de Transcrição AP-1/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Cromatina/química , Regulação da Expressão Gênica , Genoma , Ligantes , Camundongos , Receptores de Glucocorticoides/química , Elementos Reguladores de Transcrição , Fator de Transcrição AP-1/química
16.
EMBO J ; 30(15): 3028-39, 2011 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-21701563

RESUMO

Cell-selective glucocorticoid receptor (GR) binding to distal regulatory elements is associated with cell type-specific regions of locally accessible chromatin. These regions can either pre-exist in chromatin (pre-programmed) or be induced by the receptor (de novo). Mechanisms that create and maintain these sites are not well understood. We observe a global enrichment of CpG density for pre-programmed elements, and implicate their demethylated state in the maintenance of open chromatin in a tissue-specific manner. In contrast, sites that are actively opened by GR (de novo) are characterized by low CpG density, and form a unique class of enhancers devoid of suppressive effect of agglomerated methyl-cytosines. Furthermore, treatment with glucocorticoids induces rapid changes in methylation levels at selected CpGs within de novo sites. Finally, we identify GR-binding elements with CpGs at critical positions, and show that methylation can affect GR-DNA interactions in vitro. The findings present a unique link between tissue-specific chromatin accessibility, DNA methylation and transcription factor binding and show that DNA methylation can be an integral component of gene regulation by nuclear receptors.


Assuntos
Metilação de DNA , DNA/metabolismo , Elementos Facilitadores Genéticos , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Camundongos , Ligação Proteica
17.
Nat Genet ; 43(3): 264-8, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21258342

RESUMO

Development, differentiation and response to environmental stimuli are characterized by sequential changes in cellular state initiated by the de novo binding of regulated transcriptional factors to their cognate genomic sites. The mechanism whereby a given regulatory factor selects a limited number of in vivo targets from a myriad of potential genomic binding sites is undetermined. Here we show that up to 95% of de novo genomic binding by the glucocorticoid receptor, a paradigmatic ligand-activated transcription factor, is targeted to preexisting foci of accessible chromatin. Factor binding invariably potentiates chromatin accessibility. Cell-selective glucocorticoid receptor occupancy patterns appear to be comprehensively predetermined by cell-specific differences in baseline chromatin accessibility patterns, with secondary contributions from local sequence features. The results define a framework for understanding regulatory factor-genome interactions and provide a molecular basis for the tissue selectivity of steroid pharmaceuticals and other agents that intersect the living genome.


Assuntos
Cromatina/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular , Desoxirribonuclease I/farmacologia , Camundongos , Especificidade de Órgãos , Ligação Proteica/genética , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo
18.
Nat Cell Biol ; 11(9): 1093-102, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19684579

RESUMO

Studies on glucocorticoid receptor (GR) action typically assess gene responses by long-term stimulation with synthetic hormones. As corticosteroids are released from adrenal glands in a circadian and high-frequency (ultradian) mode, such treatments may not provide an accurate assessment of physiological hormone action. Here we demonstrate that ultradian hormone stimulation induces cyclic GR-mediated transcriptional regulation, or gene pulsing, both in cultured cells and in animal models. Equilibrium receptor-occupancy of regulatory elements precisely tracks the ligand pulses. Nascent RNA transcripts from GR-regulated genes are released in distinct quanta, demonstrating a profound difference between the transcriptional programs induced by ultradian and constant stimulation. Gene pulsing is driven by rapid GR exchange with response elements and by GR recycling through the chaperone machinery, which promotes GR activation and reactivation in response to the ultradian hormone release, thus coupling promoter activity to the naturally occurring fluctuations in hormone levels. The GR signalling pathway has been optimized for a prompt and timely response to fluctuations in hormone levels, indicating that biologically accurate regulation of gene targets by GR requires an ultradian mode of hormone stimulation.


Assuntos
Corticosteroides/farmacologia , Ritmo Circadiano/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Transcrição Genética/efeitos dos fármacos , Corticosteroides/sangue , Adrenalectomia , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Polimerase II/metabolismo , Ratos , Ratos Sprague-Dawley , Elementos de Resposta/genética , Fatores de Tempo
19.
J Cell Sci ; 122(Pt 3): 345-56, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19126674

RESUMO

Gene regulation often appears deterministic in the average cell population, but transcription is a probabilistic process at the single-cell level. Although many mechanisms are invoked to account for this behavior, it is difficult to determine how cell-to-cell variation in the interactions of transcription factors with target chromatin impact transcriptional output. Here, we use cells that contain a 200-copy tandem array of promoter or reporter gene units to simultaneously visualize transient interaction, equilibrium or steady-state binding of fluorescent-protein-labeled glucocorticoid receptor with its DNA response elements, the recruitment of diverse coregulators, and transcriptional output at the single-cell level. These regulatory proteins associate with target chromatin via a probabilistic mechanism that produces cell-to-cell variability in binding. The multiple steps of this process are partially independent and differ between individual regulators. The association level of each regulator influences the transcriptional output in individual cells, but this does not account for all transcriptional heterogeneity. Additionally, specific combinatorial interactions of the glucocorticoid receptor and coregulators with response elements regulate transcription at the single-cell level. Like many endogenous genes, the average array transcriptional activity evolves over time. This apparently deterministic average temporal promoter progression involves changes in the probability that specific combinatorial glucocorticoid receptor and coregulator interactions will occur on the response elements in single cells. These data support the emerging ;return-to-template' transcription model, which mechanistically unifies the observed extremely transient interactions between the transcription factor and response elements, cell-to-cell variability in steady-state association of factors with chromatin, and the resulting heterogeneous gene expression between individual cells.


Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Transcrição Genética , Animais , Linhagem Celular Tumoral , Cromatina/genética , Genes Reporter/genética , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional
20.
Endocrinology ; 150(4): 1766-74, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19131569

RESUMO

We have characterized the kinetic response of gene targets throughout the murine genome to transcriptional modulation by the glucocorticoid receptor (GR). In contrast to a model in which multiple genes are either repressed or activated during the GR response, the vast majority of responsive genes are subject to complex regulation profiles, frequently with alternate activation and repression phases. We also observe that GR binding at response elements does not always correlate with the target gene response profile. Thus, the cellular response to GR stimulation involves a highly orchestrated series of regulatory actions and not simply a binary response to hormone.


Assuntos
Receptores de Glucocorticoides/genética , Elementos de Resposta/genética , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica/genética , Cinética , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Receptores de Glucocorticoides/fisiologia , Elementos de Resposta/fisiologia , Fatores de Tempo , Ativação Transcricional/genética , Ativação Transcricional/fisiologia
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