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1.
Mol Ther ; 28(3): 820-829, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-31981492

RESUMO

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm2) compared with untreated eyes (25 ± 5/mm2; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.


Assuntos
Aquaporina 1/genética , Corpo Ciliar/metabolismo , Edição de Genes , Terapia Genética , Glaucoma/genética , Glaucoma/terapia , Animais , Aquaporina 1/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas , Dependovirus/genética , Expressão Gênica , Marcação de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Glaucoma/diagnóstico , Glaucoma/fisiopatologia , Camundongos , Retina/metabolismo , Retina/patologia , Transdução Genética , Transgenes
2.
PLoS One ; 15(1): e0227520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923266

RESUMO

Glucocorticoid (GR) and mineralocorticoid receptors (MR) are believed to classically bind DNA as homodimers or MR-GR heterodimers to influence gene regulation in response to pulsatile basal or stress-evoked glucocorticoid secretion. Pulsed corticosterone presentation reveals MR and GR co-occupy DNA only at the peaks of glucocorticoid oscillations, allowing interaction. GR DNA occupancy was pulsatile, while MR DNA occupancy was prolonged through the inter-pulse interval. In mouse mammary 3617 cells MR-GR interacted in the nucleus and at a chromatin-associated DNA binding site. Interactions occurred irrespective of ligand type and receptors formed complexes of higher order than heterodimers. We also detected MR-GR interactions ex-vivo in rat hippocampus. An expanded range of MR-GR interactions predicts structural allostery allowing a variety of transcriptional outcomes and is applicable to the multiple tissue types that co-express both receptors in the same cells whether activated by the same or different hormones.


Assuntos
Núcleo Celular/metabolismo , DNA/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Cromatina/metabolismo , Corticosterona/farmacologia , DNA/química , Dimerização , Hipocampo/metabolismo , Masculino , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Estrutura Quaternária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/genética , Alinhamento de Sequência , Ritmo Ultradiano
3.
Endocrinology ; 160(5): 1044-1056, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980716

RESUMO

Mineralocorticoid and glucocorticoid receptors (MRs and GRs) constitute a functionally important dual receptor system detecting and transmitting circulating corticosteroid signals. High expression of MRs and GRs occurs in the same cells in the limbic system, the primary site of glucocorticoid action on cognition, behavior, and mood; however, modes of interaction between the receptors are poorly characterized. We used chromatin immunoprecipitation with nucleotide resolution using exonuclease digestion, unique barcode, and single ligation (ChIP-nexus) for high-resolution genome-wide characterization of MR and GR DNA binding profiles in neuroblastoma cells and demonstrate recruitment to highly similar DNA binding sites. Expressed MR or GR showed differential regulation of endogenous gene targets, including Syt2 and Ddc, whereas coexpression produced augmented transcriptional responses even when MRs were unable to bind DNA (MR-XDBD). ChIP confirmed that MR-XDBD could be tethered to chromatin by GR. Our data demonstrate that MR can interact at individual genomic DNA sites in multiple modes and suggest a role for MR in increasing the transcriptional response to glucocorticoids.


Assuntos
Glucocorticoides/farmacologia , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , DNA/genética , DNA/metabolismo , Camundongos , Ligação Proteica , Interferência de RNA , Ratos , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Elementos de Resposta/genética
4.
Endocrinology ; 158(5): 1486-1501, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28200020

RESUMO

Glucocorticoids regulate hippocampal function in part by modulating gene expression through the glucocorticoid receptor (GR). GR binding is highly cell type specific, directed to accessible chromatin regions established during tissue differentiation. Distinct classes of GR binding sites are dependent on the activity of additional signal-activated transcription factors that prime chromatin toward context-specific organization. We hypothesized a stress context dependency for GR binding in hippocampus as a consequence of rapidly induced stress mediators priming chromatin accessibility. Using chromatin immunoprecipitation sequencing to interrogate GR binding, we found no effect of restraint stress context on GR binding, although analysis of sequences underlying GR binding sites revealed mechanistic detail for hippocampal GR function. We note enrichment of GR binding sites proximal to genes linked to structural and organizational roles, an absence of major tethering partners for GRs, and little or no evidence for binding at negative glucocorticoid response elements. A basic helix-loop-helix motif closely resembling a NeuroD1 or Olig2 binding site was found underlying a subset of GR binding sites and is proposed as a candidate lineage-determining transcription factor directing hippocampal chromatin access for GRs. Of our GR binding sites, 54% additionally contained half-sites for nuclear factor (NF)-1 that we propose as a collaborative or general transcription factor involved in hippocampal GR function. Our findings imply a dose-dependent and context-independent action of GRs in the hippocampus. Alterations in the expression or activity of NF-1/basic helix-loop-helix factors may play an as yet undetermined role in glucocorticoid-related disease susceptibility and outcome by altering GR access to hippocampal binding sites.


Assuntos
Cromatina/genética , Sequências Hélice-Alça-Hélice , Hipocampo/metabolismo , Fatores de Transcrição NFI/metabolismo , Receptores de Glucocorticoides/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Cromatina/metabolismo , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Genoma , Imunoprecipitação , Masculino , Ligação Proteica , Ratos , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Análise de Sequência , Estresse Fisiológico
5.
Biophys J ; 109(6): 1227-39, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26278180

RESUMO

The glucocorticoid receptor (GR) is a steroid-hormone-activated transcription factor that modulates gene expression. Transcriptional regulation by the GR requires dynamic receptor binding to specific target sites located across the genome. This binding remodels the chromatin structure to allow interaction with other transcription factors. Thus, chromatin remodeling is an essential component of GR-mediated transcriptional regulation, and understanding the interactions between these molecules at the structural level provides insights into the mechanisms of how GR and chromatin remodeling cooperate to regulate gene expression. This study suggests models for the assembly of the SWI/SNF-A (SWItch/Sucrose-NonFermentable) complex and its interaction with the GR. We used the PRISM algorithm (PRotein Interactions by Structural Matching) to predict the three-dimensional complex structures of the target proteins. The structural models indicate that BAF57 and/or BAF250 mediate the interaction between the GR and the SWI/SNF-A complex, corroborating experimental data. They further suggest that a BAF60a/BAF155 and/or BAF60a/BAF170 interaction is critical for association between the core and variant subunits. Further, we model the interaction between GR and CCAAT-enhancer-binding proteins (C/EBPs), since the GR can regulate gene expression indirectly by interacting with other transcription factors like C/EBPs. We observe that GR can bind to bZip domains of the C/EBPα homodimer as both a monomer and dimer of the DNA-binding domain. In silico mutagenesis of the predicted interface residues confirm the importance of these residues in binding. In vivo analysis of the computationally suggested mutations reveals that double mutations of the leucine residues (L317D+L335D) may disrupt the interaction between GR and C/EBPα. Determination of the complex structures of the GR is of fundamental relevance to understanding its interactions and functions, since the function of a protein or a complex is dictated by its structure. In addition, it may help us estimate the effects of mutations on GR interactions and signaling.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Modelos Moleculares , Receptores de Glucocorticoides/metabolismo , Algoritmos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Computadores Analógicos , Dimerização , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Mutação , Ratos , Receptores de Glucocorticoides/química , Transfecção
6.
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
7.
Mol Cell Endocrinol ; 348(2): 383-93, 2012 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-21872640

RESUMO

In recent years it has become evident that glucocorticoid receptor (GR) action in the nucleus is highly dynamic, characterized by a rapid exchange at the chromatin template. This stochastic mode of GR action couples perfectly with a deterministic pulsatile availability of endogenous ligand in vivo. The endogenous glucocorticoid hormone (cortisol in man and corticosterone in rodent) is secreted from the adrenal gland with an ultradian rhythm made up of pulses at approximately hourly intervals. These two components - the rapidly fluctuating ligand and the rapidly exchanging receptor - appear to have evolved to establish and maintain a system that is exquisitely responsive to the physiological demands of the organism. In this review, we discuss recent and innovative work that questions the idea of steady state, static hormone receptor responses, and replaces them with new concepts of stochastic mechanisms and oscillatory activity essential for optimal function in molecular and cellular systems.


Assuntos
Receptores de Glucocorticoides/fisiologia , Ciclos de Atividade , Animais , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , Glucocorticoides/fisiologia , Humanos , Modelos Moleculares , Transporte Proteico , Receptores de Glucocorticoides/metabolismo
8.
Mol Endocrinol ; 25(6): 944-54, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21511880

RESUMO

Glucocorticoid (GC) hormones are secreted from the adrenal gland in a characteristic pulsatile pattern. This ultradian secretory activity exhibits remarkable plasticity, with distinct changes in response to both physiological and stressful stimuli in humans and experimental animals. It is therefore important to understand how the pattern of GC exposure regulates intracellular signaling through the GC receptor (GR). We have previously shown that each pulse of ligand initiates rapid, transient GR activation in several physiologically relevant and functionally diverse target cell types. Using chromatin immunoprecipitation assays, we detect cyclical shifts in the net equilibrium position of GR association with regulatory elements of GC-target genes and have investigated in detail the mechanism of pulsatile transcriptional regulation of the GC-induced Period 1 gene. Transient recruitment of the histone acetyl transferase complex cAMP response element-binding protein (CREB) binding protein (CBP)/p300 is found to precisely track the ultradian hormone rhythm, resulting in transient localized net changes in lysine acetylation at GC-regulatory regions after each pulse. Pulsatile changes in histone H4 acetylation and concomitant recruitment of RNA polymerase 2 precede ultradian bursts of Period 1 gene transcription. Finally, we report the crucial underlying role of the intranuclear heat shock protein 90 molecular chaperone complex in pulsatile GR regulation. Pharmacological interference of heat shock protein 90 (HSP90) with geldanamycin during the intranuclear chaperone cycle completely ablated GR's cyclical activity, cyclical cAMP response element-binding protein (CREB) binding protein (CBP)/p300 recruitment, and the associated cyclical acetylation at the promoter region. These data imply a key role for an intact nuclear chaperone cycle in cyclical transcriptional responses, regulated in time by the pattern of pulsatile hormone.


Assuntos
Ciclos de Atividade/efeitos dos fármacos , Proteína de Ligação a CREB/genética , Corticosterona/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Hidrocortisona/farmacologia , Receptores de Glucocorticoides/genética , Fatores de Transcrição de p300-CBP/genética , Acetilação , Animais , Benzoquinonas/farmacologia , Proteína de Ligação a CREB/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Histonas/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Leupeptinas/farmacologia , Ligantes , Camundongos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Transporte Proteico/efeitos dos fármacos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Glucocorticoides/metabolismo , Elementos Reguladores de Transcrição , Transcrição Gênica , Fatores de Transcrição de p300-CBP/metabolismo
9.
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 Gênica/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
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