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1.
Cell ; 180(5): 847-861.e15, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142678

RESUMO

Early life environmental exposure, particularly during perinatal period, can have a life-long impact on organismal development and physiology. The biological rationale for this phenomenon is to promote physiological adaptations to the anticipated environment based on early life experience. However, perinatal exposure to adverse environments can also be associated with adult-onset disorders. Multiple environmental stressors induce glucocorticoids, which prompted us to investigate their role in developmental programming. Here, we report that perinatal glucocorticoid exposure had long-term consequences and resulted in diminished CD8 T cell response in adulthood and impaired control of tumor growth and bacterial infection. We found that perinatal glucocorticoid exposure resulted in persistent alteration of the hypothalamic-pituitary-adrenal (HPA) axis. Consequently, the level of the hormone in adults was significantly reduced, resulting in decreased CD8 T cell function. Our study thus demonstrates that perinatal stress can have long-term consequences on CD8 T cell immunity by altering HPA axis activity.


Assuntos
Infecções Bacterianas/imunologia , Desenvolvimento Embrionário/imunologia , Glucocorticoides/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/genética , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Dexametasona/farmacologia , Desenvolvimento Embrionário/genética , Feminino , Glucocorticoides/imunologia , Glucocorticoides/metabolismo , Humanos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Interleucina-4/farmacologia , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Neoplasias/induzido quimicamente , Neoplasias/genética , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/imunologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Receptores de Glucocorticoides/genética , Transdução de Sinais/genética
2.
Cell ; 173(4): 864-878.e29, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29681454

RESUMO

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Família 4 do Citocromo P450/deficiência , Família 4 do Citocromo P450/genética , Descoberta de Drogas , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glucocorticoides/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
3.
Cell ; 165(3): 593-605, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-27062924

RESUMO

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.


Assuntos
Fator 3-alfa Nuclear de Hepatócito/metabolismo , Cromatina/metabolismo , Desoxirribonucleases/metabolismo , Humanos , Células MCF-7 , Receptores de Estrogênio/genética , Receptores de Glucocorticoides/genética , Fatores de Transcrição/metabolismo
4.
Nat Immunol ; 19(9): 954-962, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30127438

RESUMO

Controlling the balance between immunity and immunopathology is crucial for host resistance to pathogens. After infection, activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to the production of glucocorticoids. However, the pleiotropic effects of these steroid hormones make it difficult to delineate their precise role(s) in vivo. Here we found that the regulation of natural killer (NK) cell function by the glucocorticoid receptor (GR) was required for host survival after infection with mouse cytomegalovirus (MCMV). Mechanistically, endogenous glucocorticoids produced shortly after infection induced selective and tissue-specific expression of the checkpoint receptor PD-1 on NK cells. This glucocorticoid-PD-1 pathway limited production of the cytokine IFN-γ by spleen NK cells, which prevented immunopathology. Notably, this regulation did not compromise viral clearance. Thus, the fine tuning of NK cell functions by the HPA axis preserved tissue integrity without impairing pathogen elimination, which reveals a novel aspect of neuroimmune regulation.


Assuntos
Glucocorticoides/metabolismo , Infecções por Herpesviridae/imunologia , Células Matadoras Naturais/fisiologia , Muromegalovirus/fisiologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Células Cultivadas , Feminino , Sistema Hipotálamo-Hipofisário , Imunidade Inata , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroimunomodulação , Especificidade de Órgãos , Sistema Hipófise-Suprarrenal , Receptores de Glucocorticoides/genética , Transdução de Sinais , Carga Viral
5.
Mol Cell ; 82(3): 555-569.e7, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35063133

RESUMO

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Nucleares/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Sobrevivência Celular , Células HEK293 , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Células K562 , Cinética , Simulação de Acoplamento Molecular , Proteínas Nucleares/genética , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
Mol Cell ; 82(4): 803-815.e5, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35077705

RESUMO

The hormone-stimulated glucocorticoid receptor (GR) modulates transcription by interacting with thousands of enhancers and GR binding sites (GBSs) throughout the genome. Here, we examined the effects of GR binding on enhancer dynamics and investigated the contributions of individual GBSs to the hormone response. Hormone treatment resulted in genome-wide reorganization of the enhancer landscape in breast cancer cells. Upstream of the DDIT4 oncogene, GR bound to four sites constituting a hormone-dependent super enhancer. Three GBSs were required as hormone-dependent enhancers that differentially promoted histone acetylation, transcription frequency, and burst size. Conversely, the fourth site suppressed transcription and hormone treatment alleviated this suppression. GR binding within the super enhancer promoted a loop-switching mechanism that allowed interaction of the DDIT4 TSS with the active GBSs. The unique functions of each GR binding site contribute to hormone-induced transcriptional heterogeneity and demonstrate the potential for targeted modulation of oncogene expression.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Dexametasona/farmacologia , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores de Glucocorticoides/agonistas , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
7.
Nat Immunol ; 18(5): 573-582, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28288100

RESUMO

Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8+ T cell differentiation.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Epigênese Genética , Listeriose/imunologia , Receptores de Glucocorticoides/metabolismo , Subpopulações de Linfócitos T/fisiologia , Fator de Transcrição YY1/metabolismo , Animais , Linfócitos T CD8-Positivos/microbiologia , Diferenciação Celular/genética , Biologia Computacional , Elementos Facilitadores Genéticos/genética , Perfilação da Expressão Gênica , Histonas/metabolismo , Memória Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Glucocorticoides/genética , Subpopulações de Linfócitos T/microbiologia , Fator de Transcrição YY1/genética
8.
Immunity ; 53(3): 581-596.e5, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32707034

RESUMO

Glucocorticoids (GC) are the mainstay treatment option for inflammatory conditions. Despite the broad usage of GC, the mechanisms by which GC exerts its effects remain elusive. Here, utilizing murine autoimmune and allergic inflammation models, we report that Foxp3+ regulatory T (Treg) cells are irreplaceable GC target cells in vivo. Dexamethasone (Dex) administered in the absence of Treg cells completely lost its ability to control inflammation, and the lack of glucocorticoid receptor in Treg cells alone resulted in the loss of therapeutic ability of Dex. Mechanistically, Dex induced miR-342-3p specifically in Treg cells and miR-342-3p directly targeted the mTORC2 component, Rictor. Altering miRNA-342-3p or Rictor expression in Treg cells dysregulated metabolic programming in Treg cells, controlling their regulatory functions in vivo. Our results uncover a previously unknown contribution of Treg cells during glucocorticoid-mediated treatment of inflammation and the underlying mechanisms operated via the Dex-miR-342-Rictor axis.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Inflamação/tratamento farmacológico , MicroRNAs/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Anti-Inflamatórios/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/biossíntese , Receptores de Glucocorticoides/genética , Linfócitos T Reguladores/metabolismo
9.
Immunity ; 53(3): 658-671.e6, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32937153

RESUMO

Identifying signals in the tumor microenvironment (TME) that shape CD8+ T cell phenotype can inform novel therapeutic approaches for cancer. Here, we identified a gradient of increasing glucocorticoid receptor (GR) expression and signaling from naïve to dysfunctional CD8+ tumor-infiltrating lymphocytes (TILs). Conditional deletion of the GR in CD8+ TILs improved effector differentiation, reduced expression of the transcription factor TCF-1, and inhibited the dysfunctional phenotype, culminating in tumor growth inhibition. GR signaling transactivated the expression of multiple checkpoint receptors and promoted the induction of dysfunction-associated genes upon T cell activation. In the TME, monocyte-macrophage lineage cells produced glucocorticoids and genetic ablation of steroidogenesis in these cells as well as localized pharmacologic inhibition of glucocorticoid biosynthesis improved tumor growth control. Active glucocorticoid signaling associated with failure to respond to checkpoint blockade in both preclinical models and melanoma patients. Thus, endogenous steroid hormone signaling in CD8+ TILs promotes dysfunction, with important implications for cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Glucocorticoides/metabolismo , Macrófagos/metabolismo , Melanoma Experimental/patologia , Microambiente Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linhagem Celular Tumoral , Hematopoese/imunologia , Fator 1-alfa Nuclear de Hepatócito/biossíntese , Inibidores de Checkpoint Imunológico , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/imunologia
10.
Nat Rev Mol Cell Biol ; 18(3): 159-174, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28053348

RESUMO

The glucocorticoid receptor (GR) is a constitutively expressed transcriptional regulatory factor (TRF) that controls many distinct gene networks, each uniquely determined by particular cellular and physiological contexts. The precision of GR-mediated responses seems to depend on combinatorial, context-specific assembly of GR-nucleated transcription regulatory complexes at genomic response elements. In turn, evidence suggests that context-driven plasticity is conferred by the integration of multiple signals, each serving as an allosteric effector of GR conformation, a key determinant of regulatory complex composition and activity. This structural and mechanistic perspective on GR regulatory specificity is likely to extend to other eukaryotic TRFs.


Assuntos
Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Acetilação , Animais , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Fosforilação , Domínios Proteicos , Receptores de Glucocorticoides/genética , Elementos de Resposta , Sumoilação , Transcrição Gênica
11.
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
12.
Mol Cell ; 76(4): 531-545.e5, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31706703

RESUMO

The glucocorticoid receptor (GR) is a potent metabolic regulator and a major drug target. While GR is known to play integral roles in circadian biology, its rhythmic genomic actions have never been characterized. Here we mapped GR's chromatin occupancy in mouse livers throughout the day and night cycle. We show how GR partitions metabolic processes by time-dependent target gene regulation and controls circulating glucose and triglycerides differentially during feeding and fasting. Highlighting the dominant role GR plays in synchronizing circadian amplitudes, we find that the majority of oscillating genes are bound by and depend on GR. This rhythmic pattern is altered by high-fat diet in a ligand-independent manner. We find that the remodeling of oscillatory gene expression and postprandial GR binding results from a concomitant increase of STAT5 co-occupancy in obese mice. Altogether, our findings highlight GR's fundamental role in the rhythmic orchestration of hepatic metabolism.


Assuntos
Cromatina/metabolismo , Relógios Circadianos , Ritmo Circadiano , Dieta Hiperlipídica , Gorduras na Dieta/metabolismo , Metabolismo Energético , Fígado/metabolismo , Obesidade/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Glicemia/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/sangue , Modelos Animais de Doenças , Metabolismo Energético/genética , Jejum/metabolismo , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , Gluconeogênese , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , PPAR alfa/genética , PPAR alfa/metabolismo , Período Pós-Prandial , Receptores de Glucocorticoides/deficiência , Receptores de Glucocorticoides/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Via Secretória , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Triglicerídeos/sangue
13.
Immunity ; 47(2): 214-216, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28813652

RESUMO

In this issue of Immunity,Oh et al. (2017) provide insight into the molecular effects of glucocorticoid receptor (GR) activation at a clinically relevant time point, after an inflammatory stimulus. They report that GR activation causes a global reduction in NF-κB binding, as well as time-dependent transcriptional effects.


Assuntos
NF-kappa B/imunologia , Receptores de Glucocorticoides/genética , Cromatina , Glucocorticoides , Ligação Proteica , Ativação Transcricional/efeitos dos fármacos
14.
Circ Res ; 134(10): 1306-1326, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38533639

RESUMO

BACKGROUND: Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. METHODS AND RESULTS: Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. CONCLUSIONS: Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.


Assuntos
Ritmo Circadiano , Miócitos Cardíacos , Receptores de Glucocorticoides , Animais , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Camundongos , Miócitos Cardíacos/metabolismo , Masculino , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/genética , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Conexina 43/metabolismo , Conexina 43/genética , Camundongos Knockout , Potenciais de Ação
15.
Mol Cell Proteomics ; 23(3): 100741, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38387774

RESUMO

Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for the treatment of solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic. GR is a member of the nuclear receptor family of transcription factors and heavily relies on interactions with coregulator proteins for its transcriptional activity. To elucidate the role of the GR interactome in the differential transcriptional activity of GR following treatment with the selective GR agonist and modulator dagrocorat compared to classic (ant)agonists, we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that dagrocorat and the antagonist RU486 both reduced GR interaction with CREB-binding protein/p300 and the mediator complex compared to the full GR agonist dexamethasone. Chromatin immunoprecipitation assays revealed that these changes in GR interactome were accompanied by reduced GR chromatin occupancy with dagrocorat and RU486. Our data offer new insights into the role of differential coregulator recruitment in shaping ligand-specific GR-mediated transcriptional responses.


Assuntos
Benzamidas , Cromatina , Fenantrenos , Receptores de Glucocorticoides , Humanos , Receptores de Glucocorticoides/genética , Mifepristona/farmacologia , Complexo Mediador/metabolismo , Glucocorticoides/farmacologia , Glucocorticoides/metabolismo , Dexametasona/farmacologia
16.
Nucleic Acids Res ; 52(18): e88, 2024 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-39248105

RESUMO

Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provides the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs). DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking. These results were compared to those from standard ChIP-Seq that employs sonication and to CUT&RUN which utilizes MNase to fragment the genomic DNA. Our findings indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions.


Assuntos
Imunoprecipitação da Cromatina , Cromatina , RNA Polimerase II , Fatores de Transcrição , RNA Polimerase II/metabolismo , Humanos , Cromatina/metabolismo , Cromatina/genética , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina/métodos , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , DNA/metabolismo , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Ligação a CCCTC/metabolismo , Ligação Proteica
17.
Nucleic Acids Res ; 52(2): 625-642, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38015476

RESUMO

Treatment of prostate cancer relies predominantly on the inhibition of androgen receptor (AR) signaling. Despite the initial effectiveness of the antiandrogen therapies, the cancer often develops resistance to the AR blockade. One mechanism of the resistance is glucocorticoid receptor (GR)-mediated replacement of AR function. Nevertheless, the mechanistic ways and means how the GR-mediated antiandrogen resistance occurs have remained elusive. Here, we have discovered several crucial features of GR action in prostate cancer cells through genome-wide techniques. We detected that the replacement of AR by GR in enzalutamide-exposed prostate cancer cells occurs almost exclusively at pre-accessible chromatin sites displaying FOXA1 occupancy. Counterintuitively to the classical pioneer factor model, silencing of FOXA1 potentiated the chromatin binding and transcriptional activity of GR. This was attributed to FOXA1-mediated repression of the NR3C1 (gene encoding GR) expression via the corepressor TLE3. Moreover, the small-molecule inhibition of coactivator p300's enzymatic activity efficiently restricted GR-mediated gene regulation and cell proliferation. Overall, we identified chromatin pre-accessibility and FOXA1-mediated repression as important regulators of GR action in prostate cancer, pointing out new avenues to oppose steroid receptor-mediated antiandrogen resistance.


Assuntos
Cromatina , Neoplasias da Próstata , Receptores de Glucocorticoides , Humanos , Masculino , Antagonistas de Androgênios/farmacologia , Linhagem Celular Tumoral , Cromatina/genética , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo
18.
Nucleic Acids Res ; 52(13): 7740-7760, 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-38932701

RESUMO

Androgen receptor- (AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 gene targets include the glucocorticoid receptor (GR; NR3C1) and the NE splicing factor SRRM4, which are key drivers of lineage plasticity. Thus, OC2, despite its previously described NEPC driver function, can indirectly activate a portion of the AR cistrome through epigenetic activation of GR. Mechanisms by which OC2 regulates gene expression include promoter binding, enhancement of genome-wide chromatin accessibility, and super-enhancer reprogramming. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC and support enhanced efforts to therapeutically target OC2 as a means of suppressing treatment-resistant disease.


Assuntos
Adenocarcinoma , Benzamidas , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Nitrilas , Neoplasias da Próstata , Receptores Androgênicos , Receptores de Glucocorticoides , Masculino , Humanos , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/tratamento farmacológico , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Benzamidas/farmacologia , Linhagem Celular Tumoral , Nitrilas/farmacologia , Feniltioidantoína/farmacologia , Feniltioidantoína/análogos & derivados , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Epigênese Genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/tratamento farmacológico , Animais , Linhagem da Célula/genética , Camundongos
19.
J Biol Chem ; 300(8): 107581, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39025450

RESUMO

Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T-cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for the stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.


Assuntos
Receptores de Glucocorticoides , Humanos , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células Jurkat , Proteólise , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Apoptose , Sistemas CRISPR-Cas , Linhagem Celular Tumoral
20.
J Virol ; 98(1): e0143623, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38084958

RESUMO

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.


Assuntos
Infecções por Herpesviridae , Herpesvirus Bovino 1 , Receptores de Glucocorticoides , Fator de Transcrição Sp1 , Animais , Bovinos , Camundongos , Corticosteroides/metabolismo , Dexametasona/farmacologia , DNA/metabolismo , Herpesvirus Bovino 1/fisiologia , Plicamicina/análogos & derivados , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Fator de Transcrição Sp1/metabolismo
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