RESUMEN
Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.
Asunto(s)
Proteínas de Ciclo Celular/genética , Citomegalovirus/inmunología , ADN Viral/genética , Epigénesis Genética , Histona Desacetilasas/genética , Factor B de Elongación Transcripcional Positiva/genética , Factores de Transcripción/genética , Azepinas/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzodiazepinas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/inmunología , Ciclina T/genética , Ciclina T/inmunología , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/inmunología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Replicación del ADN/efectos de los fármacos , ADN Viral/antagonistas & inhibidores , ADN Viral/inmunología , Genes Inmediatos-Precoces , Genes Reporteros , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/inmunología , Interacciones Huésped-Patógeno , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Biológicos , Factor B de Elongación Transcripcional Positiva/inmunología , Cultivo Primario de Células , Regiones Promotoras Genéticas , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , Células THP-1 , Talidomida/análogos & derivados , Talidomida/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/inmunología , Transcripción Genética , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacosRESUMEN
In contrast to the common role of histone deacetylases (HDACs) for gene repression, HDAC activity provides a required positive function for IFN-stimulated gene (ISG) expression. Here, we show that HDAC1/2 as components of the Sin3A complex are required for ISG transcriptional elongation but not for recruitment of RNA polymerase or transcriptional initiation. Transcriptional arrest by HDAC inhibition coincides with failure to recruit the epigenetic reader Brd4 and elongation factor P-TEFb due to sequestration of Brd4 on hyperacetylated chromatin. Brd4 availability is regulated by an equilibrium cycle between opposed acetyltransferase and deacetylase activities that maintains a steady-state pool of free Brd4 available for recruitment to inducible promoters. An ISG expression signature is a hallmark of interferonopathies and other autoimmune diseases. Combined inhibition of HDAC1/2 and Brd4 resolved the aberrant ISG expression detected in cells derived from patients with two inherited interferonopathies, ISG15 and USP18 deficiencies, defining a novel therapeutic approach to ISG-associated autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Regulación de la Expresión Génica/inmunología , Enfermedades Genéticas Congénitas/inmunología , Histona Desacetilasa 1/inmunología , Histona Desacetilasa 2/inmunología , Proteínas Nucleares/inmunología , Regiones Promotoras Genéticas/inmunología , Factores de Transcripción/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Proteínas de Ciclo Celular , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Células HEK293 , Células HeLa , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Humanos , Interferones/genética , Interferones/inmunología , Proteínas Nucleares/genética , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/inmunología , Factores de Transcripción/genéticaRESUMEN
Positive transcription elongation factor b (P-TEFb) is an RNA polymerase II elongation factor which exists as multiple complexes in human cells. These complexes contain cyclin-dependent kinase 9 as the catalytic subunit and different cyclin subunits-cyclin T1, T2a, T2b, or K. Cyclin T1 is targeted by the human immunodeficiency virus (HIV) Tat protein to activate transcription of the HIV provirus. Expression of this P-TEFb subunit is highly regulated in monocyte-derived macrophages (MDMs). Cyclin T1 is induced early during differentiation and is shut off later by proteasome-mediated proteolysis. Cyclin T1 can be reinduced by pathogen-associated molecular patterns (PAMPs) or HIV infection. In this study, we analyzed regulation of P-TEFb in MDMs by examining 7SK small nuclear RNA and the HEXIM1 protein; these factors associate with P-TEFb and are thought to regulate its function. 7SK and HEXIM1 were induced early during differentiation, and this correlates with increased overall transcription. 7SK expression remained high, but HEXIM1 was shut off later during differentiation by proteasome-mediated proteolysis. Significantly, the cyclin T2a subunit of P-TEFb was not shut off during differentiation, and it was not induced by activation. Induction of cyclin T1 by PAMPs was found to be a slow process and did not involve an increase in cyclin T1 mRNA levels. Treatment of MDMs with PAMPs or a proteasome inhibitor induced cyclin T1 to a level equivalent to treatment with both agents together, suggesting that PAMPs and proteasome inhibitors act at a similar rate-limiting step. It is therefore likely that cyclin T1 induction by PAMPs is the result of a reduction in proteasome-mediated proteolysis.
Asunto(s)
Ciclinas/biosíntesis , Ciclinas/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Peptidoglicano/farmacología , Procesamiento Postranscripcional del ARN/inmunología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Ciclina T , Ciclinas/genética , Infecciones por VIH/inmunología , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/inmunología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Factor B de Elongación Transcripcional Positiva/inmunología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Nuclear Pequeño/efectos de los fármacos , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/inmunología , Proteínas de Unión al ARN/efectos de los fármacos , Proteínas de Unión al ARN/inmunología , Factores de Transcripción , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Regulación hacia ArribaRESUMEN
OBJECTIVE: This study was undertaken to determine whether 7SK small nuclear RNA (snRNA), which has been proposed to function as an inhibitor of Tat cofactor P-TEFb, plays a role in transcriptional latency in T cells. DESIGN AND METHODS: The association of 7SK snRNA with P-TEFb was investigated in resting and activated peripheral blood lymphocytes (PBLs). Primary PBLs were isolated by standard methods and activated with phytohemagglutinin (PHA). Levels of 7SK snRNA were determined by Northern blotting and levels of the P-TEFb subunits cyclin-dependent kinase 9 and cyclin T1 were analyzed by immunoblotting. RESULTS: The association of 7SK snRNA with P-TEFb complexes was specific. Following activation of PBLs, the levels of 7SK snRNA increased in a manner similar to U1 and U6 snRNA, sn RNAs involved in positive aspects of cellular gene expression. Unexpectedly, the association of 7SK snRNA with P-TEFb increased dramatically following lymphocyte activation. CONCLUSION: Increased association of 7SK snRNA with P-TEFb in activated lymphocytes correlates with increased global transcription. This suggests that 7SK snRNA is unlikely to promote transcriptional latency in lymphocytes through an association with P-TEFb; it also suggests that the proposal that the association of 7SK snRNA with P-TEFb acts to inhibit transcriptional elongation needs to be re-evaluated.
