RESUMEN
BACKGROUND: Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. RESULTS: Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. CONCLUSION: These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación Viral de la Expresión Génica , Duplicado del Terminal Largo de VIH/genética , VIH-1/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/metabolismo , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Células HeLa , Humanos , Transcripción GenéticaRESUMEN
The Ras-association domain family 1 (RASSF1) gene has seven different isoforms; isoform A is a tumor-suppressor gene (RASSF1A). The promoter of RASSF1A is inactivated in many cancers, whereas the expression of another major isoform, RASSF1C, is not affected. Here, we show that RASSF1C, but not RASSF1A, interacts with betaTrCP. Binding of RASSF1C to betaTrCP involves serine 18 and serine 19 of the SS(18)GYXS(19) motif present in RASSF1C but not in RASSF1A. This motif is reminiscent of the canonical phosphorylation motif recognized by betaTrCP; however, surprisingly, the association between RASSF1C and betaTrCP does not occur via the betaTrCP substrate binding domain, the WD40 repeats. Overexpression of RASSF1C, but not of RASSF1A, resulted in accumulation and transcriptional activation of the beta-catenin oncogene, due to inhibition of its betaTrCP-mediated degradation. Silencing of RASSF1A by small interfering RNA was sufficient for beta-catenin to accumulate, whereas silencing of both RASSF1A and RASSF1C had no effect. Thus, RASSF1A and RASSF1C have opposite effects on beta-catenin degradation. Our results suggest that RASSF1C expression in the absence of RASSF1A could play a role in tumorigenesis.
Asunto(s)
Proteínas Supresoras de Tumor/metabolismo , beta Catenina/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Secuencias de Aminoácidos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Silenciador del Gen , Células HeLa , Humanos , Unión Proteica , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor/genética , beta Catenina/antagonistas & inhibidores , beta Catenina/biosíntesis , beta Catenina/genéticaRESUMEN
Although the proteasome facilitates transcription from several yeast promoters, it is unclear if its role is proteolytic or which subunits are involved. We show that the proteasome regulates the HIV-1 promoter in both proteolytic and nonproteolytic modes. In the absence of transcription factor, Tat, proteasome was associated with promoter and coding regions, and its proteolytic activity regulated the level of basal transcription emanating from the promoter. Tat switched the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1, which favors proteasome dissociation into 19S and 20S particles. Gel filtration chromatography showed that expression of both Tat and PAAF1 enhanced the abundance of a 19S-like complex in nuclear extracts. 19S, but not 20S, subunits were strongly recruited to the promoter in the presence of Tat and PAAF1 and coactivated Tat-dependent transcription. 19S components facilitated transcriptional elongation and may be involved in clearance of paused transcriptional elongation complexes from the promoter.
Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH , Células HeLa , Humanos , Regiones Promotoras Genéticas , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
ATF4 plays a crucial role in the cellular response to stress and multiple stress responses pathways converge to the translational up-regulation of ATF4. ATF4 is a substrate of the SCF(betaTrCP) ubiquitin ligase that binds to betaTrCP through phosphorylation on a DSGXXXS motif. We show here that ATF4 stability is also modulated by the histone acetyltransferase p300, which induces ATF4 stabilization by inhibiting its ubiquitination. Despite p300 acetylates ATF4, we found that p300-mediated ATF4 stabilization is independent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant ATF4-K311R. ATF4 deleted of its p300 binding domain is no more stabilized by p300 nor recruited into nuclear speckles. In consequence of ATF4 stabilization, both p300 and the catalytically inactive enzyme increase ATF4 transcriptional activity.
Asunto(s)
Factor de Transcripción Activador 4/química , Proteína de Unión a CREB/fisiología , Transcripción Genética , Factores de Transcripción p300-CBP/química , Secuencias de Aminoácidos , Western Blotting , Proteína de Unión a CREB/química , Catálisis , Línea Celular , Núcleo Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Silenciador del Gen , Vectores Genéticos , Glutatión Transferasa/metabolismo , Células HeLa , Histona Acetiltransferasas/química , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Mutación , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Transfección , Ubiquitina/química , Ubiquitina/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The human immunodeficiency virus type 1 Vpu protein acts as an adaptor for the proteasomal degradation of CD4 by recruiting CD4 and beta-transducin repeat-containing protein (betaTrCP), the receptor component of the multisubunit SCF-betaTrCP E3 ubiquitin ligase complex. We showed that the expression of a Vpu-green fluorescent fusion protein prevented the proteosomal degradation of betaTrCP substrates such as beta-catenin, IkappaBalpha, and ATF4, which are normally directly targeted to the proteasome for degradation. Beta-catenin was translocated into the nucleus, whereas the tumor necrosis factor-induced nuclear translocation of NFkappaB was impaired. Beta-catenin was also up-regulated in cells producing Vpu+ human immunodeficiency virus type 1 but not in cells producing Vpu-deficient viruses. The overexpression of ATF4 also provoked accumulation of beta-catenin, but to a lower level than that resulting from the expression of Vpu. Finally, the expression of Vpu induces the exclusion of betaTrCP from the nucleus. These data suggest that Vpu is a strong competitive inhibitor of betaTrCP that impairs the degradation of SCFbetaTrCP substrates as long as Vpu has an intact phosphorylation motif and can bind to betaTrCP.