RESUMEN
Transcription elongation is increasingly recognized as an important mechanism of gene regulation. Here, we show that microprocessor controls gene expression in an RNAi-independent manner. Microprocessor orchestrates the recruitment of termination factors Setx and Xrn2, and the 3'-5' exoribonuclease, Rrp6, to initiate RNAPII pausing and premature termination at the HIV-1 promoter through cleavage of the stem-loop RNA, TAR. Rrp6 further processes the cleavage product, which generates a small RNA that is required to mediate potent transcriptional repression and chromatin remodeling at the HIV-1 promoter. Using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq), we identified cellular gene targets whose transcription is modulated by microprocessor. Our study reveals RNAPII pausing and premature termination mediated by the co-operative activity of ribonucleases, Drosha/Dgcr8, Xrn2, and Rrp6, as a regulatory mechanism of RNAPII-dependent transcription elongation.
Asunto(s)
Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Regulación Viral de la Expresión Génica , VIH-1/genética , ARN Helicasas/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Secuencia de Bases , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , ADN Helicasas , Duplicado del Terminal Largo de VIH , Humanos , Datos de Secuencia Molecular , Enzimas Multifuncionales , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Viral/química , ARN Viral/genética , Factores de Transcripción/metabolismoRESUMEN
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
HIV-1 transactivator Tat has greatly contributed to our understanding of transcription elongation by RNAPII. We purified HIV-1 Tat-associated factors from HeLa nuclear extract and show that Tat forms two distinct and stable complexes. Tatcom1 consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL, and ELL), and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 are required for Tat transactivation. Tatcom2 is composed of CDK9, CycT1, and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SK RNA, leading to the formation of a stress-resistant 7SK snRNP particle. Besides the identification of factors required for Tat transactivation and important for P-TEFb function, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors associated in a single complex and acting at the same promoter.
Asunto(s)
Núcleo Celular/metabolismo , VIH-1/genética , ARN Viral/biosíntesis , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Línea Celular , Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , VIH-1/metabolismo , Células HeLa , N-Metiltransferasa de Histona-Lisina , Humanos , Complejos Multiproteicos , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Estrés Fisiológico , Factores de Transcripción , Factores de Elongación Transcripcional/metabolismo , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Budding yeast Cdc13, Stn1, and Ten1 form the CST complex to protect telomeres from lethal DNA degradation. It remains unknown whether similar complexes are conserved in higher eukaryotes or not. Here we isolated mammalian STN1 and TEN1 homologs and CTC1 (conserved telomere maintenance component 1). The three proteins contain putative OB-fold domains and form a complex called CST, which binds to single-stranded DNA with high affinity in a sequence-independent manner. CST associates with a fraction of telomeres consistently during the cell cycle, in quiescent cells and Pot1-knockdown cells. It does not colocalize with replication foci in S phase. Significant increases in the abundance of single-stranded G-strand telomeric DNA were observed in Stn1-knockdown cells. We propose that CST is a replication protein A (RPA)-like complex that is not directly involved in conventional DNA replication at forks but plays a role in DNA metabolism frequently required by telomeres.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Animales , Secuencia de Bases , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes , Homología de Secuencia de Aminoácido , Complejo Shelterina , Proteínas de Unión a Telómeros/químicaRESUMEN
Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase alpha inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.