RESUMEN
Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation.
Asunto(s)
ARN Polimerasa III/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores de Transcripción TFIII/metabolismo , Sitios de Unión , Línea Celular , Proliferación Celular , Proteína p300 Asociada a E1A/metabolismo , Filaminas/antagonistas & inhibidores , Filaminas/genética , Filaminas/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Polimerasa III/genética , ARN Interferente Pequeño/metabolismo , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/genética , Factores Asociados con la Proteína de Unión a TATA/antagonistas & inhibidores , Factores Asociados con la Proteína de Unión a TATA/genética , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factores de Transcripción TFIII/antagonistas & inhibidores , Factores de Transcripción TFIII/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
We carried out padlock capture, a high-resolution RNA allelotyping method, to study X chromosome inactivation (XCI). We examined the gene reactivation pattern along the inactive X (Xi), after Xist (X-inactive specific transcript), a prototype long non-coding RNA essential for establishing X chromosome inactivation (XCI) in early embryos, is conditionally deleted from Xi in somatic cells (Xi∆Xist). We also monitored the behaviors of X-linked non-coding transcripts before and after XCI. In each mutant cell line, gene reactivation occurs to ~6% genes along Xi∆Xist in a recognizable pattern. Genes with upstream regions enriched for SINEs are prone to be reactivated. SINE is a class of retrotransposon transcribed by RNA polymerase III (Pol III). Intriguingly, a significant fraction of Pol III transcription from non-coding regions is not subjected to Xist-mediated transcriptional silencing. Pol III inhibition affects gene reactivation status along Xi∆Xist, alters chromatin configuration and interferes with the establishment XCI during in vitro differentiation of ES cells. These results suggest that Pol III transcription is involved in chromatin structure re-organization during the onset of XCI and functions as a general mechanism regulating chromatin configuration in mammalian cells.
