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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. [ABSTRACT FROM AUTHOR]

RNA polymerase II (pol II) is required for the transcription of all protein-coding genes and as such represents a major enzyme whose activity is tightly regulated. Transcriptional initiation therefore requires numerous general transcriptional factors and cofactors that associate with pol II at the core promoter to form a pre-initiation complex. Transcription factor IIA (TFIIA) is a general cofactor that binds TFIID and stabilizes the TFIID-DNA complex during transcription initiation. Previous studies showed that TFIIA can make contact with the DNA sequence upstream or downstream of the TATA box, and that the region bound by TFIIA could overlap with the elements recognized by another factor, TFIIB, at adenovirus major late core promoter. Whether core promoters contain a DNA motif recognized by TFIIA remains unknown. Here we have identified a core promoter element upstream of the TATA box that is recognized by TFIIA. A search of the human promoter database revealed that many natural promoters contain a TFIIA recognition element (IIARE). We show that the IIARE enhances TFIIA-promoter binding and enhances the activity of TATA-containing promoters, but represses or activates promoters that lack a TATA box. Chromatin immunoprecipitation assays revealed that the IIARE activates transcription by increasing the recruitment of pol II, TFIIA, TAF4, and P300 at TATA-dependent promoters. These findings extend our understanding of the role of TFIIA in transcription, and provide new insights into the regulatory mechanism of core promoter elements in gene transcription by pol II. [ABSTRACT FROM AUTHOR]

Cytoskeletal filamin A (FLNA) is an important protein involved in multiple cellular processes. Previous studies have shown that FLNA can promote or inhibit cancer growth and development; however, the mechanisms underlying these events are not fully understood. Here we show that, in both 293T and SaOS2 cells, knockdown of FLNA significantly enhanced transcription of RNA polymerase (pol) III-transcribed genes except for a subset of tRNA genes. In contrast, re-expression of FLNA in an FLNA-deficient melanoma cell line (A7) repressed transcription of all pol III-transcribed genes, suggesting that FLNA inhibits pol III transcription in a cell type-specific manner. Chromatin immunoprecipitation assays revealed that the repression of pol III gene transcription by FLNA correlates with the decreased occupancy of the RNA pol III transcription machinery at promoters. Immunofluorescence microscopy and coimmunoprecipitation assays revealed that FLNA can associate with the RNA pol III transcription machinery through its actin-binding domain within nuclei. Mechanistic analysis revealed that FLNA suppresses pol III gene transcription by confining the recruitment of the RNA pol III transcription machinery at the promoters of the genes that are sensitive to the alteration of FLNA expression. These findings not only extend the understanding of FLNA function in cells but also provide novel insights into the mechanism by which FLNA represses cell proliferation. [ABSTRACT FROM AUTHOR]