Cytoskeletal Filamin A Differentially Modulates RNA Polymerase III Gene Transcription in Transformed Cell Lines

Abstract

Edited by Linda SpremulliCytoskeletal 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.Eukaryotic RNA polymerase (pol) 3 III mediates the synthesis of many non-coding RNAs that include tRNA, 5S rRNA, U6 RNA, 7SL RNA, and other small RNAs. These RNA products account for about 15% of total cellular transcripts and are involved in the regulation of protein biogenesis, RNA splicing, protein transportation, and protein-coding gene transcription (1-3). In cancer, aberrant expression of pol III products strongly correlates with the transformed state (4 -6). Abnormal high expression of pol III products in cancer is attributed to increased expression of pol III transcription factors, discharge of repression by tumor suppressors, and activation of oncogene expression (2).Cytoskeletal filamin A (FLNA) is a 280-kDa protein comprised of two N-terminal calponin homology domains and a long C-terminal rod-like domain. FLNA cross-links with F-actin through the calponin homology domains to maintain the stability of the cellular 3D network. In addition, FLNA acts as a scaffold that binds to over 90 diverse functional proteins to regulate numerous cellular processes (7-9), including cell adhesion, migration, proliferation, angiogenesis, cell signaling, DNA repair, and transcription (10 -18). It is well established that FLNA mutations cause several congenital diseases, including defects of the brain, heart, and skeleton (8). FLNA can play dual roles in cancer development; that is, FLNA can promote or inhibit cancer growth and development depending on particular circumstances (10). However, the mechanisms underlying these events are not fully understood.We sh...

The transcription factor Sp1 modulates RNA polymerase III gene transcription by controlling BRF1 and GTF3C2 expression in human cells

“…S9C). The latter result is consistent with that obtained in our previous work (37). We next examined whether knockdown of both FLNA and Sp1 affected expression of BRF1 and GTF3C2.…”

“…These data indicate that FLNA can inhibit both Sp1 expression and Pol III gene transcription in normal cell lines. Collectively, the above results, along with our previous findings (37), confirm that alteration of FLNA expression can simultaneously affect Pol III-directed gene transcription and expression of BRF1, GTF3C2, and Sp1 genes. Because BRF1 and GTFC3C2 belong to a subunit of TFIIIB and TFIIIC, respectively, and their promoters, BRF1P4 and GTF3C2P2, contain putative Sp1-binding sites, we sought to establish whether Sp1 affected the regulation of Pol III gene transcription by influencing BRF1 and TFIIIC2 expression.…”

“…Our previous work has shown that FLNA differentially modulates transcription of the Pol III-transcribed genes in transformed cell lines and that FLNA's actin-binding domain can mediate the regulation of Pol III-directed gene transcription (37). When investigating the mechanism by which FLNA regulates Pol III gene transcription, we found that FLNA depletion increased the expression of BRF1 and GTF3C2 genes in SaOS2 cell lines (Fig.…”

“…Cytoskeletal filamin A (FLNA) is involved in a variety of cellular activities, including cell migration, proliferation, and RNA polymerase I-mediated gene transcription (36). Our previous work has shown that FLNA differentially represses the expression of RNA polymerase III-transcribed genes (37). In the present study, we initially found that knockdown of FLNA enhanced the expression of TFIIB-related factor 1 (BRF1), general transcription factor III C subunit 2 (GTF3C2), and transcription factor Sp1.…”

Influenza A Virus Nucleoprotein Activates the JNK Stress-Signaling Pathway for Viral Replication by Sequestering Host Filamin A Protein

“…By cross-linking actin filaments, FLNA can finely tune the dynamic three-dimensional structure of a cell. In addition, FLNA has been reported to interact with more than 90 functionally diverse range of cellular proteins, including cytosolic effector proteins, adhesion proteins, transmembrane proteins, DNA damage repair and transcription proteins, signaling molecules, and ion channel proteins (Yue et al, 2013;Wang et al, 2016). Because of the diverse range of interacting partners, FLNA plays a role in integrating cellular mechanics as well as acts as a signaling scaffold by connecting cellular processes to the dynamic regulation of the actin cytoskeleton (Stossel et al, 2001;Feng and Walsh, 2004).…”