Investigating The Mechanism Of RNA Polymerase ? Gene Transcription Mediated By Filamin A

Filamin A(FLNA)is a kind of important mammalian cell cytoskeleton protein,encoded by genes on the X chromosome,can combine with more than 90 kinds of proteins,in transcriptional regulation,signal transduction,DNA damage repair,and maintain stable and cytoskeleton and cellular morphology play an important role in a variety of biological activities.Study shows that the FLNA mutation will lead to a mammalian heart,brain,bone,nerve,embryos and other tissue metabolism disorder and inherited diseases,and FLNA in a wide variety of cancer also plays an important role in the process,can be used as the target of clinical treatment.Our previous work showed that FLNA,as a nucleoprotein,can inhibit the transcription of RNA polymerase I(Pol I).Subsequent studies have found that FLNA differentially inhibits gene transcription directed by Pol ? in some tumor cells.But the specific regulatory mechanism is not fully clear.Therefore,in this study,we conducted an in-depth study on the transcriptional mechanism of FLNA inhibiting RNA polymerase ?.In previous studies,we through the analysis of the chromatin immune co-precipitation(ChIP assay),and compared with the 293 T cell line transfected Control shRNA,BRF1 and TF?C2 in the 5S rRNA,7SL RNA and tRNA-Met gene promoter region recruit increased significantly in 293 T FLNA knockdown stable cell lines,while promoting the transcription of these genes.Therefore,the expression of Pol ? transcription factors TF?C2 and BRF1 in FLNA knockdown and control cell lines were detected by RT-qPCR and Western Blot.The results showed that FLNA was able to inhibit the expression of TF?C2 and BRF1.Next,we used the software to analyze the protein binding sites of BRF1 and TF?C2 promoters.The results showed that there were SP1 binding sites upstream of both BRF1 and TF?C2 promoter regions.Moreover,the mRNA-seq analysis of SaOS2 FLNA knockdown stable cell lines showed that the knockdown of FLNA significantly increased the expression of SP1 mRNA.RT-qPCR and Western Blot methods were used to detect SP1 expression in SaOS2,293 T and He La FLNA knockdown and control stable cell lines.The dataconfirmed that FLNA knockdown increased the expression of SP1.When SP1 expression was further disrupted in HeLa FLNA knockdown stable cell line,Knockdown of FLNA and SP1 inhibited BRF1 and TF?C2 expression and pol ? gene transcription.Reporter assays were performed using BRF1 or TFIIC2 promoter-driving reporter expression vectors and FLNA-SP1 double knockdown and FLNA knockdown stable cell lines.The results demonstrated that knockdownof FLNA and SP1 inhibited the activities of BRF1 and TF?C2 promoters.These results suggest that FLNA can regulate the expression of BRF1 and TF?C2 and Pol ? gene transcription by changing SP1 expression.These findings not only provide a new insight into the inhibitory mechanism of Pol? gene transcription by FLNA,but also deepen our understanding of the mechanism of tumor cell proliferation.