The Role Of Transcription Factor AP2a In Gene Transcription Directed By RNA Polymerase ?

AP2a(AP2 ?)is an important transcription factor in mammalian cell,and is involved in varieties of physiological and biochemical activities.The data from the early work of this project have shown that FLNA can regulate RNA Polymerase ?-directed gene transcription and the expression of transcription factors BRF1 and TF?C2.Furthermore,mRNA-seq analysis showed that knockdown of FLNA significantly increased AP2 a mRNA expression in SaOS2 FLNA-depleted stable cell line.RT-qPCR and Western blot were used to detect AP2 a expression in SaOS2 and HeLa FLNA shRNA stable cell lines.The data confirmed that FLNA knockdown indeed increased AP2 a expression.Since FLNA can inhibit the expression of AP2 a,BRF1,TF?C2 and Pol ? genes,we asked whether AP2 a mediated gene transcription of RNA polymerase ?.In this project we aimed to investigate the role of AP2 a in Pol ? gene transcription and the mechanism by which AP2 a modulates Pol ? gene transcription.In order to achieve the research objectives,293 T and HeLa AP2 a shRNA stable cell lines was prepared,and the expression of BRF1 and TF?C2 and Pol ? genes were analyzed.The results showed that AP2 a knockdown was able to significantly reduce BRF1,TF?C2 and Pol ? genes expression,indicating that AP2 a is required for transcription of Pol ? genes.To confirm this finding,293 T cell were transfected with AP2 a expression vector,the results showed that AP2 a overexpression significantly increased BRF1,TF?C2,and Pol ? gene expression.These results confirmed that AP2 a is required for maintaining transcription of Pol ? genes.Since FLNA knockdown can enhance Pol ? gene transcription in several tumor cell lines,whether AP2 a knockdown in FLNA-depleted cell lines would suppress Pol ? gene expression was unclear.Both FLNA-and AP2a-depleted cell lines were prepared and gene expression was analyzed by RT-qPCR and Western blot.The data showed that knockdown of both FLNA and AP2 a downregulated the expression of BRF1,TF?C2 and Pol ? genes when compared with that in FLNA-depleted cell line.To understand how AP2 a regulates the expression of BRF1,TF?C2 and Pol ? genes,transcription factor binding sites in BRF1 and TF?C2 gene promoters were analyzed using online software.As expected,both BRF1 and TF?C2 promoters contain Ap2 a binding sites.Therefore,we supposed that AP2 a could regulate transcription of Pol ? genes bybinding to BRF1 and TF?C2 promoters.To test the hypothesis,BRF1 and TF?C2 promoters were respectively cloned into the reporter gene vector;the resulting vectors were transfected into stable cell lines,followed by analysis of luciferase activity.The data showed that expression of reporter gene in the AP2a-depleted cells was significantly lower than that in the cells stably expressing control shRNA.These results suggest that AP2 a can modulate expression of Pol ? genes through affecting transcription of BRF1 and TF?C2 genes.These findings not only deepen our understanding of AP2 a function in cells,but also provide a basis to further explore the mechanism of cell proliferation mediated by AP2 a.