The Roles Of EGR1 In RNA Polymerase ? And ?-directed Gene Transcription

Early growth response gene 1(EGR1)is a member of the early growth response gene family.Activation of EGR1 gene expression is associated with cell growth and differentiation.Since EGR1 binding sites are in presence of the promoter regions of many growth factor genes and protooncogenes,it suggests that EGR1 could be involved in the development of tumors.Previous studies in our laboratory have shown that Filamin A(FLNA)can be localized in the nucleolar region of cells and inhibits gene transcription directed by RNA polymerase I(Pol ?)and RNA polymerase ?(Pol ?)and cell proliferation.Recently,meseenger RNA-seq analysis using FLNA knockdown cell lines has showed that FLNA knockdown enhances EGR1 expression EGR1 at mRNA level.These results suggest that EGR1 might be involved in the regulation of gene transcription directed by Pol ? and Pol ?.Therefore,the obective of this study is to investigate role of EGR1 in Pol ? and Pol ? gene transcription and explore the mechanism by which EGR1 regulate Pol ? and Pol ? gene transcription.In order to accomplish this project,EGR1 shRNA expression lentiviral vectors were obtained by gene cloning and co-transfected along with the packaging vectors into various cell types;EGR1 knockdown stable cell lines and FLNA-EGR1 double knockdown stable cell lines were established.RT-qPCR and Western Blot were used to detect the Pol ? and Pol ? gene transcription in these stable cell lines.The results showed that EGR1 enhanced transcription of a subset of genes directed by Pol ? and Pol ?.The data from cell counting and MTT assays demonstrated that EGR1 stimulated cell proliferation.To verify the role of EGR1 in Pol ? and Pol ? gene transcription and cell proliferation,EGR1-overexpressing stable cell line was generated,and Pol ? and Pol ? gene transcription and cell proliferation were analyzed by RT-qPCR,Western Blot,cell counting and MTT assays,the results confirmed that EGR1 overexpression,indeed,increased Pol ? and Pol ? gene transcription and cell proliferation.Taken togehter,these data indicat that EGR1 can act as a positive factor to regulate Pol ? and Pol ? gene transcription and cell proliferation.In addition,the signaling pathway factors that have previously been demonstrated to regulate Pol ? and Pol ? gene transcription were anlyzed using EGR1 knockdown cell lines.The data revealed that PTEN expression was significantly increased,while RhoA expression was significantly decreased in EGR1 knockdown stable cell lines.There are no significant change for p53 expression and other proteins.These results suggest that EGR1 may regulate Pol ? and Pol ? gene transcription and cell proliferation by affecting expression of PTEN and RhoA.Collectively,we have found that EGR1 differentially regulates transcription of the genes directed by Pol ? and Pol ? and cell proliferation,and that EGR1 may regulate these activities by affecting the expression of PTEN and RhoA.The results from this study have extended the function of EGR1 in cells,and provide novel insights into the mechanism of gene transcription directed by RNA Pol ? and Pol ? and the mechanism of tumor cell proliferation.