Investigating The Role Of EGR1 In RNA Polymerase Ⅰ-directed Gene Transcription And Its Regulatory Mechanism

RNA polymerase Ⅰ(Pol Ⅰ),an important enzyme that comprises 14 subunits,is responsible for the synthesis of 45 S pre-ribosomal RNA(Pre-r RNA),which is further processed into mature 18 S,5.8S and 28 S ribosomal RNAs(r RNAs).These r RNAs account for about 60% of total transcription activity in cells.Therefore,Pol Ⅰ-directed gene transcription plays an essential role in maintaining cellular life activities.Deregulation of Pol Ⅰ gene transcription is closely correlated with tumorigenesis and cancer cell proliferation.Early growth response gene 1(EGR1)is an important transcription factor mainly involved in tissue damage,immune response and fibrosis.Recent studies have demonstrated that EGR1 is closely associated with tumor occurrence and progression,and it can promote the proliferation activity of several cancer cell types.Whether there is connection between EGR1 and Pol Ⅰ–directed transcription remains unclear.This study aimed to investigate the role of EGR1 in Pol Ⅰ-directed gene transcription and its regulatory mechanism.To determine the connection between EGR1 and Pol Ⅰ-directed transcription,,we analyzed the localization of EGR1 in cells.The result from immunofluporecence assays showed that EGR1 could co-localize with nucleolar proteins NPM1 in the nucleoli of He La,Hep G2 and AGS cells,and EGR1 protein had been detected in the purified nucleolar fration,indicating EGR1 is present in the nucleoli of these cell types.Nucleoli are the places that Pol Ⅰ transcription occurs;thus,we next determined the effect of EGR1 expression change on Pol Ⅰ-directed transcription.RT-q PCR results showed that EGR1 knockdown could inhibit the expression of Pol Ⅰ products,while EGR1 overexpression increased the expression of Pol Ⅰ transcription products,indicating that EGR1 plays a positive role in gene transcription directed by Pol Ⅰ.Whether alteration of Pol Ⅰ products mediated by EGR1 affects cell proliferation is unclear.Cell proliferation assays showed that He La and Hep G2 cell proliferation was significantly inhibited when EGR1 was stably knocked down,on the contrary,cell proliferation was enhanced when EGR1 was overexpressed.Furthermore,the presence of Pol Ⅰ transcription-specific inhibitor repressed the increase of cell proliferation and Pol Ⅰ transcription activity,indicating that EGR1 promotes cell proliferation by regulating Pol Ⅰ-directed transcription.Mechanistically,EGR1 could co-localized with transcription factor UBF and TⅠF-ⅠA in the nucleoli of He La and Hep G2 cells.The co-immunoprecipitation results confirmed that EGR1 could bind to UBF,TⅠF-ⅠA,TBP and subunit TAF1 A.We show that EGR1 binds to the r DNA promoter,and its expression change affected the activity of the r DNA promoter and the recruitments of Pol Ⅰ transcription machinery factors on the promoter.Ⅰn addition,the expression change of EGR1 affected the expression of RPA40 and TⅠFⅠA protein;meanwhile,influencing TⅠF-ⅠA m RNA and the activity of the TⅠF-ⅠA promoter,indicating that EGR1 can regulate the expression of TⅠF-ⅠA at the transcriptional or RNA processing steps.These results suggest that EGR1 modulates Pol Ⅰ-directed genes transcription by affecting expression of TⅠF-ⅠA and other factors.The findings from this study not only extend the role of EGR1 in the transcriptional regulation but also provide novel insights into the mechanisms of Pol Ⅰ-directed transcription and cell proliferation.