Study Of Regulation Of Importin 4 Gene Expression By ETS Transcription Factor

Importin 4/karyopherin 4(IPO4)which is one of nuclear import receptors plays an important role in substrate nuclear import.However,how IPO4 gene expression is regulated remains unknown.This study shows that the Ets family members ELK1 and GABP can bind to the IPO4 promoter and regulate IPO4 transcription,we identify a minimal functional promoter region of IPO4,located within-108-+118bp.Furthermore,we find that GGAA motif is essential for IPO4 promoter activity,this sequence is coincided with binding consensus of ETS transcription factors,and two members of this family,GABP and Elk1,are found to activate or repress IPO4 promoter activity.Additionally,GABPa/?1 selectively activates a minimal promoter 3 fold,ELK1 represses a minimal promoter about 70%in 293T cells,and this activation is dependent on the Ets-binding site(EBS).We also find that increased amount of GABP can gradually release repression of IPO4 by Elk1,in reverse,Elk1,which can abolish promotion via GABP,we conclude that GABP and Elk1 competes for regulation of IPO4.We certify that ELK1 and GABP bind to GGAA motif of IPO4 promoter by EMSA,They compete for binding to GGAA motif in EMSA assay.And We identify that Elk1 binds to IPO4 promoter by chromatin immunoprecipitation(ChIP)assay.Overexpression of Elk1 in HeLa cells leads to IPO4 protein downregulation,in versus,overexpression of GABP enhances IPO4 protein level.Small interfering RNA depletion of ELK1 in HeLa cells results in upregulation of IPO4 protein.For further study the function of IPO4,we knockdown IPO4 in 3T3-L1,and this can promote differentiation of 3T3-L1,this demostrates that IPO4 participate the process of preadipocyte differentiation.Thus,we have identified ELK1 and GABP as critical and functionally relevant Ets factors that regulate IPO4 expression via the Ets binding site.