The Basal Transcriptional Activation Of FGFR2 Is Mediated By Nuclear Factor Y In Osteoblast-Like Cells

Objectives:Fibroblast growth factor receptor 2(FGFR2) plays an important regulatory role in bone development.However,the regulatory mechanisms by which FGFR2 expression is controlled remain poorly understood.In this study,we focused our work on the regulation of FGFR2 promoter activation as well as the local chromosome accessibility.Methods:Firstly,the activation region of transcription was identified by DNaseâ… hypersensitive and CHART-PCR assays.Secondly,the up promoter element(UPE) of FGFR2 was determined through Dual-Luciferase reporter system.Thirdly,we used EMSA,supershift assay,chromatin immunoprecipitation and RNA interference to search for the specific transcription factor responsible for the promoter activation.Finally,the regulatory mechanism of key transcription factor to FGFR2 promoter was examined further by RNA interference.Results:DNaseâ… hypersensitive and CHART-PCR assays confined the region of proximal promoter region of FGFR2 to-143/+45.Serial deletion mutations of the 5'flanking region of FGFR2 gene based on Dual-Luciferase reporter system demonstrated the core promoter region in -86/-48bp fragment,which included a CCAAT box.The results of EMSA,supershift assay and chromatin immunoprecipitation indicated that the transcription factor NF-Y bond to the CCAAT box.Deletion of NF-Y consensus sequence resulted in the total loss of FGFR2 promoter activity. Transfection of NF-Y siRNAs in the cells substantially changed the promoter activity.Moreover, NF-Y siRNAs greatly inhibited the endogenous FGFR2 transcription level and the chromatin accessibility and H3 acetylation across the promoter.Conclusions:Taken together,our results demonstrates that interaction of NF-Y and CCAAT box determines the basal activation of FGFR2 gene promoter,and is partly caused by formation of a local open chromatin configuration across the promoter.