Nuclear factor Y is required for basal activation and chromatin accessibility of fibroblast growth factor receptor 2 promoter in osteoblast-like cells.
Fibroblast growth factor receptor 2 (FGFR2) plays an important regulatory role in bone development. However, the regulatory mechanisms controlling FGFR2 expression remain poorly understood. Here we have identified a role for the nuclear factor Y (NF-Y) in constitutive activation of FGFR2. A unique DNase I hypersensitive site was detected in the region encompassing nucleotides -270 to +230 after scanning a large range covering 33.3 kilobases around the transcription start site of FGFR2. Using a PCR-based chromatin accessibility assay, an open chromatin conformation was detected around the proximal 5' fragment of FGFR2 gene. Deletion constructs of the 5'-flanking region of FGFR2 were fused to a luciferase reporter gene. After transient transfection in C3H10T1/2, ME3T3-E1, and C2C12 as well as primary osteoblasts, a minimal region -86/+139 that is highly homologous to the human sequence and bears a CCAAT box was identified as the core promoter. Electrophoretic mobility shift assay supershift and chromatin immunoprecipitation demonstrated that the CCAAT box was the binding site for NF-Y. Deletion of NF-Y consensus sequence resulted in the total loss of NF-Y promoter activity. Overexpression of NF-Y protein and transfection of NF-Y small interfering RNAs in the cells substantially changed the promoter activity. Moreover, NF-Y small interfering RNAs greatly inhibited the endogenous FGFR2 transcription level and the chromatin accessibility and H3 acetylation across the promoter. Taken together, our results demonstrate that interaction of NF-Y at the CCAAT box is pivotal to FGFR2 gene transcription partly through the construction of a local open chromatin configuration across the promoter.
Sun
Fenyong
F
Institute of Genetic Engineering, Jinan University, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Guangzhou 510632, China.
Xie
Qiuling
Q
Institute of Genetic Engineering, Jinan University, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Guangzhou 510632, China.
Ma
Ji
J
Institute of Genetic Engineering, Jinan University, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Guangzhou 510632, China.
Yang
Songhai
S
Shaoguan Tielu Hospital, Shaoguan, 512023, Guangdong, China.
Chen
Qiongyu
Q
Institute of Genetic Engineering, Jinan University, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Guangzhou 510632, China.
Hong
An
A
Institute of Genetic Engineering, Jinan University, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Guangzhou 510632, China. Electronic address: tha@jnu.edu.cn.
D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000502:physiology*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D004247:DNA; D024202:Electrophoretic Mobility Shift Assay; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D023081:CCAAT-Binding Factor; D002843:Chromatin; D006657:Histones; D017468:Receptors, Fibroblast Growth Factor; C402244:nuclear factor Y; D016222:Fibroblast Growth Factor 2; D004247:DNA