Suppression of the human parathyroid hormone promoter by vitamin D involves displacement of NF-Y binding to the vitamin D response element.
An earlier report in the literature indicated the vitamin D response element (VDRE) in the human parathyroid hormone (hPTH) promoter could be specifically bound by an unidentified transcription factor in addition to the vitamin D receptor (VDR) complex. We confirmed that OK and HeLa cell nuclear extracts formed a specific complex with the hPTH VDRE that was insensitive to competition with other VDRE sequences. However, this factor could be competed for by a consensus NF-Y DNA-binding site, and an anti-NF-Y antibody was able to supershift the bound band. Mutational analysis indicated that the NF-Y-binding site partially overlapped the 3' portion of the VDRE. Transfection studies using an hPTH promoter construct in Drosophila SL2 cells demonstrated strong synergistic transactivation by NF-Y interactions with both the VDRE site and a previously described distal NF-Y-binding site. Finally, mobility shift studies indicated that the VDR heterodimer competed with NF-Y for binding to the VDRE sequence, and NF-Y-stimulated activity of the hPTH promoter could be suppressed in a hormone-dependent manner when the VDR heterodimer complex was coexpressed in SL2 cells. In summary, these findings establish the presence of a proximal NF-Y-binding site in the hPTH promoter and highlight the potential for synergism between distal and proximal NF-Y DNA elements to strongly enhance transcription. Furthermore, findings suggest that the repressive effects of vitamin D on hPTH gene transcription may involve displacement of NF-Y binding to the proximal site by the VDR heterodimer, which subsequently attenuates synergistic transactivation.
Koszewski
Nicholas J
NJ
Division of Nephrology, Bone and Mineral Metabolism and Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky 40536-0298, USA. njhosz0@uky.edu
Alimov
Alexander P
AP
Park-Sarge
Ok-Kyong
OK
Malluche
Hartmut H
HH
D000818:Animals; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D019281:Dimerization; D004330:Drosophila; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry; D010281:Parathyroid Hormone / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D020218:Response Elements; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014807:Vitamin D / Q000378:metabolism*
D023081:CCAAT-Binding Factor; D009841:Oligonucleotides; D010281:Parathyroid Hormone; D011994:Recombinant Proteins; D014807:Vitamin D; D004247:DNA