Dissection of differentially regulated (G+C)-rich promoters of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene

The PTH/PTH-related peptide (PTHrP) receptor (PTHR) is required for normal skeletal development, and a wide array of physiological responses mediated by PTH and PTHrP. We have previously identified three promoters, P1-P3, whi ch control human PTHR gene transcription. P2 and P3 are (G+C)-rich, functio n in a number of tissues, lie within the same CpG island, and display many hallmarks of housekeeping promoters. However, they are differentially regul ated during development as P2, but not P3, functions in fetal tissues. Here , we have used both stably and transiently transfected human osteoblast-lik e cells to delineate regions of P2 and P3 required for promoter activity. D eletion analyses performed in stably transfected cells indicated that seque nces extending from -91 to -12 relative to the transcription start site wer e required for function of the P2 promoter. No negative regulatory elements were detected in P2. Tn contrast, deletion of an A-rich region of P3 exten ding from -147 to -115 was required for optimal basal activity, suggesting that this sequence acts as a repressor of P3. Strikingly, however, whereas the A-rich region also functioned as a negative element when inserted upstr eam of the (G+C)-rich P2 promoter, it enhanced expression from the thymidin e kinase promoter, suggesting that its function depends on other transcript ion factors bound to promoter sequences. Fine deletion of P3 sequences prox imal to -115 implicated Sp1 motifs and downstream initiation sites in P3 fu nction. These studies indicate that function of P2 and P3 is controlled by ubiquitously expressed transcription factors and raise the possibility that P3 activity is repressed during fetal development.