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, which control human PTHR gene transcription. P2 and P3 are (G+C)-rich, function in a number of tissues, lie within the same CpG island, and display many hallmarks of housekeeping promoters. However, they are differentially regulated during development as P2, but not P3, functions in fetal tissues. Here, we have used both stably and transiently transfected human osteoblast-like cells to delineate regions of P2 and P3 required for promoter activity. Deletion analyses performed in stably transfected cells indicated that sequences extending from -91 to -12 relative to the transcription start site were required for function of the P2 promoter. No negative regulatory elements were detected in P2. In contrast, deletion of an A-rich region of P3 extending 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 upstream of the (G+C)-rich P2 promoter, it enhanced expression from the thymidine kinase promoter, suggesting that its function depends on other transcription factors bound to promoter sequences. Fine deletion of P3 sequences proximal to -115 implicated Spl motifs and downstream initiation sites in P3 function. 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.

Methylation patterns of human parathyroid hormone (PTH)/PTH-related peptide receptor gene promoters are established several weeks prior to onset of their function.

Expression of the human parathyroid hormone (PTH)/PTH-related peptide receptor (PTHR) gene is controlled by three promoters, P1-P3. P1 functions specifically in kidney, whereas P2 is ubiquitously active. P3 is also widely active, although more so in kidney than other tissues. However, only P2 functions at midgestation. We examined the role of methylation in controlling PTHR promoter activity. Function of all promoters was inhibited by CpG methylation in vitro. Significantly, P1 is selectively hypomethylated in adult kidney in vivo, strongly suggesting that demethylation is required for renal P1 function. Moreover, this pattern is established by 11. 75 weeks of fetal age, several weeks prior to the onset P1 activity. P3 is unmethylated at midgestation, although it is inactive at this stage of development, and thus exhibits characteristics of both tissue-specific and ubiquitously active promoters. These results show that adult methylation patterns of P1 and P3 are established several weeks prior to their induction, indicating that their function requires factors expressed late in development.

Developmental upregulation of human parathyroid hormone (PTH)/PTH-related peptide receptor gene expression from conserved and human-specific promoters.

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR) functions in skeletal development and mediates an array of other physiological responses modulated by PTH and PTHrP. PTHR gene transcription in mouse is controlled by two promoters: P1, which is highly and selectively active in kidney; and P2, which functions in a variety of tissues. P1 and P2 are conserved in human tissue; however, P1 activity in kidney is weak. We have now identified a third human promoter, P3, which is widely expressed and accounts for approximately 80% of renal PTHR transcripts in the adult. No P3 activity was detected in mouse kidney, indicating that renal PTHR gene expression is controlled by different signals in human and mouse. During development, only P2 is active at midgestation in many human tissues, including calvaria and long bone. This strongly suggests that factors regulating well conserved P2 control PTHR gene expression during skeletal development. Our results indicate that human PTHR gene transcription is upregulated late in development with the induction of both P1 and P3 promoter activities. In addition, P2-specific transcripts are differentially spliced in a number of human cell lines and adult tissues, but not in fetal tissues, giving rise to a shorter and less structured 5' UTR. Thus, our studies show that both human PTHR gene transcription and mRNA splicing are developmentally regulated. Moreover, our data indicate that renal and nonrenal PTHR gene expression are tightly coordinated in humans.

Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene: analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type 1b.

Expression of the PTH/PTH-related peptide (PTHrP) receptor (PTHR) in the mouse is controlled by at least two promoters. The downstream promoter (P2) is ubiquitously expressed, whereas expression of the upstream promoter (P1) is largely restricted to kidney. These observations may provide a genetic basis for a human PTH resistance syndrome, pseudohypoparathyroidism type 1b (PHP1b), in which renal, but not osseous, signaling by PTH is defective. We, therefore, cloned and characterized the 5'-end of the human PTHR gene and found that its organization is very similar to that of the mouse. Transcription initiation sites of human P1 and P2 promoters are in similar, but not identical, positions to those of the mouse gene. The identification of a human P2 promoter is significant because no P2-specific human PTHR complementary DNAs have been isolated to date. Southern analysis of genomic DNA from seven PHP1b patients did not reveal any rearrangements in proximal promoter regions or exons encoding 5'-untranslated region sequences. No significant sequence differences were found in clones of normal and patient DNAs encompassing proximal promoter sequences, and untranslated region and signal sequence exons. Thus, in the seven PHP1b patients analyzed, no defects were identified that would influence initiation site selection, stability, or splicing of renal PTHR transcripts. These data indicate that the genetic defect(s) in PHP1b in these patients lies in distal enhancer elements of the gene, in an essential transcriptional regulator, or in some as yet unidentified cofactor required for renal PTH signaling.