Parathyroid hormone (PTH) and calcitriol (1,25(OH)2D3) are key participants in the maintenance of calcium homeostasis in the body. Previous work has demonstrated that 1,25(OH)2D3, acting through its nuclear receptor (VDR), can directly suppress transcription of the PTH gene and DNA sequences that mediate this action have been identified. The broad objective of the proposed study is to elucidate the molecular basis for the effects of the steroid-receptor complex on suppressing PTH gene transcription. The hypothesis to be tested is that the transcriptional potential of the VDR complex is intrinsically coupled to the DNA sequence of the specific binding site. In this model, the DNA binding site acts as an allosteric effector to modulate the conformation of the VDR complex and thereby coordinate the ability of specific factors to associate with this complex to produce the desired transcriptional response. The proposed research will concentrate on a DNA sequence from the avian PTH (cPTH) gene previously characterized by the PI.
The specific aims of the proposed study include to 1) examine conformational changes that occur in the receptor binding to this negative sequence versus other DNA sequences known to elicit positive transcriptional responses to 1,25(OH)2D3 and 2) identify proteins that interact with the VDR complex as it drives expression from the cPTH response element and characterize their capacity to mediate repression of gene transcription by the vitamin in biochemical assays. Conformational changes in receptor structure will be monitored by protease mapping experiments that contrast VDR complexes bound to repressor and enhancer sequences. DNA-bending and phasing array analyses will also be conducted to determine if complex binding to these sequences results in an altered ability to bend DNA or in the orientation of the DNA bend. Parallel studies will identify and characterize protein factors that associate with an intact VDR complex as it drives expression from the cPTH DNA element in a yeast one-hybrid screen. Cloned proteins identified from this screen will be analyzed for their ability to mediate interactions with both negative and positive DNA response elements in transient transfection studies, pull-down assays, DNA affinity chromatography experiments and electrophoretic mobility shift assays. Investigation of the factors involved in vitamin D-dependent repression of PTH gene transcription will make significant contributions to our overall understanding of the less well-studied area of negative gene transcription by nuclear proteins. Moreover, it will provide new opportunities for investigating the underlying causes to the resistance to 1,25(OH)2D3 observed in patients with secondary hyperparathyroidism associated with impaired renal function, with the possibility of devising treatment modalities that focus on the factors identified in this study.
