The vitamin D receptor (VDR) is a regulatory factor that modulates the transcriptional activity of a subset of the functional genome in response to calcitriol. Activity is initiated through direct binding of the VDR to DNA sequences located within sites with regulatory capability, and for most target genes involves heterodimeric co-participation with retinoid X receptor (RXR). Recent studies have revealed that alterations in the transcriptional activity of target genes in response to the VDR/RXR heterodimer involve the recruitment of additional multi-protein coregulator complexes that display unique and diverse activities critical to transcriptional modulation. These activities are enzymatic in nature and result in the modification of chromatin structure, control entry and activation of RNA polymerase II, process RNA transcripts, and abrogation receptor signaling. Although considerable detail has emerged regarding the activities of these complexes in other systems, little is known regarding their specific roles in calcitriol-mediated transactivation at the specific target gene level. The potential diversity of actions of these complexes in mediating calcitriol signaling, however, raises interesting questions with regard to this hormone's mechanism of action. The goals of this proposal are to study the downstream transcriptional activating events in bone cells integral to calcitriol-modulated transcription, focusing on endogenous 25OHD3-24-hydroxylase (Cyp24) and osteopontin (OPN) genes as genes with contrasting regulatory processes. The proposal is comprised of three specific aims.
Aim 1 : Investigate the dynamics of transcriptional processes that are associated with the activation of genes by calcitriol. We will utilize a variety of techniques including hormone-binding and VDR upregulation assays, chromatin immunoprecipitation (ChlP) approaches and mRNA analyses to explore VDR/RXR interaction, endogenous DNA binding, coactivator recruitment and transcriptional cycling, events that occur during activation of Cyp 24 and OPN gene expression.
Aim 2 : Contrast the molecular mechanisms inherent to Cyp24 and OPN activation by vitamin D, assess how these mechanisms are impacted by basal gene activity, and determine how these properties can influence gene selectivity. We will utilize the assays described above to characterize the """"""""resting and activation states"""""""" of the two promoters with regard to heterologous transactivator participation, coactivator/corepressor balance, and histone modification.
Aim 3 : Identify the intranuclear events that are integral to VDR inactivation and signal termination at the Cyp24 promoter. We plan to identify the mechanism through which the VDR is targeted for inactivation and evaluate the specific roles of ubiquitinylation and the 26S proteasome in this process. These studies will advance our understanding of the mechanism of action of calcitrol and are likely to provide new approaches to the therapeutic utilization of calcitriol and its analogs in the treatment of bone disease and cancer.
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