The long-term goal of this proposal is to define functionally and structurally how the vitamin D3 receptor (VDR) acts as both an activator and repressor of transcription initiation. To do so, we will define the molecular basis for the regulation of two genes we have recently identified as transcriptional targets of VDR, interleukin-2 (IL-2) and p21 WAF1, CIPI. These genes encode proteins that play critical roles in mediating vitamin D3's effect on growth inhibition in T cells and terminal differentiation of myeloid leukemic cells, respectively. Il-2 transcription is repressed, and p21 transcription is induced, by liganded VDR. In addition, we will delineate the crystal structure of VDR and its heterodimeric partner RXR, and define its mode of action as a transcriptional regulator through the development of a receptor- and ligand-responsive cell-free system. This work will enable us to further define regulation by VDR and vitamin D3 as paradigms for positive and negative control.
Our aims are to: 1. Characterize the DNA recognition elements for VDR-mediated p21 gene activation and Il-2 repression. We will define the DNA elements mediating both induction and repression, and propose how these elements influence the transcriptional activity of VDR in response to vitamin D3. 2. Identify protein partners of VDR required for p21 activation and Il-2 repression, and define the nature of the interactions. We will identify interacting partners making direct contacts with VDR and that are involved in mediating both the positive and negative regulation. The nature of the interactions, as well as specific regions in VDR required for activation and repression, will be defined. 3. Determine the mechanism of VDR transcriptional activation. VDR transcriptional enhancement will be recapitulated in crude extracts, and reconstituted with purified components. The precise point at which VDR acts during transcription initiation will be defined, and its function on chromatin assembled templates analyzed. The mechanistic role of the ligand in VDR transcriptional activation will be clarified. 4. Describe structural determinants of VDR function. We will pursue the three-dimensional structures of VDR-RXR as complexes on and off DNA, and in the presence and absence of ligand, collaboratively with two protein crystallography groups.
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