Bone is a dynamic tissue which is remodeled throughout life as a result of the biological activities of osteoblasts and osteoclasts. The production and survival of these cells as well as their metabolic activities are all highly regulated by local and systemic factors that include growth modulators, cytokines and steroid and peptide hormones. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) together with parathyroid hormone play an integral role in many of these processes. Indeed, 1,25(OH)2D3 is known to influence the cellular activity of mature osteoblasts and to modulate the proliferation and differentiation of early precursors. Despite a general description of vitamin D action on bone, the mechanisms that permit orchestration of these activities by 1,25(OH)2D3 at the genome-wide level remain unclear. Striking technological advances have emerged which now permit such evaluations. As a consequence, we propose three specific aims.
Specific Aim 1 : To identify the 1,25(OH)2D3-independent and -dependent vitamin D receptor (VDR) cistromes and their properties in osteoblast precursors and to assess the changes that occur to these cistromes during differentiation. Genome-wide chromatin immunoprecipitation (ChIP)-DNA microarray (chip) (ChIP-chip) and gene expression techniques together with a series of bioinformatic tools will be used to identify and contrast the VDR cistromes in osteoblast precursors and in fully differentiated osteoblasts. The genome-wide properties and nature of these cistromes will be assessed.
Specific Aim 2 : To explore the molecular mechanisms associated with the regulation by VDR of selected gene targets in both osteoblast precursors and differentiated osteoblasts. DNA fragment and BAC clone analyses, together with ChIP-chip analysis using customized tiled arrays will be used to assess the role and mechanisms of action of VDR at representative classes of target genes in undifferentiated and differentiated osteoblasts.
Specific Aim 3 : To establish principles for the regulation of gene expression by 1,25(OH)2D3 and its receptor in osteoblasts at the genome-wide level. ChIP-chip approaches will be used to extend, genome-wide, the general features of VDR-mediated gene regulation identified in Aims 1 and 2 in osteoblast precursors and fully differentiated osteoblasts. The role of 1,25(OH)2D3 in the regulation of transcription factor co-recruitment, epigenetic modification and RNA polymerase II modulation will be assessed. The research proposed herein is likely to provide novel insight into the gene targets and underlying mechanisms that mediate the pleiotropic activities 1,25(OH)2D3 at the genome level in osteoblasts. The studies will also provide new information relevant to the design, development and potential therapeutic application of new vitamin D analogs.
Vitamin D plays significant roles both in the maintenance of mineral homeostasis and in the control of cellular growth and function. The studies herein seek to enhance our understanding of the mechanisms that underlie vitamin D action in bone cells such that better and more selective medicines can be created.
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