Dynamic Mechanisms of Vitamin D-Induced Gene Expression

描述（由申请人提供）：骨是一种动态组织，在整个生命过程中，由于成骨细胞和破骨细胞的生物活性而被重塑。这些细胞的产生和存活以及它们的代谢活动都受到局部和全身因素的高度调节，包括生长调节剂、细胞因子、类固醇和肽激素。1,25-二羟基维生素D3 （1,25(OH)2D3）和甲状旁腺激素在许多这些过程中起着不可或缺的作用。事实上，已知1,25(OH)2D3影响成熟成骨细胞的细胞活性，并调节早期前体的增殖和分化。尽管对维生素D对骨骼的作用有一般的描述，但在全基因组水平上由125 (OH)2D3协调这些活动的机制仍不清楚。现在出现了惊人的技术进步，使这种评价成为可能。因此，我们提出了三个具体目标。特异性目的1：鉴定成骨细胞前体中1,25(OH) 2d3依赖性和依赖性维生素D受体（VDR）囊基质及其特性，并评估这些囊基质在分化过程中发生的变化。全基因组染色质免疫沉淀(ChIP)-DNA微阵列（ChIP- ChIP）和基因表达技术以及一系列生物信息学工具将被用于鉴定和对比成骨细胞前体和完全分化成骨细胞中的VDR基质。基因组范围的特性和性质将被评估。具体目的2：探讨VDR调控成骨细胞前体和分化成骨细胞中选定基因靶点的分子机制。DNA片段和BAC克隆分析，以及使用定制瓷砖阵列的ChIP-chip分析将用于评估VDR在未分化和分化成骨细胞中对代表性靶基因的作用和作用机制。具体目的3：建立成骨细胞中1,25(OH)2D3及其受体在全基因组水平上调控基因表达的原理。ChIP-chip方法将用于在全基因组范围内扩展Aims 1和2中鉴定的成骨细胞前体和完全分化成骨细胞中vdr介导的基因调控的一般特征。将评估1,25(OH)2D3在转录因子共募集、表观遗传修饰和RNA聚合酶II调节中的作用。本文提出的研究可能为在成骨细胞基因组水平上介导1,25(OH)2D3的多效性活性的基因靶点和潜在机制提供新的见解。这些研究还将为新的维生素D类似物的设计、开发和潜在的治疗应用提供新的信息。公共卫生相关性：维生素D在维持矿物质平衡和控制细胞生长和功能方面发挥着重要作用。本文的研究旨在增强我们对维生素D在骨细胞中作用机制的理解，从而创造出更好、更有选择性的药物。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.