Dynamic Mechanisms of Vitamin D-Induced Gene Expression

描述（由申请人提供）：维生素D受体（VDR）是一种调节因子，可调节功能基因组子集对骨化三醇的转录活性。活性是通过VDR与具有调控能力的位点内的DNA序列直接结合而启动的，对于大多数靶基因来说，它涉及与类视黄醇X受体（RXR）的异源二聚体共同参与。最近的研究表明，靶基因对VDR/RXR异源二聚体的转录活性的改变涉及额外的多蛋白共调节复合物的募集，这些复合物显示出对转录调节至关重要的独特和多样化的活性。这些活性本质上是酶促的，导致染色质结构的修饰，控制RNA聚合酶II的进入和激活，处理RNA转录物，以及消除受体信号。尽管这些复合物在其他系统中的活性已经有了相当多的细节，但对于它们在骨化三醇介导的特定靶基因水平上的转激活中的具体作用知之甚少。然而，这些复合物在介导骨化三醇信号传导中的潜在多样性，引发了关于这种激素作用机制的有趣问题。本研究的目的是研究骨化三醇调控的骨细胞下游转录激活事件，重点研究内源性25ohd3 -24-羟化酶（Cyp24）和骨桥蛋白（OPN）基因作为具有不同调控过程的基因。这项建议包括三个具体目标。目的1：研究与骨化三醇激活基因相关的转录过程的动力学。我们将利用多种技术，包括激素结合和VDR上调试验，染色质免疫沉淀（ChlP）方法和mRNA分析来探索VDR/RXR相互作用，内源性DNA结合，共激活子募集和转录循环，cyp24和OPN基因表达激活过程中发生的事件。目的2：对比维生素D激活Cyp24和OPN的内在分子机制，评估这些机制如何受到基础基因活性的影响，并确定这些特性如何影响基因选择性。我们将利用上述分析来表征两种启动子在异源反激活子参与、辅激活子/辅抑制子平衡和组蛋白修饰方面的“静息和激活状态”。目的3：确定在Cyp24启动子处VDR失活和信号终止不可或缺的核内事件。我们计划确定VDR失活的靶向机制，并评估泛素化和26S蛋白酶体在这一过程中的具体作用。这些研究将促进我们对骨化三醇作用机制的理解，并可能为骨化三醇及其类似物在骨病和癌症治疗中的应用提供新的途径。

DESCRIPTION (provided by applicant): 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.