MECHANISMS OF GENE CONTROL BY THE VITAMIN D3 RECEPTOR

维生素 D3 受体的基因控制机制

• 批准号: 2518325
• 负责人: LEONARD P FREEDMAN
• 金额: $ 20.96万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518325
• 关键词: 1,25 dihydroxycholecalciferol; T lymphocyte; cell cycle proteins; cell differentiation; cell free system; cell growth regulation; chromatin; crystallization; gene induction /repression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; interleukin 2; myeloid stem cell; nucleic acid sequence; protein structure function; reporter genes; retinoate; retinoid binding proteins; site directed mutagenesis; transcription factor; vitamin D receptors; vitamin analog

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.

该提议的长期目标是在功能上定义 在结构上，维生素D3受体（VDR）如何充当激活剂 和转录启动的阻遏物。 为此，我们将定义 调节两个基因的分子基础我们最近已经鉴定出 作为VDR，白介素-2（IL-2）和P21 WAF1的转录靶标，CIPI。 这些基因编码在介导维生素中起关键作用的蛋白质 D3对T细胞的生长抑制作用和终末分化的影响 髓样白血病细胞。 IL-2转录被压抑，并且 配体VDR诱导P21转录。 此外，我们将 描述VDR及其异二聚体伴侣RXR的晶体结构， 并将其作用方式定义为转录调节器 受体和配体反应性无细胞系统的开发。 这 工作将使我们能够进一步将VDR和维生素D3的调节定义为 阳性和阴性对照的范例。我们的目标是： 1。表征VDR介导的P21基因的DNA识别元件 激活和IL-2抑制。 我们将定义介导的DNA元件 归纳和镇压，并提出这些要素如何影响 VDR对维生素D3的转录活性。 2。确定P21激活所需的VDR的蛋白质伴侣和IL-2 抑制并定义相互作用的性质。 我们将确定 与VDR直接联系的互动合作伙伴涉及 在介导正调节和负调节时。 的本质 相互作用以及激活所需的VDR中的特定区域 和镇压将被定义。 3。确定VDR转录激活的机制。 VDR 转录增强功能将在粗提取物中概括，并且 用纯化的组件重构。 VDR的确切点 将定义在转录启动过程中的行为，并在 分析的染色质组装模板。 配体的机械作用 在VDR转录激活中将阐明。 4。描述VDR功能的结构决定因素。 我们将追求 VDR-RXR的三维结构作为络合物开启和关闭DNA，以及 配体的存在和不存在，与两种蛋白质合作 晶体学组。