Nuclear Receptor Co-Activators in the Lung

描述（由申请人提供）：这项工作的长期目标是研究核受体共激活因子在介导肺发育和呼吸上皮细胞表面活性剂蛋白B （SP-B）基因表达的关键信号通路中的功能作用。共激活因子包括p160家族成员（SRC-1， ACTR和TIF2）和CBP/p300具有组蛋白乙酰转移酶（HAT）活性，已知在基因激活，细胞分化和增殖中发挥重要作用。将利用生物化学/分子生物学和条件转基因小鼠模型的最新方法。具体目的是：1)：表征H441细胞中SP-B染色质乙酰化，以验证SP-B启动子/增强子区域的组蛋白在转录激活过程中过度乙酰化的假设。将进行染色质交联和染色质免疫沉淀（CHIP）测定；2)：表征核受体共激活因子与TTF-1之间的反式相互作用，以验证核受体共激活因子直接与TTF-1相互作用以控制靶SP-B基因表达的假设。通过免疫共沉淀、Western blot、GST下拉和哺乳动物双杂交系统鉴定核受体共激活物和TTF-1的精确相互作用域；3)：核受体共激活因子对TTF-1乙酰化的表征，以验证核受体共激活因子在肺上皮细胞中乙酰化TTF-1的假设。将鉴定特异性核受体共激活剂（如SRC-1、ACTR、CBP/p300）乙酰化TTF-1。TTF-1上精确的乙酰化赖氨酸残基将通过结构域作图和位点特异性诱变来确定；4)：在多西环素可调节的转基因小鼠中鉴定显性负RAR，以验证核受体共激活剂对肺气肿肺发育和呼吸上皮细胞恢复/重塑至关重要的假设。

DESCRIPTION (provided by applicant): The long-term goals of this work are to study the functional roles of nuclear receptor co-activators in mediating signaling pathways critical to lung development and surfactant protein B (SP-B) gene expression in respiratory epithelial cells. Co-activators including p160 family members (SRC-1, ACTR and TIF2) and CBP/p300 possess histone acetyltransferase (HAT) activity and are known to play important roles in gene activation, cell differentiation and proliferation. The state of the art approaches of biochemical/molecular biology and conditional transgenic mouse models will be utilized. The specific aims are: 1): Characterization of SP-B chromatin acetylation in H441 cells to test hypothesis that histones in the SP-B promoter/enhancer region are hyperacetylated during transcriptional activation. Chromatin cross-linking and chromatin immunoprecipitation (CHIP) assays will be performed; 2): Characterization of trans-interactions between nuclear receptor coactivators and TTF-1 to test hypothesis that nuclear receptor co-activators interact directly with TTF-1 to control target SP-B gene expression. Co-immunoprecipitation, Western blot, GST pulldown and mammalian two-hybrid system will be performed to identify the precise interacting domains of both nuclear receptor co-activators and TTF-1; 3): Characterization of TTF-1 acetylation by nuclear receptor co-activators to test hypothesis that nuclear receptor co-activators acetylate TTF-1 in lung epithelial cells. Specific nuclear receptor co-activators (e.g. SRC-1, ACTR, CBP/p300) acetylating TTF-1 will be identified. The precise acetylated lysine residues on TTF-1 will be identified by domain mapping and site-specific mutagenesis; 4): Characterization of dominant negative RAR in doxycycline-regulatable transgenic mice to test hypothesis that nuclear receptor co-activators are critical to lung development and recovery/remodeling of respiratory epithelial cells from emphysema.