Nuclear Receptor Co-Activators in the Lung

描述（由申请方提供）：本研究的长期目标是研究核受体辅激活因子在介导对肺发育和呼吸道上皮细胞表面活性蛋白B（SP-B）基因表达至关重要的信号通路中的功能作用。 包括p160家族成员（SRC-1、ACTR和TIF 2）和CBP/p300的共激活因子具有组蛋白乙酰转移酶（HAT）活性，并且已知在基因激活、细胞分化和增殖中起重要作用。 将利用生物化学/分子生物学和条件性转基因小鼠模型的最新方法。 具体目标是：1）：H441细胞中SP-B染色质乙酰化的表征，以检验SP-B启动子/增强子区域中的组蛋白在转录激活期间被过度乙酰化的假设。 将进行染色质交联和染色质免疫沉淀（CHIP）试验; 2）：表征核受体共激活因子与TTF-1之间的反式相互作用，以检验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。 将进行免疫共沉淀、Western印迹、GST pulldown和哺乳动物双杂交系统以鉴定核受体共激活剂和TTF-1的精确相互作用结构域; 3）：通过核受体共激活剂表征TTF-1乙酰化以检验核受体共激活剂在肺上皮细胞中乙酰化TTF-1的假设。 将鉴定乙酰化TTF-1的特异性核受体共激活剂（例如SRC-1、ACTR、CBP/p300）。 TTF-1上精确的乙酰化赖氨酸残基将通过结构域定位和位点特异性诱变来鉴定; 4）：表征强力霉素可调节的转基因小鼠中的显性负性RAR，以检验核受体共激活剂对肺发育和肺气肿的呼吸上皮细胞的恢复/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.