Cloning of nuclear receptor specific co-factor by phage display system

类固醇、类维生素a、甲状腺激素、维生素D3和类前列腺素的核激素受体包括一个大的序列特异性转录因子家族。它们通过调节基因转录在发育、分化和体内平衡中发挥着不同的作用。核受体被认为与调节受体与基础转录机制组分相互作用的共调节蛋白一起介导其转录效应。迄今为止，已经分离并鉴定了几个辅助因子。最近的生物化学和遗传学研究支持核心组蛋白的超乙酰化是基因激活的特征，相反，组蛋白去乙酰化参与转录抑制。核受体共抑制因子SMRT和N-CoR与Sin3和组蛋白去乙酰化酶蛋白形成复合物，表明组蛋白去乙酰化介导的染色质重塑可能是受体介导的抑制机制。发现CBP和p300与人组蛋白乙酰转移酶蛋白P/CAF有功能相互作用。因此，CBP/p300及其相关蛋白P/CAF似乎通过靶向修饰染色质结构，在配体依赖性转录调控和功能中发挥关键作用。然而，这些辅助因子在核受体中是常见的。本项目的目的是利用一种新的克隆系统鉴定选择性核受体辅因子。为了鉴定潜在的共调节因子，我们采用了T7噬菌体展示系统。与谷胱甘肽- s转移酶融合的受体被用来分离能够结合受体的蛋白。来自cDNA的肽在每个噬菌体颗粒表面表达，通过与靶核受体结合和穿过无关受体来扩增和富集靶cDNA。

Nuclear hormone receptors for steroids, retinoids, thyroid hormone, vitamin D3, and prostanoids comprise a large family of sequence-specific transcription factors. They play diverse roles in development, differentiation and homeostasis by modulating gene transcription. Nuclear receptors thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription machinery.To date several co-factors have been isolated and characterized. Recent biochemical and genetic studies support the notion that hyperacetylation of core histones is a characteristic of gene activation, and, conversely, histone deacetylation is involved in transcriptional repression. Nuclear receptor co-repressors SMRT and N-CoR form complexes with Sin3 and histone deacetylase proteins, suggesting that chromatin remodeling by histone deacetylation is a possible mechanism for receptor mediated repression. It was found that CBP and p300 interact functionally with a human histone acetyltransferase protein P/CAF. Therefore, it appears that CBP/p300 and its associated protein P/CAF play a pivotal role in the ligand dependent transcriptional regulation and function through targeted modification of the chromatin structure. These co-factors, however, are common among the nuclear receptors. The purpose of this project is to identify the selective nuclear receptor cofactors using a novel cloning system.To identify potential coregulators, we applied the T7 phage display system. The receptor fusioned with glutathione-S-transferase were used to isolate proteins capable of binding the receptor. The peptide from cDNA are expressed on the surface of each phage particle, and target cDNA can be amplified and enriched by binding to target nuclear receptor and passing-through irrelevant receptor.