Determining the interactome of the transcriptional coregulator LCoR

转录因子(TF)作为共调节蛋白(共激活因子或共抑制因子)的信标，为RNA PolII及其辅助因子的招募准备启动子区域(激活)，或者创造一个难以与PolII结合(抑制)的染色质环境。共调节因子的数量比转录因子少，可以被多类转录因子招募。*在White Lab中，以核受体雌激素受体α的配体结合域(一种激素依赖的转铁蛋白)为诱饵，在双杂交筛选中鉴定出转录共抑制物LCOR(配体依赖的共抑制物)。LCOR早在胚胎发育的两细胞阶段就有表达，并在成人中广泛表达。它最初的特征是通过招募CtBP辅阻遏子和组蛋白去乙酰基酶来抑制核受体对激素依赖的反式激活；例如，切除乳腺癌细胞中的LCOR导致孕酮刺激的基因表达增加，这与其强烈抑制孕激素受体功能的能力一致。然而，随后的工作表明LCOR参与了多种转录因子的转录抑制。我们发现，LCOR直接与Kruppel-like factor6(KLF6)相互作用，有助于抑制KLF6靶基因；LCOR直接与辅阻遏子KAP-1相互作用，有助于通过KAP-1相关的转录因子(如ZBRK-1)抑制靶基因。*最近，我们使用了Mader实验室共同开发的一种新的、强大的生物信息学工具(Mistic；最小生成树推断聚类)来分析LCOR的表达模式，该工具为在大型数据库中可视化和探索RNA-Seq基因表达相关性提供了一个直观的界面。这些分析表明，LCOR在不同组织类型的样本中的表达与涉及转录调控的一系列迷人的因素密切相关。这些包括转录因子REST，以及与细胞周期调节有关的转录复合体的成分，转录延伸，以及与DNA修复有关的蛋白质，极大地扩大了潜在的LCOR辅助因子的范围。**建议的计划*建议的研究计划旨在利用尖端功能基因组学和生物信息学技术的组合来表征LCOR“交互作用组”，然后进行生化验证实验，以确定LCOR在所确定的复合体中的功能作用。对与LCOR相互作用的因子及其相关蛋白质复合体的表征将为LCOR控制的生化过程提供大量的见解。*我们的具体目标如下：*项目1和2.我们将使用Mistic分析LCOR在不同的RNA-Seq转录本数据库中的共表达模式。我们还将分析细胞系转录本中的表达谱，以确定在不同组织中观察到的簇的模型系统。在这些研究之后，将进行生物化学验证LCOR与可能的新辅助因子的功能相互作用的实验。*项目3和4。我们将通过ChIPseq分析确定含LCOR与DNA的复合体在基因组中的分布。我们将分析在TF结合部位富集芯片区域，以确定共表达和/或相互作用的TF是否正在将LCOR招募到DNA。还将调查LCOR占用的位点上共表达和/或相互作用的辅助因子的相关性。我们还将在对照细胞和LCOR耗竭细胞中进行比较基因表达谱研究，以确定LCOR是否在其调节区调节包含LCOR相互作用因子的基因集的活性。

Transcription factors (TFs) act as beacons for recruitment of coregulatory proteins (coactivators or corepressors) that prepare promoter regions for recruitment of RNA pol II and its cofactors (activation) or, alternatively, create a chromatin environment refractory to pol II binding (repression). Coregulators are fewer in number than transcription factors and can be recruited by multiple classes of TFs. *Transcriptional corepressor LCoR (ligand-dependent corepressor) was identified in the White lab in a 2-hybrid screen using the ligand binding domain of the nuclear receptor estrogen receptor alpha, a hormone-dependent TF, as a bait. LCoR is expressed as early as the two-cell stage of embryonic development and widely expressed in the adult. It was initially characterized for its capacity to repress hormone-dependent transactivation by nuclear receptors through recruitment of CtBP corepressors and histone deacetylases; e.g. ablation of LCoR in breast cancer cells leads to elevated progesterone-stimulated gene expression, consistent with its capacity to strongly inhibit progesterone receptor function. However, subsequent work has implicated LCoR in transcriptional repression by multiple classes of TFs. We found that LCoR interacts directly with Kruppel-like factor 6 (KLF6) and contributes to repression of KLF6 target genes, and that it interacts directly with corepressor KAP-1 and contributes to repression of target genes by KAP-1-associated TFs such as ZBRK-1. *Recently, we analyzed LCoR expression patterns using a novel, powerful bioinformatics tool (MiSTIC; Minimum Spanning Trees Inferred Clustering) co-developed in the Mader lab, which provides an intuitive interface for visualizing and exploring RNA-Seq gene expression correlations in large databases. These analyses revealed that LCoR expression is correlated strongly across samples in different tissue types with a fascinating cluster of factors implicated in transcriptional regulation. These include the TF REST, along with components of transcription complexes implicated in cell cycle regulation, and transcription elongation, as well as proteins implicated in DNA repair, greatly expanded the range of potential LCoR cofactors. **Proposed program*The program of research proposed is designed to characterize the LCoR "interactome" using a combination of cutting edge functional genomics, and bioinformatics techniques, followed by biochemical validation experiments to determine the functional role of LCoR in the complexes identified. Characterization of the factors that interact with LCoR and the protein complexes with which it is associated will provide numerous insights into the biochemical processes that LCoR controls. *Our specific aims are as follows:*Projects 1 and 2. We will analyze using MiSTIC the co-expression patterns of LCoR in different databases of RNA-Seq transcriptomes. We will also analyze expression profiles in transcriptomes of cell lines in order to identify model systems for the clusters observed in different tissues. These studies will be followed by experiments to biochemically validate the functional interactions of LCoR with putative novel cofactors.*Projects 3 and 4. We will determine the genome-wide distribution of LCoR-containing complexes with DNA by ChIPseq analysis. We will analyze ChIP regions for enrichment in TF binding sites to determine whether co-expressed and/or interacting TFs are recruiting LCoR to DNA. Association of co-expressed and/or interacting cofactors on LCoR-occupied sites will also be investigated. We will also perform comparative gene expression profiling studies in control and LCoR-depleted cells to determine whether LCoR modulates the activity of sets of genes containing LCoR-interacting TFs in their regulatory regions.