Determining the interactome of the transcriptional coregulator LCoR

转录因子（TF）作为募集辅调节蛋白（辅激活因子或辅抑制因子）的信标，这些辅调节蛋白为募集RNA pol II及其辅因子（激活）准备启动子区域，或者，创造一个对pol II结合难治的染色质环境（抑制）。辅调节因子的数量比转录因子少，可以被多种类型的转录因子募集。 白色实验室使用核受体雌激素受体α（一种雌激素依赖性TF）的配体结合结构域作为诱饵，在双杂交筛选中鉴定了转录辅阻遏物LCoR（配体依赖性辅阻遏物）。LCoR早在胚胎发育的两细胞阶段就表达，并在成体中广泛表达。其最初的特征在于其通过募集CtBP辅阻遏物和组蛋白脱乙酰酶抑制核受体的孕酮依赖性反式激活的能力;例如，乳腺癌细胞中LCoR的消融导致孕酮刺激的基因表达升高，与其强烈抑制孕酮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样因子6（KLF 6）相互作用，并有助于抑制KLF 6靶基因，它直接与辅阻遏物KAP-1相互作用，并有助于通过KAP-1相关的TF（如ZBRK-1）抑制靶基因。 最近，我们使用马德尔实验室共同开发的一种新的、功能强大的生物信息学工具（MiSTIC;最小生成树推断聚类）分析了LCoR表达模式，该工具为可视化和探索大型数据库中的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表达在不同组织类型的样本中与一组涉及转录调控的迷人因子密切相关。这些包括TF REST，沿着与细胞周期调控和转录延长有关的转录复合物组分，以及与DNA修复有关的蛋白质，极大地扩展了潜在LCoR辅因子的范围。 拟议方案 提出的研究计划的目的是使用尖端功能基因组学和生物信息学技术的组合来表征LCoR“相互作用组”，然后通过生物化学验证实验来确定LCoR在所确定的复合物中的功能作用。表征与LCoR相互作用的因子及其相关的蛋白质复合物将为LCoR控制的生化过程提供许多见解。 我们的具体目标如下： 项目1和2。我们将使用MiSTIC分析不同RNA-Seq转录组数据库中LCoR的共表达模式。我们还将分析细胞系转录组中的表达谱，以确定在不同组织中观察到的簇的模型系统。这些研究之后将进行实验，以生物化学验证LCoR与推定的新型辅因子的功能相互作用。 项目3和4。我们将通过ChIPseq分析确定含LCoR的复合物与DNA的全基因组分布。我们将分析ChIP区域在TF结合位点的富集，以确定共表达和/或相互作用的TF是否将LCoR募集到DNA中。还将研究LCoR占据位点上共表达和/或相互作用辅因子的相关性。我们还将在对照和LCoR耗尽的细胞中进行比较基因表达谱研究，以确定LCoR是否调节在其调控区含有LCoR相互作用TF的基因组的活性。

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.