Determining the interactome of the transcriptional coregulator LCoR

确定转录共调节因子 LCoR 的相互作用组

• 批准号: RGPGP-2014-00084
• 负责人: White, John
• 金额: $ 3.57万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Group
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=668461
• 关键词: Determining; interactome; transcriptional; coregulator; LCoR

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.

转录因子（TFS）充当募集核调节蛋白（共激活剂或核心蛋白）的信标，这些蛋白质（共激活剂或核压剂）准备启动子区域募集RNA POL II及其辅助因子（激活），或者，或者，或者，或者，在POL II结合（抑制）中产生了染色质环境环境折射。核心节的数量比转录因子少，可以由多个类TF招募。 *使用核受体雌激素受体受体α的配体结合结构域（一种激素依赖性TF），在白色实验室中在白色实验室中鉴定了转录核心lcOR（配体依赖性核心）作为诱饵。 LCOR早在胚胎发育的两细胞阶段就表达，并在成年人中广泛表达。最初，它的特征是其通过募集CTBP核压子和组蛋白脱乙酰基酶来抑制核受体激素依赖性反式激活的能力。例如LCOR在乳腺癌细胞中的消融导致孕激素刺激的基因表达升高，这与其强烈抑制孕酮受体功能的能力一致。但是，随后的工作已将LCOR牵涉到多个TFS类的转录抑制作用。我们发现LCOR与Kruppel样因子6（KLF6）直接相互作用，并有助于抑制KLF6靶基因，并且它与Corepressor Kap-1直接相互作用，并有助于KAP-1相关TF（例如ZBRK-1）抑制靶基因。 *最近，我们使用一种在Mader Lab中开发的新型，功能强大的生物信息学工具（Mimister;最小跨越树的最小跨树）分析了LCOR表达模式，该工具提供了一个直观的界面，该界面可在大数据库中可视化和探索RNA-SEQ基因表达相关性。这些分析表明，在不同组织类型的样品中，LCOR表达与与转录调控有关的引人入胜的因素簇密切相关。其中包括TF休息，以及与细胞周期调节有关的转录复合物和转录伸长的成分，以及与DNA修复有关的蛋白质，大大扩展了潜在的LCOR辅因子的范围。 **提出的计划*提出的研究计划旨在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“ Interactome”，然后进行生化验证实验，以确定LCOR在所鉴定的复合物中的功能作用。与LCOR相互作用的因素和与之相关的蛋白质复合物相互作用的特征将为LCOR控制的生化过程提供许多见解。 *我们的具体目的如下：*项目1和2。我们将在RNA-SEQ转录组不同数据库中使用Mistic使用LCOR的共表达模式进行分析。我们还将分析细胞系转录组中的表达谱，以识别在不同组织中观察到的簇的模型系统。这些研究将进行实验，以生化验证LCOR与假定的新型辅助因子的功能相互作用。我们将分析芯片区域在TF结合位点富集，以确定共表达和/或相互作用的TF是否正在募集LCOR到DNA。还将研究在LCOR占用位点上共表达和/或相互作用的辅助因子的关联。我们还将在对照和缺乏LCOR的细胞中进行比较基因表达研究研究，以确定LCOR是否调节其调节区域中包含LCOR相互作用TF的基因集的活性。