Determining the interactome of the transcriptional coregulator LCoR

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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.