The surprising relationship between macrophages and insulin resistance provides a promising interface in which to apply our emerging understanding of the molecular mechanisms of nuclear receptor actions and recent progress in defining the underlying strategies of transrepression. In Project 2, we will focus on NCoR/SMRT corepressor complexes as transcriptional checkpoints controlling both ligand-dependent regulation of gene expression by nuclear receptors and the activities of signal-dependent transcription factors that drive inflammatory programs of gene expression. We will better define the molecular mechanisms and roles of the TBLi, TBLRi and GPS2 components of N-CoR corepressor complexes in the regulation of AP1/NF-kB target genes and we will use genome-wide location analyses (GWLA) to investigate the roles of these proteins in positive and negative regulation of macrophage and adipocyte gene expression.
Three Specific Aims are proposed.
Specific Aim i will test the hypothesis that N-CoR/SMRT complexes regulate inflammatory responses that contribute to insulin resistance and are targets of anti-diabetic actions of PPARy agonists. These studies will be performed in collaboration with Units i and 3 using mice reconstituted with N-CoR-/- or SMRT-/- fetal liver hematopoietic progenitor cells.
Specific Aim 2 will investigate the hypothesis that the TBLi/TBLRi exchange complex is regulated by signal-specific phosphorylation of TBLRi/TBLi. We will investigate the protein kinase control of corepressor complex dismissal from AP-1 and NF-kB target genes, and the role of these events in PPARy-mediated activation of positively regulated genes and transrepression of inflammatory response genes.
Specific Aim 3 will explore the role of GPS2 and KIAA1787 in JNK-dependent gene activation/repression events and to test the hypothesis that GPS2 is required for normal insulin sensitivity based on observations that JNK-expression and activity are consistently elevated in diet-induced obesity models and that AP-1 activity is constitutively increased on a subset of gene targets in N-CoR-/- macrophages. These studies will utilize a combination of single cell nuclear microinjection of siRNAs, an ultra-sensitive, multiplexed RNA quantification method (RASL) and ChlP-DASL to define roles of GPS2 in signal-dependent activation of inflammatory response genes.
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