ChlP-Chip analysis and characterization of nuclear receptor and co-regulator complexes by massspectroscopy represent two general approaches that are used to define genomic targets and molecularmechanisms of nuclear receptor action. However, the availability of adequate antibodies remains a majorlimitation. Even for some of the most widely studied nuclear receptors and co-regulators, there are nosuitable antibodies that provide the necessary levels of enrichment required for robust identification ofgenomic binding sites on most ChlP-Chip platforms. In the case of proteomics approaches, epitope tags arethe preferred method of affinity purification in order to avoid potential effects of protein-specific antibodies onprotein-protein interactions of interest. In this project, we will generate a generic set of reagents that willenable rapid, high affinity isolation of all human and mouse nuclear receptors (NRs) and a subset ofco-regulators (Co-Rs). This will be accomplished by constructing vectors that direct expression of nuclearreceptors or co-regulators containing an N-terminal tag that is a substrate for the E. coli biotin ligase, BirA.Introduction of these vectors into mouse or human cell lines that express BirA results in their efficientbiotinylation, enabling subsequent purification with streptavidin affinity matrices. We will validate thisapproach by developing macrophage cell lines that express biotin-tagged versions of the NRs found to benormally expressed in macrophages, based on the NURSA expression dataset. We will then performChlP-ChIP analysis of a subset of tagged nuclear receptors and co-regulators in these cell lines to definetheir genomic locations on a large scale. As time and resources permit, we will expand utilization of thesevectors to include biotin tagging of additional co-regulators and expression of tagged proteins in other celltypes that are relevant to metabolic syndrome and cardiovascular disease. This project will enable studiesof NRs and Co-Rs in cell types that play critical roles in metabolic syndrome and cardiovascular disease,and provide resources to the community that will have broad application to diverse areas of biology andhuman diseases in which NRs represent potential therapeutic targets.
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