ChlP-Chip analysis and characterization of nuclear receptor and co-regulator complexes by mass spectroscopy represent two general approaches that are used to define genomic targets and molecular mechanisms of nuclear receptor action. However, the availability of adequate antibodies remains a major limitation. Even for some of the most widely studied nuclear receptors and co-regulators, there are no suitable antibodies that provide the necessary levels of enrichment required for robust identification of genomic binding sites on most ChlP-Chip platforms. In the case of proteomics approaches, epitope tags are the preferred method of affinity purification in order to avoid potential effects of protein-specific antibodies on protein-protein interactions of interest. In this project, we will generate a generic set of reagents that will enable rapid, high affinity isolation of all human and mouse nuclear receptors (NRs) and a subset of co-regulators (Co-Rs). This will be accomplished by constructing vectors that direct expression of nuclear receptors 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 efficient biotinylation, enabling subsequent purification with streptavidin affinity matrices. We will validate this approach by developing macrophage cell lines that express biotin-tagged versions of the NRs found to be normally expressed in macrophages, based on the NURSA expression dataset. We will then perform ChlP-ChIP analysis of a subset of tagged nuclear receptors and co-regulators in these cell lines to define their genomic locations on a large scale. As time and resources permit, we will expand utilization of these vectors to include biotin tagging of additional co-regulators and expression of tagged proteins in other cell types that are relevant to metabolic syndrome and cardiovascular disease. This project will enable studies of 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 and human diseases in which NRs represent potential therapeutic targets.
NIH ExPorter
NIH ExPorter