This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of the research in my laboratory is to dissect the cellular activities of NR4A nuclear receptors in the genetic model organism C. elegans. The cellular activities of NR4A, including mechanisms of transcriptional regulation and interactions with signal transduction pathways, are poorly understood. Previous studies have supported our central hypothesis that NHR-6, the NR4A ortholog in C. elegans, has distinct, dualistic functions in regulating cell proliferation and cell differentiation during organogenesis.
The aims of the current project are focused on identifying the specific pathways by which NHR-6 regulates these distinct processes. We are addressing this question through two specific aims: 1) Identify NHR-6 target genes using whole-animal chromatin immunoprecipitation (ChIP);and 2) Identify candidate signaling pathways regulating NHR-6 using genetic interaction studies. For the first aim, we have been successful in optimizing a protocol for performing whole-animal ChIP, a technique that to date has not been extensively performed for C. elegans. Using a functional GFP-tagged NHR-6 fusion, we have been successful in initial attempts to IP NHR-6-bound DNA. Additionally, we have also generated the necessary genetic reagents (stable transgenic lines) for the analysis. Additional IP experiments are underway with the goal of analyzing the DNA using ChIP-seq. A long-term plan for this study will be to use these approaches to identify proliferation- and differentiation-specific NHR-6 target genes. For the second specific aim, we have established an RNAi-based genetic interaction screen. The screen is a combinatorial approach where RNAi against nhr-6 is combined with RNAi against candidate signaling genes. The screen is underway and has identified candidate interactors. We plan to identify and characterize these candidate signaling pathways with the goal of identifying proteins that regulate distinct activities of NHR-6.
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