For my postdoctoral work, I wanted to pursue my earlier interest in how organisms sense their environment to regulate gene networks, within a multicellular genetic model system. I was looking for not only the right project, but also the best training environment to prepare me to run an independent research group. My postdoctoral position in Dr. Keith Yamamoto’s lab involves identifying mechanisms by which a single transcription factor can regulate genes differently in specific tissues. The Yamamoto lab studies nuclear hormone receptors, an important class of metazoan-specific transcription factors that regulate virtually all aspects of metazoan physiology and development. They govern essential physiologic pathways and processes, and their activity is commonly gated through binding of small lipophilic ligands, making them excellent targets for pharmaceutical intervention. I focused on the C. elegans nuclear hormone receptor, NHR-25, a transcription factor conserved across all major phyla. NHR-25 is expressed throughout C. elegans development and in multiple tissues, and plays roles in fat metabolism, development, molting, and asymmetric cell divisions in epithelial tissues.
I used NHR-25 to explore the roles of post-translational modification in nuclear receptor activity. In collaboration with Masako Asahina (ASCR/UCSF), I examined the roles of SUMO (small ubiquitin-like modifier) in modulating NHR-25 function in development, using the C. elegans vulva as a model organ (Ward, Bojanala et al., 2013). SUMO is a reversible, highly dynamic modification with similar enzymology to ubiquitin and regulates protein activity, localization, and protein complex composition. We identified SUMO as an NHR-25 interacting protein through an unbiased yeast two-hybrid screen aimed to uncover novel regulators of NHR-25. In vitro biochemistry revealed that NHR-25 is sumoylated, highlighting the ability of yeast two-hybrid screens to identify SUMO-substrate interactions as well as classic non-covalent interactions. We demonstrated that sumoylation of NHR-25 is important for ensuring correct cell fate promotion or maintenance during vulval development and that NHR-25 forms a SUMO-dependent gradient over the course of vulval development. Mechanistically, sumoylation restricts NHR-25 in vivo and in heterologous cell culture assays, potentially through regulating DNA binding.
In seeking candidate NHR-25-regulated target genes to optimize ChIP-seq experiments, I analyzed microarrays performed in Dr. Kaveh Ashrafi’s lab (Ward et al., 2014). Despite a mutually suppressive genetic relationship, inactivation of nhr-25 or an acyl-CoA synthetase gene (acs-3) produced highly similar transcriptomes (Mullaney et al., 2010; Ward et al., 2014). The identities of the up-regulated genes and our functional follow-up studies strongly suggested that both acs-3 and nhr-25 mutants suffer from enhanced susceptibility to pathogens and stresses, work greatly aided by collaboration with Jonathan Ewbank's group at CIML. The findings that acs-3 and nhr-25 had distinct sensitivities to various stresses and that some of the defects were exacerbated in the double mutants is most consistent with the notion that different mechanisms underlie the pathogen sensitivity defects of these two mutants.
In collaboration with Dan Dickinson and Bob Goldstein (UNC), we developed a methodology to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination (Dickinson et al., 2013). We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outlined a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. See Dan's Weebly site for great overview. I then built on this technology to develop an approach that allowed selection-free introduction of epitopes into the C. elegans genome (Ward, 2014). By selecting for repair of a temperature-sensitive allele of pha-1, I found that one can enrich for knock-ins at other genomic loci. This enrichment is further stimulated by inactivation of non-homologous endjoining by cku-80 RNAi. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.