Shen, Yongquan et al. (2015) International Worm Meeting "Complex regulation of the GLI transcription factor tra-1 in C. briggsae.."

Ellis, Ronald, Shifman, Yelena, Zapfe, Emmalisa, Shen, Yongquan

[International Worm Meeting, 2015]

The transcription factor tra-1 is the sole nematode homolog of the Gli proteins, and is a key regulator of sexual identity from Caenorhabditis to Pristionchus. In C. elegans, tra-1(lf) mutations are recessive and cause XX animals to develop as males, whereas gf mutations are dominant and cause XO animals to become females. Elucidating how C. briggsae tra-1 is regulated is critical for understanding hermaphroditic development. Thus, we are using genome editing to produce and study specific Cbr-tra-1 mutations.First, C. briggsae and its close relatives make two tra-1 transcripts that differ only at the 5'-end. The v181 mutation creates a frameshift in an early exon that eliminates both TRA-1A and TRA-1B, so it defines the null phenotype. As in C. elegans, the XX and XO null mutants develop as males but produce oocytes late in life. By contrast, the v182 mutation only eliminates TRA-1A. Since its phenotype resembles that of v181, we conclude that TRA-1A plays the predominant role in sex determination.Second, C. briggsae has a small open reading frame in its 5'-UTR that is conserved in C. elegans. The v247 mutation bypasses a stop codon in this open reading frame, causing dominant feminization of the germ line and partial masculinization of the soma. We infer that this ORF regulates TRA-1 activity.Third, C. elegans TRA-1 is cleaved in hermaphrodites to produce a repressor that shuts down male genes. We isolated C. briggsae tra-1(v197), a frameshift near the cleavage site that removes the C-terminus, and found that smg-5; tra-1(v197) animals produce a truncated from of the protein and develop as females. Thus, the cleaved form of TRA-1 represses male fates in C. briggsae, as it does in C. elegans.Fourth, we used genome editing to insert a FLAG tag in the middle of TRA-1. The tagged protein directs normal development, is cleaved in hermaphrodites, and is predominately found in the nuclei of developing germ cells.Finally, the WDR-5 proteins work with TRA-1 in C. elegans (Li and Kelly, 2014). Surprisingly, knocking down both Cbr-wdr-5.1 and Cbr-wdr-5.2 causes animals of both sexes to make oocytes, whereas similar mutations cause C. elegans to make sperm. Thus, the role of the WDR-5 proteins in the sperm/oocyte decision appears to have changed dramatically during recent evolution. We are studying how these chromatin regulators interact with TRA-1.

Wei, Qing et al. (2013) International Worm Meeting "Using TALENs to create new genetic model systems."

Shen, Yongquan, Ellis, Ronald E, Chen, Xiangmei, Wei, Qing, Shifman, Yelena

[International Worm Meeting, 2013]

With the advent of genome sequencing and RNA interference, there has been an explosion in the number of model organisms used for evolutionary research. However, most studies are hindered by their exclusive reliance on reverse genetic approaches. The ability to carry out sophisticated forward genetic screens would allow the unbiased detection of new genes in these species, and greatly increase their usefulness. Unfortunately, the sets of genetic markers, balancing chromosomes and tools that make research so rapid with Drosophila melanogaster or Caenorhabditis elegans took decades to develop. For example, although C. briggsae has been studied for almost 20 years, so far random screens have produced useful sets of marker genes for only four of its six chromosomes. Since TALENs can induce mutations in any gene, we thought they might dramatically speed up this process. For this approach to work, several assumptions had to be tested. First, how frequently do mutations in a C. briggsae gene give a phenotype similar to that of C. elegans? Second, can the position of nonsense mutations in one species help in the design of target sites in another? Third, can incomplete genome sequences and SNP maps be used in conjunction with synteny to identify good marker genes? In just a few months, we have created morphological mutants on the remaining C. briggsae chromosomes. Most have useful phenotypes that resemble those of C. elegans nonsense mutants, such as unc-40 and dpy-5 on LGI, unc-34 and unc-51 on LGV or unc-1 on X. However, some have more severe phenotypes; for example, C. briggsae unc-54 and dpy-11 mutants are barely viable, whereas the corresponding C. elegans mutants are healthier. We did isolate an inframe deletion of dpy-11 and missense mutation of unc-54 for use as markers. We also created him-8 mutants that produce numerous males by non-disjunction, and smg-5 mutants that eliminate the nonsense-mediated decay system. Thus, TALENs can be used to adapt model organisms for genetic research.