Analyzing Germline-Specific Expression in Caenorhabditis elegans

Maintaining cells in an undifferentiated totipotent state is essential for initiating developmental programs that lead to a fully formed organism in each generation and for maintaining immortal germ cells across generations. Caenorhabditis elegans is a powerful genetic model organism to study early germ cell development due to the animal’s transparency and the ability to screen for mutant phenotypes. However, our ability to use standard techniques to study gene expression using fluorescent reporter genes has been limited due to germline-specific silencing mechanisms that repress transgenes. Therefore, we lack even basic knowledge of how expression is regulated in C. elegans germ cells. In this study, we develop methods to overcome these silencing mechanisms by using a class of noncoding DNA, called Periodic An/Tn Clusters (PATCs), to prevent transgene silencing in the germline. We use these improved tools to test the proposed role of putative germline-specific regulatory DNA motifs and the role a periodic TT signal within germline promoters. We fused GFP to the promoter of a germline expressed gene (pcn-1), which is enriched for PATCs and contains a germline-specific motif (TTAAAG). Our results show that despite enrichment and phylogenetic conservation, the TTAAAG motif is not required for germline expression. To test additional motifs and periodic TTs, we have designed a system that will allow us to test synthetic gene fragments for bi-directional germline expression. These tools will allow us to rapidly test motif redundancy, motif spacing, and TT periodicity using gfp and rfp signals in the germline and will enable experiments aimed at understanding the role of germline regulatory elements.