Investigating the role of Sumo in piRNA-mediated germline heterochromatin maintenance in C.elegans

PROJECT SUMMARY Germline immortality is essential for species survival. Evidence from plants to animals suggests germline immortality depends on small RNA pathways that promote genome stability. In Caenorhabditis elegans germline, small RNAs that interact with the PIWI Argonaute protein PRG-1—called piRNAs—initiate silencing of foreign sequences that is then maintained by Worm Argonautes (WAGOs). Nuclear WAGO-9 binds the nucleosome remodeling and deacetylase (NuRD) complex via histone deacetylase (HDA-1), which initiates heterochromatin formation on piRNA targets. piRNA pathway defects activate expression of foreign sequences and disrupt germ cell development and fertility. How collaboration between the piRNA pathway and heterochromatin machinery is regulated to promote germline immortality is unclear. In the C. elegans germline, assembly of HDA-1 into the NuRD complex depends on the small ubiquitin-like modifier (SUMO). In worms expressing mutant HDA-1 that cannot be SUMOylated, piRNA-mediated silencing, germline heterochromatin formation, and fertility are lost. Interestingly, HDA-1 SUMOylation is not needed for its assembly into the NuRD complex in somatic embryo. Together, these findings suggest that the piRNA pathway relies on SUMO to co-opt NuRD complex assembly in the germline for silencing of piRNA targets. This proposal seeks to explore how germline NuRD complex assembly and its recruitment by the piRNA pathway are regulated. Aim 1 will explore the role of SUMOylated and germline-specific factors in preventing default (SUMO independent) assembly of the NuRD complex in germline. The highly-conserved MRG-1 chromodomain protein is essential for piRNA-mediated silencing and is SUMOylated. Preliminary data suggest SUMOylated MRG-1 in germline renders HDA-1 function to be SUMO-dependent. Co-immunoprecipitation assays will be used to determine whether SUMOylated MRG-1 prevents unmodified HDA-1 from assembling into the germline NuRD complex. Because SUMOylated MRG-1 is abundant in embryonic tissue, this aim will also use mass spectrometry and immunofluorescence assays to identify potential germline-specific factors interacting with SUMOylated MRG-1 to render germline HDA-1 function SUMO dependent. Aim 2 will determine whether HDA- 1 SUMOylation promotes interaction with WAGO-9. Putative SUMO-interacting motifs of WAGO-9 will be mutated and the effect on piRNA-mediated silencing and HDA-1interaction will be determined. This aim will also computationally analyze published data characterizing strength of piRNA-target interactions to explore how the piRNA pathway may utilize its targeting of transcripts to enact downstream silencing via heterochromatin. This work will reveal how highly conserved small RNA and chromatin factors coordinate to promote germline immortality, and provide the fellow with training in genetics, microscopy, biochemistry, and bioinformatics to prepare them for a future career as an independent investigator.