Argonaute/small RNA pathways promote germline pluripotency. For example, in C. elegans, the PIWI Argonaute, PRG-1, engages thousands of endogenously encoded piRNAs and localizes within peri-nuclear nuage (P-granules) to scan mRNAs as they exit the nucleus. We previously identified ZNFX-1 as a highly conserved protein required for the stable inheritance of both silenced and active epigenetic states. ZNFX-1 contains two domains, a helicase domain and several copies of a putative Zinc finger domain also found in a second highly conserved protein NFX-1. In
znfx-1 helicase mutants, 22G-RNAs lose their uniform distribution along their targets and accumulate toward the 5' ends of mRNAs. Thus ZNFX-1 appears to utilize its helicase activity to position RdRP toward the 3' ends of mRNAs to ensure uniform targeting. Interestingly, however, mutations predicted to abrogate helicase activity remain viable and fertile while mutations that delete both the helicase and the cysteine rich domain are germline mortal. Using the cysteine rich domain as bait a yeast 2 hybrid assay identified
znfx-1 itself and
nfx-1, suggesting that this domain promotes homotypic interactions. Several other putative Zinc finger proteins were returned from the screen, including the Zinc finger protein
row-1, a CBP homolog
zk1010.10, a TCF19 homolog
c01g6.5, and
lin-36. The list also includes proteins lacking putative DNA binding domains and with no known domains, such as TTF2 homolog
f54e12.2 and worm specific genes
c49g7.3,
c29e4.13, and
f53a9.1. The apparent association with several putative transcription factors raises the possibility that ZNFX-1 docks these factors in the peri-nuclear zone to coordinate small RNA and transcriptional silencing. We have begun to mutate these genes in a strain containing an epigenetically silenced reporter. Currently, we have observed desilencing of the reporter in worms with lesions in
k07c5.6 (SLU7) and in
zk1010.10 (CBP variant), and we have failed to observe altered expression patterns in worms with mutations in
f54e12.2 (TTF2),
c01g6.5 (TCF19), or
row-1. Moving forward, we plan to test the remaining candidates named above. We will also search for germline mortal synthetic interactions with our
znfx-1 helicase mutants and for in vivo binding to ZNFX-1 protein. From these experiments, we hope to develop a better understanding of how the ZNFX-1 pathway acts to promote fertility.