The germline deploys both small-RNA and chromatin-based mechanism to program epigenetic inheritance. Yet how small-RNA pathways such as the piRNA pathway establish repressive chromatin marks at target genes is still poorly understood. The transition from active to silent chromatin is thought to require the removal of activating histone marks such as H3K9 Acetylation, and the installation of silencing marks such as H3K9 trimethylation. Several of our studies have recently converged on components of a highly conserved nucleosome remodeling and histone deacetylase (NuRD) complex, (including MEP-1, HDA-1/HDAC, and LET-418/Mi2) as important factors in promoting both fertility and the sensitivity of germline genes to epigenetic silencing. For example, we have identified MEP-1 and HDA-1 as proteins that bind directly to PIE-1. PIE-1 also binds and recruits UBC-9 which conjugates the small-ubiquitin like peptide SUMO to its targets. We show that PIE-1 itself is Sumoylated and also facilitates the Sumoylation of HDA-1. Sumoylation of HDA-1 is required for a transition from high to lower levels of 'activating histone marks' at the transition from mitosis to meiosis in the adult gonad. For example, Lysine to Arginine (K>R) mutations that abolish HDA-1 Sumoylation, although viable and fertile, fail to transition from high to low levels of H3K4 methylation and H3K9 Acetylation at the onset if meiosis. Independently, an RNAi based screen for factors that de-silence a piRNA sensitive GFP transgene, identified the SUMO gene,
smo-1, as a desilencing factor. Consistent with this finding, CRISPR induced lesions, including a hypomorphic allele of
smo-1, 3xFLAG::
smo-1, and a temperature-sensitive (ts) allele of
ubc-9 also caused de-silencing of the reporter. Using a 10XHIS::SUMO in co-IP and Mass-Spectrometry studies we confirmed the Sumoylation of HDA-1, MEP-1, PIE-1, LET-418 and several other conserved factors. Furthermore, depletion of HDA-1, LET-418, or MEP-1 using the auxin-degron system prevents piRNA-dependent silencing. More interestingly, animals expressing a HDA-1 K>R protein that is refractory to Sumoylation, potently desilence a piRNA reporter. And importantly, a K>R HDA-1::SMO-1 fusion protein made by inserting the SUMO orf before the stop codon of HDA-1 restores transgene silencing. Finally, although we have not yet tested all of the NuRD factors for mutator activity, we have found that hypomorphic alleles of MEP-1 exhibit increased expression of transposon mRNA and cause a high frequency of spontaneous transposition. Taken together these findings suggest that PIE-1 expression in the meiotic zone, promotes the Sumoylation and activation of HDA-1, and that this upregulation is important for the piRNA dependent regulation of chromatin silencing in the germline.