Highlights d Putative histone methyltransferases SET-25 and SET-32 establish heritable silencing d SET-25 and SET-32 are dispensable for silencing maintenance in subsequent generations d Secondary siRNA abundance does not correlate with silencing phenotypes d set-32 and set-25 mutants exhibit aberrant H3K9me3, fertility, and lifespan

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Chromatin regulators contribute to the developmental control of gene expression. In the nematode Caenorhabditis elegans, the roles of chromatin regulation in development have been explored in several contexts, including vulval differentiation. The synthetic multivulva (synMuv) genes are regulators of vulval development in C. elegans and the proteins encoded by these genes include components of several histone modification and chromatin remodelling complexes. By inhibiting ectopic expression of the epidermal growth factor (LIN-3) in the nematode hypodermis, the synMuv genes prevent inappropriate vulval induction. In a forward genetic screen for modifiers of the expression of a hypodermal reporter gene, we identified a mutation that results in increased expression of the reporter. This mutation also suppresses ectopic vulval induction in synMuv mutants and we have consequently named the affected gene suppressor of synthetic multivulva-1 (sumv-1). We show that SUMV-1 is required in the hypodermis for the synMuv phenotype and that loss of sumv-1 function suppresses ectopic expression of lin-3 in synMuv mutant animals. In yeast two-hybrid assays SUMV-1 physically interacts with SUMV-2, and reduction of sumv-2 function also suppresses the synMuv phenotype. We identified similarities between SUMV-1 and SUMV-2 and mammalian proteins KAT8 NSL2 and KAT8 NSL3, respectively, which are components of the KAT8/MOF histone acetyltransferase complex. Reduction of function of mys-2, which encodes the enzymatic component of the KAT8/MOF complex, also suppresses the synMuv phenotype, and MYS-2 physically interacts with SUMV-2 in yeast two-hybrid assays. Together these observations suggest that SUMV-1 and SUMV-2 may function together with MYS-2 in a nematode KAT8/MOF-like complex to antagonise the activity of the synMuv genes.

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Summary 13Some epigenetic modifications are inherited from one generation to the next, providing a potential 14 mechanism for the inheritance of environmentally acquired traits. Transgenerational inheritance of 15 RNA interference phenotypes in Caenorhabditis elegans provides an excellent model to study this 16 phenomenon, and whilst studies have implicated both chromatin modifications and small RNA 17 pathways in heritable silencing their relative contributions remain unclear. Here we demonstrate 18 that the histone methyltransferases SET-25 and SET-32 are required for the establishment of a 19 transgenerational silencing signal but not for long-term maintenance of this signal between 20 subsequent generations, suggesting that transgenerational epigenetic inheritance is a multi-step 21 process with distinct genetic requirements for establishment and maintenance of heritable silencing. 22Furthermore, small RNA sequencing reveals that the abundance of secondary siRNAs (thought to be 23 the effector molecules of heritable silencing) does not correlate with silencing phenotypes. 24Together, our results suggest that the current mechanistic models of epigenetic inheritance are 25 incomplete. 26