SET-25 is a H3K9 methyltransferase necessary for the establishment of transgenerational epigenetic inheritance in C. elegans. In a SET-25 null strain, animals exposed to an RNAi stimulus are unable to pass silencing on to their progeny to the same degree as wild-type animals. Interestingly, some SET-25 null progeny can still inherit silencing, and these animals are then able to pass silencing on to their offspring at a level similar to wild-type.
With only an annotated SET domain, structural predictions and homology searches using SET-25 have identified a putative chromodomain, hypothesised to be involved in protein-protein or protein-nucleic acid interactions, and an n-terminal intrinsically disordered region. In this study, we generated transgenic strains with mutations in each of these domains and used them to investigate their individual contribution to H3K9me3 deposition with CUT&RUN, and their role in localisation using fluorescence microscopy. We found that, though both the chromodomain and SET domain are important for the deposition of H3K9me3, mutations to the SET domain did not significantly affect protein localisation. In contrast, mutations to the chromodomain appeared to decrease localisation specificity and foci formation, suggesting it is necessary for the targeting of specific loci for silencing.