RNAi-mediated gene silencing in Caenorhabditis elegans can be inherited across multiple generations. It is known that chromatin readers and writers play a role in the extent and duration of silencing. Still, the complete mechanisms and requisite protein structures are yet to be elucidated. SET-25 is a histone-3 tri-methyllysine-9 transferase required to establish transgenerational epigenetic inheritance. This protein is named after its writer domain, a SET domain, and we propose it also contains a chromodomain, a well-conserved methyllysine reader domain. The multidomain interplay between these two structures and an N-terminal disordered region promotes the inheritance of RNAi signals even when the environmental stimulus is removed.
Using structural prediction tools, such as AlphaFold, we have generated constructs of the putative chromodomain to explore the structure-function relationship. The presence of this chromodomain also more closely aligns the C. elegans protein with predicted homologs, including the drosophila SU(VAR)3-9 and human SUV39H1, the latter being previously implicated as an RNA-binding protein. We have also generated mutant strains which systematically ablate the function of different domains to examine their in vivo effect on transgenerational epigenetic inheritance phenotypes and global H3K9me3 levels.