Constitutive heterochromatin makes up 20-50% of animal genomes and is associated with the silencing of gene expression, including that of repetitive elements. It is enriched for repressive histone modifications H3K9me2 and/or H3K9me3, which act as a binding sites for chromodomain-containing proteins related to HP1. Heterochromatin-induced gene silencing is an important modulator of developmental transitions, and defects in heterochromatin have been linked to a variety of human diseases, including cancer. The mechanisms through which heterochromatin is established and maintained have been extensively studied in the single-celled yeast Saccharomyces pombe, but they are not well understood in animals. We seek to identify and functionally dissect components of constitutive heterochromatin in C. elegans. Previous work in our lab identified a network of five factors that genetically interact and co-localise with H3K9me2 at repetitive elements (
met-2,
set-25,
let-418,
lin-13 and
hpl-2; McMurchy et al, 2017). These heterochromatin factors regulate transposable element repression, gene expression, DNA repair, fertility and growth in C. elegans, in close collaboration with small RNA pathways. In order to expand this network, we carried out multiple genetic interaction screens using RNAi to discover genes whose knockdown could enhance or suppress the phenotypes of heterochromatin-defective mutants. Strains we screened include mutants of H3K9 methyltransferases (
met-2,
set-25), HP1 orthologues (
hpl-1,
hpl-2), and HP1-interacting genes (
lin-61,
lin-13,
tdp-1). We identified genetic interactions through RNAi, using a panel of 2288 RNAi clones that target genes known or predicted to encode nuclear localised proteins. The screens identified shared as well as unique enhancers and suppressors of the heterochromatin-defective mutants and highlighted components of ubiquitylation, SUMOylation, RNA splicing and chromatin remodelling pathways, with most hits having orthologues in humans. Our results expand knowledge of the network of functional players in heterochromatin formation and function in a metazoan organism and identify targets for in depth studies in C. elegans and human cells.