Constitutive heterochromatin is an epigenetic compartment of animal genomes that is important for gene regulation, chromosome architecture and the maintenance of genome integrity. It is associated with the silencing of gene expression and the repression of repetitive element activity. Constitutive heterochromatin is also an important modulator of development and becomes dysregulated in human cancers. However, the mechanisms by which it is formed and functions in animals, and how it interacts with other processes in the nucleus, are not well understood.

In this work, I defined a network of functional components of constitutive heterochromatin in Caenorhabditis elegans through conducting a set of genetic interaction screens. I began by designing a high throughput screening approach using RNAi to find genetic enhancers and suppressors of the growth and fertility defects of heterochromatin-defective mutants. Using an RNAi sub-library targeting 2309 genes encoding nuclear proteins, I then screened seven heterochromatin mutants for genetic interactors. I identified 289 enhancers and 89 suppressors, which form a highly interconnected network, with 75% interacting with more than one heterochromatin mutant. Genetic enhancers included components of chromatin modifying complexes, ubiquitination and sumoylation pathways, and transcription and RNA processing factors. Some of these factors were required for silencing of a heterochromatic reporter, suggesting roles in gene silencing. Genetic suppressors were enriched for a broad range of chromatin modifying factors, many of which are associated with active transcription.

I next characterised the changes in gene and repetitive element expression shown by eight different heterochromatin mutants. As for genetic interactions, I found that shared alterations were common, and I identified candidates that may be responsible for the slow growth of some heterochromatin mutant strains. Alongside the upregulation of repetitive elements, I observed three major gene expression changes: the ectopic expression of germline genes in somatic tissues, the downregulation of metabolic pathways and the induction of diverse stress- response pathways. The promoters of some upregulated germline genes are marked by histone H3 lysine 9 dimethylation (H3K9me2), consistent with direct repression, whereas deregulation of the metabolic and stress response pathway genes appears indirect as their promoters generally lack H3K9me2.

I investigated the mechanisms by which genetic suppressors rescue the growth defect of heterochromatin mutants through assessing the effect of their knockdown on gene and repetitive element expression in the hpl-1;hpl-2 double mutant, which lacks both HP1 orthologues. The loss of diverse genetic suppressors corrected shared groups of gene and repetitive element expression alterations, including the upregulation of stress response pathways. This suggests that suppression is through the restoration of normal gene expression programmes rather than through induction of additional changes that counteract those of heterochromatin mutants.

Overall, my work expands the known network of functional components of constitutive heterochromatin in the context of a developing animal. I reveal how activating and silencing chromatin modifying pathways are balanced to prevent genome deregulation. Finally, I implicate the activation of stress response pathways in the physiological defects of heterochromatin-defective animals. These results hold relevance for the understanding and potential treatment of diseases such as cancer which show defective heterochromatin function.