The question of how our genomes are regulated to produce different cell types in a reproducible manner, is key to understanding both healthy life and disease. Our laboratory has shed light on the role of a specific ubiquitous histone modification, histone H3 lysine 9 methylation, in the establishment and maintenance of heterochromatin. We use the nematode C. elegans, which has only 2 histone methyltransferases (HMTs) that deposit this conserved modification, rather than the 6 found in man. These two HMTs are members of conserved enzyme families. We have shown that the two C. elegans H3K9 methyltransferases (MET-2 and SET-25) have essential but distinct functions in ensuring genomic stability, and we have shown that the C. elegans mutant lacking a functional H3K9me-HMT MET-2, requires tumor suppressors BRCA-1 and BRCA-2, and other factors that stabilize replication forks such as the ATR-checkpoint kinase and RAD-51, for viability (Padeken et al., 2019). Loss of MET-2 leads to early aging and sterility in worms. We hypothesize that both of these phenomena can be traced to the promiscuous transcription of repeat DNA; this leads to aberrant RNA:DNA hybrids or R-loops that cause irreparable damage when encountered by replication forks. Further molecular and biochemical studies of the MET-2 enzyme identified novel interaction partners, one of which ensures the proper nuclear localization and functional activity of MET-2 (Delaney et al., 2019). This work also highlighted a critical role for MET-2 during temperature stress. A subsequent microscopy-based screen identified the factors LIN-65 and ARLE-14 as being essential for allowing MET-2 to form functional condensates and be targeted to specific genes and repeats. We also identified additional factors involved in the targeting of the second HMT, which deposits H3K9me3, namely a nuclear Argonaut which binds RNA and recruits the Dicer enzyme. This allowed us to connect the small RNA pathway to SET-25 targeting. A second targeting pathway for SET-25 depends on the H3K9me2 reader LIN-61. The two factors contribute to the H3K9me3-mediated silencing of endogenous viral transposons embedded in the genome.
We then studied the role of spatial organization of heterochromatin in differentiated tissues – namely gut and muscle. We have shown that a lamin mutation causes deterioration of muscle in worms, recapitulating a human disease (Harr et al., 2020) and we have shown that in intestinal cells, the disruption of a strict sequestration of a euchromatic factor, CBP/p300, away from heterochromatin, leads to an activation of repressed tissue specific genes and loss of intestine-cell identity (Cabianca et al., 2019). Intriguingly, this depends on another histone H3 methylation mark, H3K36me2 or me3, and its reader MRG-1. Finally we have discovered a new principle of silencing that depends on selective nuclear degradation of transcripts. We showed that a complex called LSM2-8 – coupled with an RNA degrading machine (XRN-2) degrades specifically messages arising from genes repressed by the so-called Polycomb pathway, a system that maintains another histone mark on conditionally silent genes, histone H3K27me3. This mRNA degradation is the first proof that select mRNA turnover in the nucleus communicates to the compaction state of chromatin on genes. This has relevance for the innate immunity genes in man.
Our most important contribution from our ERC funded program of research was to show that continued expression of the H3K9 HMT, MET-2, is necessary for the maintenance of nondividing, differentiated tissues, such as muscle. We could show that MET-2 continuously restores H3K9me2 to repress germline and genes specific to other tissues, thereby allowing muscle cells to maintain their identity as muscle. This is achieved by preventing transcription factor access. Depending on the tissue type and stage of development, the loss of MET-2 provokes the derepression of different sets of genes, reflecting the transcription factor profile of that stage/cell type. This indicates that although H3K9me marks are dispensible for development in general, they are essential for long-term tissue maintenance.
