H3K9me is dispensable for C. elegans development but essential for genome integrity

Epigenetic mechanisms as key regulators of chromatin biology have been the focus of intensive research over the past 20 years. It has become clear that epigenetic pathways play a major role in the pathology of numerous diseases ranging from neurodegenerative repeat expansion diseases to certain cases of cancer.
One of the best-studied epigenetic marks is the methylation of histone 3 on lysine 9 (H3K9me). H3K9 methylation plays a major role in silencing parts of the genome. H3K9me domains encompass a broad variety of sequences, ranging from single genes to repetitive elements. In addition to its function in transcriptional repression, H3K9me is implicated in chromosome segregation and the maintenance of genome integrity. Lately the H3K9me mark received even more attention due to an identified role in mis-silencing of tumor suppressor genes during the development of cancer. Consequently, some of the first drugs to manipulate H3K9me are tested as cancer therapies.
Studying H3K9me in complex multicellular organisms has so far proven to be difficult. Both mice and Drosophila have at least five histone methyl transferase (HMTs) enzymes that are essential and partially redundant, allowing only for the study of partial reductions in H3K9me. In the nematode C. elegans our lab identified the two methyl transferases, essential for all H3K9 methyltaion throughout development MET-2 and SET-25.
Astonishingly C. elegans is viable and fertile in the complete absence of H3K9me. This allowed us to use the met-2 set-25 mutant to characterize the development of a multicellular organism in the absence of this central heterochromatic mark and ask: what is the main role of H3K9me in C. elegans and is there a functional difference between H3K9me3 and H3K9me1, me2 states.
The major site of H3K9 methylation are repetitive elements (RE).
Chapter 2 will therefore give an overview of the current state of knowledge on RE classes found in the genome, how they are controlled and what danger they pose. A special focus is put on the interplay of different epigenetic silencing mechanisms, with H3K9me at its center, that ensure repeat silencing at all stages of development.
Chapter 3 contains the majority of the experimental work characterizing the role of H3K9me in C. elegans. Starting with the observation of a striking increase of DNA damage checkpoint dependent apoptosis in met-2 set-25 germlines, we identify increased mutagenesis specifically in the context of derepressed RE. We suggest that RNA:DNA hybrids that accumulate on derepressed RE drive these mutations by provoking conflicts with the DNA replication machinery.
In the second experimental part (Chapter 4) we closely analyze the distinct contribution of H3K9me2 and me3 to the roles identified in Chapter 3. We identify a partially interdependent role of MET-2 and SET-25, making SET-25 dependent on, and at the same time, redundant with MET-2 at the majority of its target loci. Similarly, the majority of H3K9me associated phenotypes are not, or only mildly observed in worms lacking H3K9me3, arguing for very similar abilities in silencing of H3K9me2 and me3. Besides quantitative differences in the number of transposable elements and genes depending on MET-2 or SET-25 for their transcriptional silencing, tandem repeats depended exclusively on MET-2. Interestingly, the identified silencing pathways occur in different nuclear sub-compartments. In contrast to our previous model we found that SET-25 dependent silencing occurs all over the genome, while MET-2 repressed regions are enriched at the nuclear periphery. The tethering of endogenous heterochromatic sequences was also found to completely depend on the function of MET-2 and was independent of SET-25. We speculate that peripheral localization is involved in the MET-2 silencing function.
In the same study we also take an additional unbiased approach to identify previously overlooked roles of H3K9me by performing a whole genome synthetic lethality screen with the met-2 set-25 double mutant. The hits were then further tested in each of the single mutants, met-2 and set-25, showing exclusive genetic interaction with met-2. Finding many factors shown or suggested to be involved in RNA:DNA hybrid prevention and DNA damage repair, we conclude that the role of H3K9me in genome integrity described in Chapter 3 as one of its most important roles in C. elegans.
This thesis thereby provides evidence how repetitive elements derepression in the absence of H3K9me can lead to the occurrence of spontaneous DNA damage, putting a special emphasis on the danger of tandem repeat expression.