UCLA

The microrchidia (MORC) family of proteins are widely conserved eukaryotic and prokaryotic organisms. MORCs are GHKL (Gyrase, HSP90, Histidine Kinase, MutL) ATPases. Despite evidence that they are involved in gene silencing and genome compaction in multiple eukaryotic organisms, it is unknown what is their direct contribution to gene regulation in vivo or how they act on a molecular level. To elucidate how MORCs act to maintain gene silencing, I determined that MORCs are GHKL ATPases that form multimers and regulate a unique subset of genes. I show that MORCs with functional CW histone mark reader domains can be targeted to chromatin by recognition of histone tails, and validate MORC3 localization in vivo. From a structural analysis perspective, we also show that MORCs dimerize at the N terminus upon ATP binding, and that they are DNA binding proteins which show length-dependent binding preferences. MORCs prefer to bind to longer DNA templates over short DNA templates, and they also have little sequence specificity with regards to their binding specificity. Using single molecule studies, we show that C. elegans MORC-1 compacts DNA by loop trapping it; MORC-1 also compacts nucleosome templates. These single molecule studies also clarify the role of ATP metabolism in MORC action. We show that ATP stimulates the rate of compaction on DNA, and is further stimulated by an ATP non-hydrolysable analog, demonstrating that nucleotide binding impacts MORC-1 stability on DNA. We also show that MORC-1 phase separates in vitro and during DNA compaction, MORC-1 forms large foci that are topologically entrapped on DNA. The foci mirror the 1,6 hexanediol resistant puncta observed in vivo. Their resistance to 1,6 hexanediol treatment is likely because MORC-1 puncta are topologically entrapped and use DNA as a scaffold.

MORCs thus depend on alternative mechanisms in order to bind to specific regions of chromatin for the same mechanistic process, which culminates in genome compaction. Altogether, my thesis reveals novel mechanistic insights into the MORC family. I propose a model whereby MORCs are targeted in vivo by either their own CW domains or other protein factors, trap loops of DNA at that locus to compact and silence the gene, and multimerize on themselves to create a phase separated environment that enforces genome compaction by inducing DNA compaction.