In species that use sex chromosomes to differentiate between its sexes, there is a difference in sex chromosome number and the number of gene copies which results in unequal gene dosage. In species that cannot tolerate this imbalance in gene dosage, the dosage compensation process is applied in which the gene expression levels between the two sexes are made equal. In mammals, female somatic cells (XX) have one X chromosome inactivated to equalize to the single X chromosome in male somatic cells (XY). In C. elegans, hermaphrodites (XX) downregulate both X chromosomes by half to equalize to the single X chromosome in their male counterparts (XO).

Dosage compensation in C. elegans is conducted primarily by the Dosage Compensation Complex (DCC), a multimeric set of proteins including a CondensinIDC complex and several additional proteins that bind, compact and downregulate the X chromosomes in hermaphrodites. One of the non-condensin proteins in the DCC is DPY-21, a Histone 4 Lysine 20 (H4K20)- specific demethylase. DPY-21 binds to the hermaphroditic X chromosomes and converts H4K20 di-methylation (H4K20me2) into mono-methylation (H4K20me1). This results in the enrichment of the repressive H4K20me1 mark on the X and the transcriptional downregulation of the X chromosomes. Although DPY-21 is part of the DCC, it localizes to the X chromosomes at a later stage of embryogenesis and does not bind mitotic chromosomes, contrasting to the other DCC components. Additionally, DPY-21 has been shown to have a DCC-independent role in the germline where dosage compensation in unrequired. This drives the question of the regulation of DPY-21 and its interactors in addition to the DCC.

The first part of my thesis is the characterization of DPY-21 regulation through phosphorylation. Using CRISPR/Cas9 site-directed mutagenesis, I generated a dpy-21 phosphomutant C.elegans strain by mutating the phosphosites of DPY-21 into Alanines to render them non-phosphorylatable. Through physiological assays, I found that the dpy-21 phosphomutant C. elegans strain has defects in embryogenesis and larval stages. In embryogenesis, the phosphomutations disrupts DPY-21’s localization to the X chromosomes and delays the enrichment of H4K20me1 to the X chromosomes. Through biochemical and imaging analysis, I demonstrate that the global levels of 4K20me1 is increased in the phosphomutant strain, suggesting that the mutant protein may be hyperactive. At the larval stages, the dpy-21 phosphomutant strain exhibits developmental delay and increased propensity to form dauers. These defects are resolved by adulthood which implies that DPY-21’s activity is most critical in the embryonic and larval stages.

The second part of my work revealed novel interactors of DPY-21. I conducted an immunoprecipitation-mass spectrometry (IP-MS ) on DPY-21 to establish its interacting partners and found the kinase SPK-1 to be a top interactor of DPY-21. In spk-1 mutants, there is delayed H4K20me1 enrichment on the X chromosomes in embryogenesis which ultimately results in disrupted H4K20me1 enrichment on the X chromosomes in adults. This shows that SPK-1 may regulate DPY-21 although this may be indirect. Taken together, my work has characterized a novel dpy-21 mutant for the field that leads to its hyperactivity in generating H4K20me1 globally as well as the novel interactor SPK-1 that may regulate DPY-21.