Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila...

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20 null mutants are inviable, we found that an R634G mutation in Set8 predicted from in vitro experiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivo and is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20 null, and Set8R634G mutants. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.