View Program - 63rd Annual Drosophila Research Conference

Generation and maintenance of specific chromatin domains are essential for proper genome regulation, cell cycle progression, and organismal development. Chromatin is a complex of DNA wound around histone proteins which have N-terminal tails that can be chemically modified post-translationally (PTMs). Proteins termed “readers” bind histone PTMs and either directly or as part of multi-protein complexes alter chromatin structure to control critical genome functions. One such reader discovered in Drosophila melanogaster and called Lethal (3) malignant brain tumor (L(3)mbt) functions as a tumor-suppressor in larval brains. L(3)mbt has been implicated in chromatin condensation and transcriptional regulation. Previous in vitro studies showed that L(3)mbt and its human homolog preferentially bind histone H4 that is methylated on lysine 20 (H4K20me). However, in vivo studies have shown that L(3)mbt does not exclusively co-localize with H4K20me throughout the genome; thus, how L(3)mbt interacts with chromatin and exerts its functions remains incompletely understood. Determining whether L(3)mbt depends on H4K20me for binding the genome in vivo has never been addressed in the most direct way – by mutating H4K20. With our histone gene replacement platform in Drosophila, we generated H4K20A and H4K20R mutants to assess the consequence of eliminating H4K20me on L(3)mbt interaction with the genome. In order to perform this experiment, we built GFP- and FLAG-tagged alleles of l(3)mbt at the endogenous locus using CRISPR-Cas9 and the Scarless Gene Editing system. These alleles complement a null mutation of l(3)mbt, indicating that the epitope-tagged proteins are fully functional. We assessed L(3)mbt’s cytological localization in wildtype and H4K20-mutant animals via confocal microscopy using anti-GFP and anti-FLAG antibodies. In wildtype larval brains, imaginal discs, and adult ovaries, L(3)mbt is predominantly nuclear during interphase with much of the protein residing in the nucleoplasmic compartment. During mitosis, L(3)mbt disperses throughout the cell after nuclear envelope breakdown and does not localize to condensed mitotic chromosomes. In H4K20-mutant animals, we detected a small decrease in total L(3)mbt signal without any change in L(3)mbt localization. These results indicate that we are unable to use cytology alone to make conclusions about whether H4K20me is necessary for L(3)mbt chromatin binding. Therefore, we will next use approaches like CUT&RUN to determine the association of these epitope-tagged L(3)mbt proteins with the genome in wildtype and H4K20-mutant animals at a high resolution.

Determining how H4K20 methylation contributes to L(3)mbt recruitment to chromatin