539A Poster - 07. Chromatin, epigenetics and genomics
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Aaron T. Crain; Robert J. Duronio

Affiliation: University of North Carolina at Chapel Hill, Chapel Hill, NC

Keywords:
d. histone variants and modifications; s. cell competition

The mono-methylation of lysine 20 of histone H4 (H4K20me1) is involved in numerous processes necessary for cell proliferation, including DNA replication, gene expression, and chromosome condensation during mitosis. These functions of H4K20me1 have been largely derived from interpreting the phenotypes resulting from manipulation of the activity of Set8, the lysine methyltransferase responsible for depositing H4K20me1. However, such interpretations are complicated by studies demonstrating that Set8 has several non-histone substrates with essential roles in cell proliferation, such as p53 and PCNA, as well as non-catalytic functions. To directly assess the contribution of H4K20me in cell proliferation, we engineered a novel system for generating mosaic tissues containing clones of histone mutant cells. We first created a new, precise deletion of the replication dependent histone locus on chromosome 2 (HisC) that is marked with Act5c-dsRed. This allele was then combined with an established transgenic histone replacement platform in which a transgene containing a synthetic histone gene array capable of complementing homozygous HisC deletions is integrated on chromosome 3. These transgenes are engineered to express either wild type (WT) or mutant (e.g. H4K20A or H4K20R) histones. We then use FLP/FRT-mediated mitotic recombination to produce dual-color mosaic tissue in the Drosophila eye, where one color (green) labels cells expressing wild-type histones and the other color (red) labels cells expressing only mutant histones. Thus, we can easily visualize and quantify competitive proliferation of histone mutant cells adjacent to wild-type cells. Strikingly, we found that both H4^K20A and H4^K20R mutant cells can proliferate adjacent to wild-type cells, achieving 34% and 22% of the adult eye, respectively, compared to 42% for H4^WT. In contrast, we found that Set8^null mutant cells cannot proliferate adjacent to wild-type cells and die. We interpret the lack of proliferation and death of Set8^null cells as likely due to H4K20me-independent mechanisms, and thus conclude that the role of H4K20me in cell proliferation remains unknown. To discover the functions of H4K20me in cell proliferation we will utilize our newly established mosaic system to perform a forward genetic screen for modifiers of proliferation of H4K20 mutant cells. This work establishes the first platform for performing forward genetics using histone mutant cells in animals.