The Effects of Histone Double-Mutants on Mitotic Recombination in D. melanogaster

Double-stranded breaks (DSBs) are detrimental lesions that lead to genome instability and threaten cell vitality, replication, and growth. Such lesions can be repaired by pathways such as non-homologous end-joining (NHEJ) and homologous recombination (HR). Mitotic Recombination (MR) is a form of HR that occurs in mitosis, and although it is a high-fidelity process that is commonly done to repair DSBs, a rare phenomenon known as a Mitotic Crossover Event (MCO) may occur. This may potentially lead to loss of heterozygosity (LOH), a condition that leads to the loss of the only functional copy of a gene or homozygosity of a mutant gene and may result in tumorigenesis. It is unknown how chromatin organization affects the occurrence of MCOs. States of chromatin organization are organized by the nucleosome, which is composed of 2 of each histone protein H2A, H2B, H3, and H4, and DNA. The addition of either an acetyl or methyl group in the histone tails, particularly on the amino acid Lysine, is essential to DNA replication, gene expression, silencing, and DNA repair protein recruitment, and is done in a process called Post-Translational Modification (PTM). Previous experiments using histone mutants showed that substituting lysine with either arginine (R) or alanine (A) in H4K20 inhibits its PTMs by methyltransferases, increased the rates of MCO in the Drosophila male germline. Later analysis of MCO location identified hotspots within 3 regions in pericentric heterochromatin. It is unknown whether the rate of MCO relative to the H4K20 single mutants would increase, decrease, or remain constant with the addition of a second histone mutation. We hypothesized that the rate of MCO events of the double histone mutants would generate a higher rate of MCO events in comparison to the H4K20R single mutant, and that the locations of MCO hotspots located for H4K20 would not change for the double-histone mutants. To test our hypothesis, we used a genetic assay to measure MCO in the male germline. MCO can be scored in the progeny by looking at the combination of the phenotypic markers, which are recessive. To analyze MCO location in the double-mutants, we performed an allele-specific PCR targeting the regions identified in the H4K20A and H4K20R single-mutants. From these experiments, the double-mutants H4K20A/H3K36R and H4K20R/H3K36R did not show significant changes in rates of MCO. However, there was a statistically significant increase in the rate of MCO in H4K20R/H3K4R, H4K20R/H3K9R, and H4K20R/H4K16R double-mutants in comparison to H4K20R single-mutants. The genetic assay showed that all the events in the double-mutants tested are in the region that spans the centromere. Analysis ofMCO region for H4K20R/H3K4R, H4K20R/H3K9R, and H4K20R/H4K16R showed no MCO occurring on chromosome 3R. This work provides insights into the role of histone PTMs on MCO and how the combination of specific mutants increases chromosome fragility. The understanding of this process is important to the medical field because MCO can lead to loss of heterozygosity, a hallmark of many common cancers such as lymphoma, breast cancer, and Glioblastoma Multiforme, a lethal form of pediatric brain cancer.