Uncovering a link between H2Av and the cell cycle during early Drosophila embryonic development
Histones in the nucleus associate with DNA and aid in condensing it into chromatin. They also play fundamental roles in the transition between active and inactive chromatin states. Some canonical histones are replaced in nucleosomes by histone variants, providing another layer of complexity in regulating gene expression and epigenetic silencing. In Drosophila melanogaster, the sole H2A variant is H2Av. Previous work has shown that H2Av along with H2A and H2B are sequestered on lipid droplets (LDs) by the protein Jabba. For H2Av, LD interactions are transient, and H2Av molecules continuously exchange between LDs through the cytoplasm. This exchange buffers H2Av availability and reduces the rate of H2Av import into the nucleus. Jabba mutants lack this buffering ability and overaccumulate H2Av in the nucleus. Jabba mutants also display a shortened nuclear cycle 13 and increased nuclear falling. Using embryos containing two copies of H2Av-Dendra in addition to endogenous H2Av, nuclear cycle length was measured via live imaging. Nuclear falling was quantified by Hoechst staining and analysis through Amira. This revealed that these Jabba phenotypes arise from nuclear H2Av overaccumulation. In order to uncover H2Av’s role in cell cycle timing and nuclear falling, we are currently mutating different regions of H2Av and measuring nuclear cycle length via live imaging and quantifying nuclear falling via Hoechst staining and analysis through Amira. Site direct mutagenesis to generate 2X H2AvS137A, 2X H2Av reveal that H2A.X function is not required for the speed-up phenotype observed but is necessary for nuclear falling. A Chk1 heterozygous mutant in an H2Av-Dendra background was generated to measure nuclear cycle length. This revealed that partial checkpoint inactivation causes nuclear cycle 13 speedup and staining with Hoechst revealed increased nuclear falling in these embryos.