Developmental and cell type specific histone gene expression patterns

About This Grant

PROJECT SUMMARY: Histone proteins are critical for the compaction and organization of newly synthesized genomes. Histone protein concentrations help regulate the timing of rapid cell divisions in the early animal embryo. Misregulation is developmentally lethal: histone overexpression leads to extra or asynchronous cell divisions, while reduced histone expression leads to cell cycle arrest. As embryos develop, cell division dramatically slows. This change in the cell cycle leads to radically differing demands for histone transcripts and variable rates of histone gene expression. Cells must quickly alter their histone expression dynamics to match their cycling needs. Unequal demand for histone transcripts is perpetuated later in development as diverse cell types acquire novel proliferative potentials. Because of the ubiquitous necessity of histone proteins, genomes often carry many copies of each histone that are nearly identical in sequence, which may be clustered or distributed. This creates a unique regulatory problem in differentiating cells because histone genes are indiscernible from each other. A longstanding, major assumption in the field is that histone genes are largely regulated in concert: all histone genes are expressed or silenced to the same degree. I aim to fill this fundamental knowledge gap by tracing histone transcripts to both gene and locus of origin to determine how identical histone genes are expressed throughout development to maintain proper proliferative potential. I will test my hypothesis in two specific aims: In Aim 1, I will test how clustering of histone genes affects expression by engineering a transgenic D. melanogaster that includes sequence variation in histone coding regions. This allows for the tracing of transcripts to their gene of origin. I will perform RNA sequencing to compare mRNA dynamics from individual histone genes through an embryonic time course as the cell cycle lengthens. These experiments will determine how the expression patterns of individual genes within a cluster change with dynamic cell division requirements. In Aim 2, I will test how separating histone genes between loci alters regulation by utilizing the natural genomes of Drosophila virilis and D. simulans/transgenic D. melanogaster hybrids, whose genomes carry two histone loci of different sizes (D. virilis) or similar sizes (hybrids). I will leverage natural histone coding sequence variations in both species to perform single nucleotide variant fluorescent in situ hybridization, visualizing locus-specific transcriptional patterns in differentiating neural cells. These experiments will define regulatory strategies that attenuate histone expression as cells differentiate and the cell cycle slows. Overall, these aims illustrate how identical sets of histone genes are differentially regulated during early animal development, which is crucial to our understanding of developmental cellular differentiation and proliferation. I designed these experiments, in collaboration with my sponsor and co-sponsor, to address critical knowledge gaps in our field, develop expertise in a broad array of techniques, and provide me with the training opportunities to become an independent researcher.