Using Photoswitchable Proteins to Assess Chromatin Stability

Abstract

Nucleosome modifications in the genome influence local heterochromatin state. Varied examples of epigenetics rely on transgenerational effects and heritability of expression states between cell (mitotic) or organismal (meiotic) generations. Most of the current focus is on transgenerational inheritance of expression states determined by methylation of lysine-4 and lysine-9 sites of histone H3. We will use photoswitchable protein Dendra2 fused to histone protein H3 to test the nature of histone-based heritability in the germline stem cell niche of male Drosophila melanogaster. Photoswitching provides a solid time-point measure of which H3 proteins are present at the time of UV exposure and which proteins are new at the time of photography. This will provide an opportunity to monitor chromosome-scale histone stability, recycling, and turnover, to monitor chromosome movement in live nuclei, and to critically test histone inheritance through cell division. Photoswitching allows us to generate a snapshot of histone behavior and elucidate the contribution of expression state modification to transgenerational effects. Here, we present background, recent progress in creation and future experimentation.