Spatiotemporal Control of the Epigenome via Photoactivatable Nuclear Localization

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DESCRIPTION (provided by applicant): Abstract Gene activation and silencing via epigenetic modifications is important for determining cell fate and is critical for the proper development of multicellular organisms. Determining the relative importance of various epigenetic markers in specific developmental processes can be challenging because these modifications are often dynamic and vary between cell and tissue types. Common approaches such as a gene knockout of an enzyme required for epigenetic modifications can be used to assess the global importance of the modification, but do not provide a straightforward approach for testing the importance of the modification at specific loci, cell types and developmental time points. Here, we aim to create a general approach for the control of epigenetic modifications that is reversible and can be applied at specific times in development to a specified set of cells. In preliminary studies we have developed a light activatable protein that localizes to the cytoplasm in the dark, but enters the nucleus when the cell is illuminated with blue light. Our hypothesis is that this switch, called LANS for Light Activatable Nuclear Shuttle, can be used to control the activity of proteins that must be in the nucleus to be functional. As proof of concept, we have shown that we can use LANS to control the activity of a transcription factor in yeast with light. Here, we wil explore if light activated nuclear localization can be used to control enzymes and scaffolding proteins required for histone and DNA methylation and acetylation. To target predetermined loci, we will fuse LANS with naturally occurring and engineered DNA binding domains. We will test the LANS switch in mammalian cell culture, and we will explore whether the switch can be used to manipulate cell fate in C. elegans via light mediated control of a histone deacetylase.