Blue Light Control of CREB via A Designed Dominant Negative

Abstract

Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Direct optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe a protein engineering approach that generated opto-DN-CREB, a blue light controlled inhibitor of the endogenous transcription factor CREB. We created opto-DN-CREB fusing the dominant negative inhibitor A-CREB to the photoactive yellow protein (PYP). A light driven change in the conformation of PYP interferes with coiled-coil formation between A-CREB and the bZIP domain of CREB, thereby enabling CREB homodimerization and DNA binding. We characterized the optogenetic control of CREB function in vitro, in cultured HEK293T cells, and in primary cultured neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Specific dominant negative inhibitors exist for numerous endogenous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events.