Summary 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. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue light controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events.

Photo-controlled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photo-controlled DNA binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from H. halophila (Morgan et al., J. Mol. Biol. 2010, 399:94-112). Here we report a structure-based attempt to improve the degree of photoswitching observed with this chimeric protein. Using computational design tools PoPMuSiC 2.0, Rosetta, Eris and bCIPA we identified a series of single and multiple point mutations that were expected to stabilize the folded dark state of the protein and thereby enhance the degree of photoswitching. While a number of these mutations, particularly those that introduced a hydrophobic residue at position 143, did significantly enhance dark-state protein stability as judged by urea denaturation studies, dark-state stability did not correlate directly with the degree of photoswitching. Instead, the influence of mutations on the degree of photoswitching was found to be related to their effects on the degree to which DNA binding slowed the pB to pG transition in the PYP photocycle. One mutant, K143F, caused a ~10-fold slowing of the photocycle and also showed the largest difference in apparent K d for DNA binding − 3.5-fold lower upon irradiation. This change in apparent K d causes a 12-fold enhancement in fraction bound DNA upon irradiation due to the cooperativity of DNA binding by this family of proteins. The results highlight the strengths and weaknesses of current approaches to a practical problem in protein design as well as suggesting strategies for further improvement of designed photo-controlled transcription factors. Keywordscoiled-coil; bZIP; photo-control; photoactive yellow protein; PYP; optogenetics; photoisomerization; genetically encoded; LOV domain Photo-control of transcriptional processes in living cells may help to elucidate the roles of location and timing of gene expression in spatiotemporally complex settings such as occur during development and during normal functioning of the nervous system (1). Naturally occurring photo-controlled transcription factors are known (2,3), however, they are often multi-component systems so that engineering them for use as tools for the photo-control of transcription is not straightforward. Alternatively, naturally occurring light-dependent protein-protein interactions may be co-opted to photo-control transcription. Quail and Correspondence to: G. Andrew Woolley, awoolley@chem.utoronto.ca. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 February 22. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript colleagues used a phytochrome-GAL4-DNA-binding-domain fusion and a PIF3-GAL4-activation-domain fusion to photo-control the expression of genes that contain a promoter with a GAL4 binding site (4). A similar approach using the transactivator VP16 fused to the LOV domain of FKF1 and the ...

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Light-switchable proteins offer numerous opportunities as tools for manipulating biological systems with exceptional degrees of spatiotemporal control. Most designed light-switchable proteins currently in use have not been optimised using the randomisation and selection/screening approaches that are widely used in other areas of protein engineering. Here we report an approach for screening light-switchable DNA-binding proteins that relies on light-dependent repression of the transcription of a fluorescent reporter. We demonstrate that the method can be used to recover a known light-switchable DNA-binding protein from a random library.