4 research outputs found
Optogenetic inhibitor of the transcription factor CREB
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
Optogenetic inhibitor of the transcription factor CREB
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
Optical Control of Protein–Protein Interactions via Blue Light-Induced Domain Swapping
The design of new optogenetic tools
for controlling protein function
would be facilitated by the development of protein scaffolds that
undergo large, well-defined structural changes upon exposure to light.
Domain swapping, a process in which a structural element of a monomeric
protein is replaced by the same element of another copy of the same
protein, leads to a well-defined change in protein structure. We observe
domain swapping in a variant of the blue light photoreceptor photoactive
yellow protein in which a surface loop is replaced by a well-characterized
protein–protein interaction motif, the E-helix. In the domain-swapped
dimer, the E-helix sequence specifically binds a partner K-helix sequence,
whereas in the monomeric form of the protein, the E-helix sequence
is unable to fold into a binding-competent conformation and no interaction
with the K-helix is seen. Blue light irradiation decreases the extent
of domain swapping (from <i>K</i><sub>d</sub> = 10 ÎĽM
to <i>K</i><sub>d</sub> = 300 ÎĽM) and dramatically
enhances the rate, from weeks to <1 min. Blue light-induced domain
swapping thus provides a novel mechanism for controlling of protein–protein
interactions in which light alters both the stability and the kinetic
accessibility of binding-competent states