A Photodeactivatable Antagonist for Controlling CREB-Dependent Gene Expression

Abstract

A novel photodeactivation strategy for controlling gene expression has been developed based on light-induced activation of cAMP response element binding protein (CREB). Light-induced cleavage of the photoresponsive protecting group of an antagonist of CREB binding protein (CBP) results in photocleaved products with weak binding affinity for CBP. This photodissociation reaction enables protein-protein interactions between CBP and CREB that trigger the formation of a multiprotein transcription complex to turn gene expression "on". This enables irradiation of antagonist-treated HEK293T cells to be used to trigger temporal recovery of CREB-dependent transcriptional activity and endogenous gene expression under photolytic control.

Figure 1

Four photolytic strategies for the optochemical spatiotemporal control of protein activity. (a) Photolytic release of a caged agonist to turn protein activity “on”. (b) Photolytic release of a caged antagonist to turn protein activity “off”. (c) Photolytic cleavage of a photodeactivatable antagonist to turn protein activity “on”. (d) Photolytic cleavage of a photodeactivatable antagonist results in protein–protein interactions affording a complex that turns “on” protein activity and gene expression.

Figure 2

Design of photodeactivatable CBP inhibitors (PCIs). (a) Caged CBP inhibitor strategy for photochemical downregulation of CREB-mediated gene expression. (b) Photocleavage of the o-nitrobenzyl PPG of a PCI affords CI-1 that exhibits weak CBP binding affinity. (c) Photocleavage of a CBP bound antagonist (PCI) results in upregulation of CREB-mediated gene expression. (d) CBP inhibitors, naphthol AS-E and 666-15.^,

Figure 3

Photolysis of PCIs. HPLC analyses of the photolytic cleavage products of 50 μM samples of (a) PCI-1 and (b) PCI-2 in 20 mM HEPES buffer (pH 7.4). Time course of the photolysis reactions of (c) PCI-1 and (d) PCI-2 to produce CI-1. Light intensity: 4 mW cm^–2, λ = 365 nm ±5 nm. Error bars denote ± SD (N = 3).

Figure 4

CREB inhibitory activities of PCIs before and after photolysis. Inhibition of CREB activity using (a) PCI-1 and (b) PCI-2, with both data sets presented in comparison to CI-1 and PCI photoproducts. Relative luminescence values are reported as a ratio of Firefly:Renilla luciferase luminescent intensities. Photolysis of PCIs (100 μM) carried out using UV illumination (10 mW cm^–2, λ = 365 ± 5 nm) for 10 min in Hanks’s buffered saline solution (HBSS). Error bars denote ± SD (N = 3).

Figure 5

Light-mediated recovery of CREB activity in PCI-treated HEK293T cells. CREB-mediated gene expression levels after treatment of cells with 6 μM PCI-1 and PCI-2 before and after illumination (λ_ex = 365 nm, 10 mW cm^–2 for 5 min) in HBSS (1% DMSO). Error bars denote ± SD (N = 3). Significance evaluated using the Student t-test.

Figure 6

Results of qRT-PCR experiments used to determine NR4A2 gene expression levels in HEK293T cells. CREB-dependent NR4A2 mRNA expression levels of HEK293T cells treated with 6 μM PCI-1 and PCI-2, before and after illumination (λ_ex = 365 nm, 10 mW cm^–2 for 5 min) in DMEM (0.6% DMSO). Error bars denote ± SD (N = 3). Significance evaluated using the Student t-test.