Engineering Optogenetic Control of Endogenous p53 Protein Levels

Abstract

The transcription factor p53 is a stress sensor that turns specific sets of genes on to allow the cell to respond to the stress depending on its severity and type. p53 is classified as tumor suppressor because its function is to maintain genome integrity promoting cell cycle arrest, apoptosis, or senescence to avoid proliferation of cells with damaged DNA. While in many human cancers the p53 gene is itself mutated, there are some in which the dysfunction of the p53 pathway is caused by the overexpression of negative regulators of p53, such as Mdm2, that keep it at low levels at all times. Here we develop an optogenetic approach to control endogenous p53 levels with blue light. Specifically, we control the nuclear localization of the Mmd2-binding PMI peptide using the light-inducible export system LEXY. In the dark, the PMI-LEXY fusion is nuclear and binds to Mdm2, consenting to p53 to accumulate and transcribe the target gene p21. Blue light exposure leads to the export of the PMI-LEXY fusion into the cytosol, thereby Mdm2 is able to degrade p53 as in the absence of the peptide. This approach may be useful to study the effect of localized p53 activation within a tissue or organ.

A light way for nuclear cell biologists

“…Importantly, methods based on nuclease-dead Cas9 (dCas9), Zinc fingers or TALENs allow controlling transcription of selected endogenous genes. This is true also for those methods whereby an intact, natural TF is light-regulated, for instance NFAT ( 112 ) or p53 ( 49 , 113 ). However, in this case, all target genes of the TF are, at least potentially, activatable—some may not be activated due to missing post-translational modifications on the TF, which may require pathway activation, or specific TF dynamics ( 19 , 114 ).…”

“…In the case of optogenetic gene expression systems, the interaction between the DBD and the TAD is controlled through light-inducible heterodimerizers (see above). Other methods to control gene expression consist in controlling with light: (i) homodimerization, since several TFs contact their cognate DNA response element in dimeric form ( 85 , 88 , 89 , 96 , 98 , 111 ); (ii) a post-translational modification that regulates the localization or activity of the TF ( 37 , 94 , 112 ); (iii) the nuclear localization of the TF ( 48–51 ) and (iv) the stability of the TF ( 113 ).…”

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

“…Although the development of these system required substantial efforts in protein engineering, this versatile strategy enables the light-dependent protein translocation in a simpler way by fusing the biofunctional domain to the LINuS or LEXY. In addition to optical control of gene expression by manipulating the localization of transcription factor [ 20 , 21 ], LINuS and LEXY systems have also been used in optogenetic regulation of cofilin-1 for controlling F-actin assembly [ 99 ], manipulating of the endogenous p53 protein levels [ 100 ], and optogenetic translocation of TEV protease for manipulating protein degradation [ 101 ].…”

Lights up on organelles: Optogenetic tools to control subcellular structure and organization

“…Once darkness is restored, the Jα helix regains its coiled configuration and again masks the effector domain (Crosson & Moffat, 2001; Konold et al, 2016; Peter, Dick, & Baeurle, 2010). A powerful application of this strategy is the photocaging of subcellular localization motifs, which have proven useful in controlling protein levels within specific subcellular compartments such as the nucleus (Wehler & Di Ventura, 2019).…”

“…Blue light induces the display of the export signal and prompts LEXY or LINX-tagged POIs to bind exportins and leave the nucleus. LEXY has been employed for light inducible export of both the tumor suppressor p53 and the p53-Mdm2/ MdmX inhibitor (PMI) which, when localized in the nucleus, decreases p53 degradation (Niopek et al, 2016;Wehler & Di Ventura, 2019). Nuclear export can also be controlled with chromatin-anchored LEXY designed to bind CRM1 (exportin-1) in the photoactivated state, thereby preventing CRM1-mediated protein export in the light (Niopek et al, 2016).…”