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Engineering optogenetic control of p53

Wehler, Pierre

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Abstract

Cells respond to external and internal stimuli mostly with changes in transcription of target genes. Gene expres- sion is mediated by transcription factors, central nodes in highly regulated signalling networks. In multiple sig- nalling pathways, different stimuli converge on the same transcription factor, yet induce different cell fates. Re- cently, the dynamics of transcription factors have been identified to play a key role in converting the received trigger into the appropriate response. It is proposed that the same transcription factor induces different gene expression programs purely depending on its dynamics. One example of particular interest is the transcription factor p53. Under physiological conditions, p53 levels are kept low by a tightly regulated network. Upon stress, p53 levels increase and show a pulsatile or sustained behaviour, depending on the type and severityof the stress. Interestingly, it was proposedthat p53 dynamics dic- tate which downstream gene expression programs are initiated. However, many questions remain open, such as whether the dynamics are sufficient to drive differential gene expression, or whether stress-induced post- translational modifications of p53 and other interacting factors play a role alongside p53 dynamics. Moreover, it is still unknown, how p53 dynamics are eventually translated into a specific target gene expression programs. The p53 signalling and regulatory network is both, highly complex and dynamic. Understanding this network requires targeted dissection using specific and precise methods. To address these requirements, I employed op- togenetic methods, as light possesses unmatched spatial and temporal resolution. In contrast to chemical per- turbation methods, light as a trigger is non-invasive and has a superior specificity. Here, I used optogenetics to reconstitute various p53 dynamics, by controlling the levels and localization of p53, and I investigated the out- come of these manipulations in the absence of upstream stress. Specifically,Iachievedlight-mediatedcontrolofendogenousp53levelsanditsactivity. TheE3ubiquitinligase Mdm2isknowntobethemainregulatorofp53levels. Icoulddemonstratethatthep53-Mdm2inhibitorypeptide (PMI) inhibits p53 degradation in vivo , and that its effect on p53 levels is localisation-dependent, and only occurs when PMI is present in the nucleus. To control the localization of PMI with light I used a light-inducible nuclear export system (LEXY). LEXY is a versatile protein tag that harbours an engineered As LOV2 domain exposing a nuclear export sequence(NES) uponbluelightillumination, leadingto rapidnuclearexport ofthetaggedamino acid sequence, and re-import into the nucleus once the cells are not subjected to blue light anymore. By fusing PMI to LEXY, I could obtain light-mediated control over localisation of PMI-LEXY and thus degradation of p53, resulting in elevated p53 levels. Additionally, expression of the p53 target gene p21 was increased, showing that not only p53 protein levels are increased, but also that p53 is transcriptionally active. In a second approach, I used LEXY to control the localization of an exogenously expressed p53 with light. I could show that I can repeatedly accumulate p53 tagged with LEXY in and out of the nucleus, effectively gener- ating p53 pulses akin to those obtained under certain stress conditions. I generated a stable cell line expressing p53-LEXY under an inducible promoter, allowing robust expression of p53-LEXY. p53-LEXY is transcriptionally ac- tive, andcaninducetranscriptionofmultiplep53targetgenesintheabsenceofstress. Moreover, p53-LEXYcould induce the terminal cell fate senescence. However, prolonged blue light exposure and application of more com- plex illumination patterns resulted in impaired translocation and cellular stress. Reduction of the light intensity to prevent phototoxicity corresponded to losing the ability to control p53-LEXY with light. Inducing genotoxic stress to stimulate p53 activity increased the expression of some p53 target genes in the stable cell line, yet re- vealed high variability between biological replicates, questioning the utility of this cell line.

Item Type: Dissertation
Supervisor: Di Ventura, Prof. Dr. Barbara
Date of thesis defense: 9 April 2019
Date Deposited: 16 Apr 2019 13:25
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Institute of Pharmacy and Molecular Biotechnology
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