<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Engineering optogenetic control of p53"^^ . "Cells respond to external and internal stimuli mostly with changes in transcription of target genes. Gene expres-\r\nsion is mediated by transcription factors, central nodes in highly regulated signalling networks. In multiple sig-\r\nnalling pathways, different stimuli converge on the same transcription factor, yet induce different cell fates. Re-\r\ncently, the dynamics of transcription factors have been identified to play a key role in converting the received\r\ntrigger into the appropriate response. It is proposed that the same transcription factor induces different gene\r\nexpression programs purely depending on its dynamics.\r\nOne example of particular interest is the transcription factor p53. Under physiological conditions, p53 levels\r\nare kept low by a tightly regulated network. Upon stress, p53 levels increase and show a pulsatile or sustained\r\nbehaviour, depending on the type and severityof the stress. Interestingly, it was proposedthat p53 dynamics dic-\r\ntate which downstream gene expression programs are initiated. However, many questions remain open, such\r\nas whether the dynamics are sufficient to drive differential gene expression, or whether stress-induced post-\r\ntranslational modifications of p53 and other interacting factors play a role alongside p53 dynamics. Moreover,\r\nit is still unknown, how p53 dynamics are eventually translated into a specific target gene expression programs.\r\nThe p53 signalling and regulatory network is both, highly complex and dynamic. Understanding this network\r\nrequires targeted dissection using specific and precise methods. To address these requirements, I employed op-\r\ntogenetic methods, as light possesses unmatched spatial and temporal resolution. In contrast to chemical per-\r\nturbation methods, light as a trigger is non-invasive and has a superior specificity. Here, I used optogenetics to\r\nreconstitute various p53 dynamics, by controlling the levels and localization of p53, and I investigated the out-\r\ncome of these manipulations in the absence of upstream stress.\r\nSpecifically,Iachievedlight-mediatedcontrolofendogenousp53levelsanditsactivity. TheE3ubiquitinligase\r\nMdm2isknowntobethemainregulatorofp53levels. Icoulddemonstratethatthep53-Mdm2inhibitorypeptide\r\n(PMI) inhibits p53 degradation in vivo , and that its effect on p53 levels is localisation-dependent, and only occurs\r\nwhen PMI is present in the nucleus. To control the localization of PMI with light I used a light-inducible nuclear\r\nexport system (LEXY). LEXY is a versatile protein tag that harbours an engineered As LOV2 domain exposing a\r\nnuclear export sequence(NES) uponbluelightillumination, leadingto rapidnuclearexport ofthetaggedamino\r\nacid sequence, and re-import into the nucleus once the cells are not subjected to blue light anymore. By fusing\r\nPMI to LEXY, I could obtain light-mediated control over localisation of PMI-LEXY and thus degradation of p53,\r\nresulting in elevated p53 levels. Additionally, expression of the p53 target gene p21 was increased, showing that\r\nnot only p53 protein levels are increased, but also that p53 is transcriptionally active.\r\nIn a second approach, I used LEXY to control the localization of an exogenously expressed p53 with light. I\r\ncould show that I can repeatedly accumulate p53 tagged with LEXY in and out of the nucleus, effectively gener-\r\nating p53 pulses akin to those obtained under certain stress conditions. I generated a stable cell line expressing\r\np53-LEXY under an inducible promoter, allowing robust expression of p53-LEXY. p53-LEXY is transcriptionally ac-\r\ntive, andcaninducetranscriptionofmultiplep53targetgenesintheabsenceofstress. Moreover, p53-LEXYcould\r\ninduce the terminal cell fate senescence. However, prolonged blue light exposure and application of more com-\r\nplex illumination patterns resulted in impaired translocation and cellular stress. Reduction of the light intensity\r\nto prevent phototoxicity corresponded to losing the ability to control p53-LEXY with light. Inducing genotoxic\r\nstress to stimulate p53 activity increased the expression of some p53 target genes in the stable cell line, yet re-\r\nvealed high variability between biological replicates, questioning the utility of this cell line."^^ . "2019" . . . . . . . "Pierre"^^ . "Wehler"^^ . "Pierre Wehler"^^ . . . . . . "Engineering optogenetic control of p53 (PDF)"^^ . . . 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