Gertuko biotinilazioa E2F7 transkripzio faktorearen interaktoma deskribatzeko estrategia gisa: TurboID sistemaren garapena

Abstract

E2F transkripzio faktoreek (E2F1-8) prozesu biologiko eta gaixotasun ugaritan parte hartzen dute. E2F7ri dagokionez, bere funtzio nagusia geneen adierazpena erreprimitzea dela deskribatu dugu, baina bere ekintza-mekanismoa ezagutzeke dago. Lan honetan, E2F7ren interaktoreak aztertzeko TurboID biotina ligasaren bidezko proteinen gertuko biotinilazio-sistema baten garapena deskribatzen dugu. Horretarako, TurboID-E2F7 ekoizten duten zelula-lerro egonkorrak garatu ditugu. Gure datuek berretsi dute TurboID-E2F7 fusio-proteina zelulen nukleoan kokatzen dela batik bat, E2F7 proteina basatia bezala. Aldiz, bere interaktoreak zitoplasman zein nukleoan daudela ikusi dugu. Gainera, lan honetan garatu den estrategia E2F7ren interaktoreak identifikatzeko sistema baliagarria dela frogatu dugu.

E2F transcription factors (E2F1-8) play an important role in several biological processes and diseases. Regarding E2F7, we have shown that its main role is to repress expression of protein- and microRNA-coding genes, but little is known about its mechanism. In this work, we describe the development of a proximity biotinylation approach based on the TurboID biotin ligase in order to study E2F7 interactors. With this aim, we generated stable cell lines that express TurboID-E2F7 in response to doxycycline. Our data confirm that TurboID-E2F7 localizes mainly in the nucleus, similarly to the wild-type E2F7, and describe the presence of E2F7 interactors in both the nucleus and the cytoplasm. In addition, we show that the TurboID strategy is suitable for identification of E2F7 interactors.