Characterization of the circadian chromatin landscape using a novel CRISPR/Cas9-guided proximity-labelling technique

Descripción del proyecto

Mapeo del reloj interno del cuerpo

La mayoría de los organismos vivos poseen relojes internos que se sincronizan con las estaciones o el ciclo de día y noche. El funcionamiento normal de estos ciclos de veinticuatro horas (o circadianos) es vital para los humanos, y una alteración continua de su ritmo provoca enfermedades graves. Se desconoce en gran medida el proceso real de la transmisión circadiana. El proyecto CLOCK prevé utilizar un método de etiquetado CRISPR/Cas9-APEX (CASPEX) para mapear tres importantes secuencias reguladoras circadianas y buscar nuevos modificadores del reloj. Se prevé que los resultados aportarán más conocimientos para tratar la elevada incidencia en la Unión Europa de patologías relacionadas con la alteración del ritmo circadiano como el cáncer, la diabetes y las enfermedades cardiovasculares y neurodegenerativas.

Objetivo

To stay in synchrony with environmental cycles, most living organisms possess endogenous clocks. Circadian clocks are molecular oscillators present in most mammalian cells that drive circadian (~24 h) rhythms of a wide range of molecular, physiological and behavioral functions. Circadian clocks are essential for health. In humans their dysregulation (e.g. caused by shift work, jet lag etc.) has been associated with the development of multiple pathologies (e.g. cancer, metabolic diseases like diabetes and obesity as well as cardiovascular and neurodegenerative diseases) prevalent in the European Union.

One central aspect of molecular oscillator function is the tight regulation of circadian transcription. Over the years, several proteins and cis-regulatory enhancer elements (i.e. specific sequences located around the promoter region, e.g. E-box, RRE, D-box) have been shown to be essential for circadian transcription. However, mainly because of technical limitations, those studies focused on few regulators and have left many gaps in the understanding of the dynamics of the circadian transcription. Therefore, this project proposes to use state-of-the-art quantitative genomic-locus proteomics to provide the first comprehensive and unbiased characterization of the rhythmic protein binding at key circadian regulatory regions – a key regulatory node of circadian clock function Using a CRISPR/Cas9-APEX labelling method (CASPEX), we will first characterize the circadian chromatin landscape of the three main circadian regulatory regions (i.e. E-boxes, RREs, D-boxes). We expect to find new clock modifiers, whose role for circadian rhythm generation will be investigated in a subsequent part of the project, using an RNAi-based secondary screen. Overall, this project will provide novel insights in the circadian oscillator mechanism in humans, which is essential for developing better treatment strategies for circadian clock-associated disorders.