Mechanical Basis of Genome Organisation through INO80

Descripción del proyecto

Descifrar la base mecánica de la regulación génica

Los nucleosomas son las unidades básicas de la cromatina y están formados por ADN enrollado alrededor de proteínas histonas. El posicionamiento de los nucleosomas es un proceso dinámico promovido por la interacción entre la secuencia de ADN, los complejos de cromatina y otros factores, lo que influye en qué regiones del ADN están accesibles para la transcripción. Nuevas pruebas sugieren que este posicionamiento también depende de las propiedades mecánicas del ADN en las regiones promotoras de los genes. El proyecto MebaGINO, financiado por las acciones Marie Skłodowska-Curie, tiene por objeto investigar los mecanismos biofísicos que determinan el posicionamiento de los nucleosomas, centrándose en la enzima INO80 de remodelación de la cromatina. Los investigadores utilizarán datos experimentales para crear un modelo biofísico que permita explicar cómo las señales mecánicas controlan la remodelación de la cromatina.

Objetivo

Recent breakthroughs found that mechanical features of gene promoters influence nucleosome positioning through ATP-dependent chromatin remodeling enzymes. This finding challenges classical models of DNA-sequence-dependent histone recruitment. While mechanisms for the translation of DNA’s physical parameters to nucleosome positions are debated, their experimental validation is still pending. MebaGINO aims to address this fundamental gap in knowledge. Taking advantage of recent advances in time-resolved CryoEM, I will answer the following research question:

What is the biophysical mechanism underlying the influence of DNA mechanics on nucleosome positioning by INO80?

I will use the INO80 chromatin remodeler as a model system to understand how nucleosomes are positioned adjacent to gene promoters featuring specific mechanical patterns. Firstly, I will interrogate how the nucleosome translocation process will be stalled through substitution of ATP with ATP-analogues on DNA with varying stiffness. From these experiments, I will obtain molecular structures via single-particle CryoEM. Next, I will obtain molecular structures from short-lived states using time-resolved CryoEM. For this, I will contribute to the development of an automated platform for CryoEM sample freezing which will be made available to the scientific community via EMBL's Electron Microscopy facility. Finally, I will build a biophysical model explaining the influence of DNA mechanics on INO80’s translocation process. Thus, I will provide novel views onto the mechanical basis of gene regulation which are relevant beyond fundamental research as for example during cancer development.

During this project, I will receive extensive training on cutting-edge structural biology instrumentation while contributing with my nanotechnology skills. Combined with my biophysics background, this fellowship will enable me to establish an interdisciplinary research group focused on structural biophysics.