Mechanical Basis of Genome Organisation through INO80

Descrizione del progetto

Decodificare le basi meccaniche della regolazione genica

I nucleosomi sono le unità fondamentali della cromatina e sono costituiti da DNA avvolto attorno alle proteine istoniche. Il posizionamento dei nucleosomi è un processo dinamico guidato dall’interazione tra sequenza del DNA, complessi della cromatina e altri fattori, che influenza quali regioni del DNA risultano accessibili alla trascrizione. Nuove prove indicano che questo posizionamento dipende anche dalle proprietà meccaniche del DNA nelle regioni dei promotori genici. Con il supporto del programma di azioni Marie Skłodowska-Curie, il progetto MebaGINO si propone di studiare i meccanismi biofisici che regolano il posizionamento dei nucleosomi, concentrandosi sull’enzima INO80 che rimodella la cromatina. I ricercatori utilizzeranno i dati di laboratorio per generare un modello biofisico in grado di spiegare in che modo gli stimoli meccanici controllano il rimodellamento della cromatina.

Obiettivo

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.