Visualising how proteins fold DNA into topologically associating domains in single human cells

Descripción del proyecto

Ultraestructura tridimensional de la organización del ADN en células individuales

Comprender cómo se regula la información genética en las células constituye un reto fascinante de la biología moderna. Los estudios hologenómicos han demostrado la existencia de una organización jerárquica del genoma mediante proteínas de organización, que regulan procesos fundamentales como la transcripción, la replicación y la reparación del ADN. Los dominios de asociación topológica (TAD, por sus siglas en inglés) son componentes básicos funcionales de la organización de la cromatina por encima del nivel de nucleosoma. Los equipos de microscopía de superresolución disponibles hoy día permiten estudiar estructuras genómicas y su dinámica «in situ» a una resolución de 10 nm, un tamaño similar al de un nucleosoma. El objetivo del proyecto ChromaSTORM, financiado con fondos europeos, es dilucidar los principios de la organización de los TAD en células humanas mediante un método basado en la obtención de imágenes para visualizar su componente genómico, así como las proteínas estructurales. El proyecto proporcionará la primera descripción tridimensional de esta superestructura de cromatina.

Objetivo

How can phenotypic variations emerge from cells that carry the same genetic information? It is one of today’s great challenges to understand how genetic information is modulated inside the cell. Over the last decade, insight from genome-wide proximity-based ligation approaches revealed that the genome is organised in a hierarchical manner with the help of structuring proteins, and that this spatial organisation regulates the core functions of the genome, such as transcription, replication and repair. In this context, topologically associating domains (TADs) were identified as fundamental and functional building blocks of chromatin organisation above the nucleosome level. Despite its vital importance, our current understanding of the spatial organisation of TADs remains largely enigmatic. With the advent of super-resolution microscopy, tools are now available for studying genomic structures and their functional dynamics in situ at a resolution of 10 nm which corresponds to the size of a few nucleosomes.
The goal of this project is to reveal the principles of TAD organisation in human cells. To achieve this, I will employ a multidisciplinary imaging-based approach. I will simultaneously visualise the DNA backbone of TADs and architectural proteins involved in TAD structure applying 3D super-resolution microscopy. I will focus on the key TAD organisers CTCF and Cohesin, as well as on Mediator and Condensin II. Additionally, I will directly study their individual structuring function for TADs by their acute depletion. Integrating these data with quantitative measurements of absolute protein copy numbers, I will derive a data-driven model of inner TAD organisation in cells. These studies will provide the first 3D description of this fundamental chromatin super-structure and will further our understanding of genome architecture which is a prerequisite for understanding genome function.