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

Project description

3D ultrastructure of DNA organisation in single cells

Understanding how genetic information is regulated inside the cell represents an intriguing challenge of modern biology. Genome-wide studies have revealed a hierarchical genome organisation by structuring proteins, which regulates fundamental processes such as transcription, replication and DNA repair. Topologically associating domains (TADs) are functional building blocks of chromatin organisation above the nucleosome level. The now available super-resolution microscopy set-ups allow the study of genomic structures and their dynamics in situ at a resolution of 10 nm, which is comparable to the size of a nucleosome. The aim of the EU-funded ChromaSTORM project is to reveal the principles of TAD organisation in human cells using an imaging-based approach to visualize its genomic component as well as the architectural proteins. The project will provide the first 3D description of this central chromatin super-structure.

Objective

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.

Coordinator

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Net EU contribution
€ 174 806,40
Address
Meyerhofstrasse 1
69117 Heidelberg
Germany

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Region
Baden-Württemberg Karlsruhe Heidelberg, Stadtkreis
Activity type
Research Organisations
Links
Total cost
€ 174 806,40