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

Project Information

ChromaSTORM

Grant agreement ID: 842542

DOI

10.3030/842542

Project closed

EC signature date 10 April 2019

Start date 1 September 2020

End date 31 August 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 174 806,40

EU contribution

€ 174 806,40

Coordinated by

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany

Periodic Reporting for period 1 - ChromaSTORM (Visualising how proteins fold DNA into topologically associating domains in single human cells)

Reporting period: 2020-09-01 to 2022-08-31

Summary of the context and overall objectives of the project

Our body is composed of a multitude of cells with different functions like blood cells, skin cells and neuronal cells. Yet the genetic information in all our cells is the same. How can different phenotypes emerge from cells carrying the identical genetic sequence? It is one of today’s great challenges to understand how genetic information is modulated inside the cell nucleus. Over the last decade, research has revealed that our genome is organised on multiple levels with the help of structuring proteins, and that this spatial organisation regulates the core functions of the genome resulting in specialised cell types. In this context, topologically associating domains (TADs) were identified as fundamental and functional building blocks of chromatin organisation playing important roles in gene regulation and DNA replication. TADs are self-interacting regions with the size of a few hundreds of kilobases (kb). These domains are organised by the two structuring proteins Cohesin and CTCF, and are supposed to be formed by a mechanism called ‘DNA loop extrusion’. Most of our current insight is based on bulk measurements using genome-wide proximity-based ligation approaches.
In order to shed light on the spatial organisation of single TADs together with their structuring proteins in single cells this project set out to establish a novel imaging approach using super-resolution microscopy. Recent advances in this technology enable the visualisation of these fine scale genomic structures and their functional dynamics in 3D in single cells at nanometre resolution.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first step of the project, a labelling method was established suitable for the labelling of DNA and protein components of chromatin and their simultaneous visualisation. For this, generic TAD labelling by incorporation of fluorescent thymidine analogs was combined with nanobody staining of proteins. In the second step, the super-resolution imaging conditions were optimised using a custom-built Stochastic Optical Reconstruction Microscopy (STORM) set-up allowing for dual-colour 3D imaging. Next, dual-colour 3D data was acquired for single TADs together with CTCF and Cohesin, respectively. Endogenous and homozygous human cultured knock-in cell lines were employed allowing for a quantitative image analysis as all proteins of interest expressed by these cell lines carried a tag. To assess the structuring function of Cohesin imaging data of generic TADs after acute depletion of Cohesin as well as of the Cohesin unloader WAPL was acquired. In a final step, a computational image analysis pipeline was established to extract the following information from the data sets: (i) volume and shape of single TADs, (ii) protein concentration, (iii) how many proteins co-localise with single TADs, (iv) where do these proteins localise in respect to the TAD volume, and (v) how do TAD volume and shape change after acute depletion of Cohesin and WAPL.
The results of the project were disseminated at multiple international conferences and internal seminars of the host institute.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The methodological approaches which were developed in this project are beyond state-of-the-art in several aspects: (i) Conventionally, fluorescence imaging is performed of either DNA or protein but not of both components at the same time due to very different labelling protocols. The native nature of the chosen generic TAD labelling method is directly applicable with immunofluorescence-based protein staining techniques. (ii) Dual-colour 3D super-resolution imaging is performed with a quantitative analysis read-out. This is achieved by using homozygous knock-in cell lines and by employing a reference cell line to calibrate the STORM signals. (iii) Typically, chromosome conformation capture methods are used to study chromatin organisation giving only insight into the organisation of the DNA. The developed integrative imaging approach enables the visualisation of both components of chromatin, DNA and proteins, in the 3D space and thus offers insight into how structuring proteins organise DNA on the level of single TADs for the first time.
In the future, this approach can be used not only to study chromatin folding but also to study other biochemical processes taking place at the level of TAD organisation such as transcription and replication. This image-based quantitative analysis of single TAD organisation provides important complementary information to the field of chromatin organisation. In the future, our understanding of genome function has large potential for advancing medical treatments, especially in the field of personalised medicine.

Human cultured cell with generic TAD labelling and Cohesin staining.