Revealing the architecture of replicated human chromosomes by sister-chromatid-sensitive fluorescence in situ hybridisation

Project Information

MicroChrom

Grant agreement ID: 101103258

DOI

10.3030/101103258

Project closed

EC signature date 17 April 2023

Start date 1 August 2024

End date 31 July 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 199 440,96

Coordinated by

INSTITUT FUER MOLEKULARE BIOTECHNOLOGIE GMBH
Austria

Periodic Reporting for period 1 - MicroChrom (Revealing the architecture of replicated human chromosomes by sister-chromatid-sensitive fluorescence in situ hybridisation)

Reporting period: 2024-08-01 to 2026-07-31

Summary of the context and overall objectives of the project

After DNA replication, each chromosome consists of two identical copies, called sister chromatids. Their organisation is important for correct chromosome segregation and for accurate DNA repair. In particular, sister chromatids provide an ideal template for repairing DNA double-strand breaks because they contain the same genetic information. However, because the two sister chromatids have essentially identical DNA sequences, it has been difficult to study their individual three-dimensional organisation in cells.

MicroChrom aimed to overcome this limitation by developing a sister-chromatid-sensitive fluorescence in situ hybridisation method (scsFISH), an imaging approach that uses fluorescent probes to label specific regions of DNA. The goal was to distinguish and trace the two sister chromatids separately in individual human cells. The project then used this method to study how sister chromatids are organised, how their organisation varies between individual cells, and how chromosome structure changes when DNA double-strand breaks are induced at defined genomic sites.

The project addresses a basic question in chromosome biology: how replicated chromosomes are organised in three dimensions and how this organisation contributes to accurate DNA repair. Understanding these processes is important because errors in DNA repair can lead to mutations and chromosome alterations that can contribute to cancer development.

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

A highly multiplexed scsFISH workflow was successfully established. The method combines automated microscopy, microfluidics and computational image analysis and allows the two sister chromatids to be distinguished from each other. It reaches a spatial precision of approximately 30 nm and supports around 90 sequential hybridisation rounds, allowing many positions along a chromosome to be analysed within the same experiment.

scsFISH was first applied to several genomic regions in unperturbed cells and after altering proteins involved in chromosome organisation. Individual cells showed strong variability in chromosome conformation, while average patterns were similar to those obtained with sequencing-based methods that measure chromosome contacts across large cell populations. The sister-specific nature of scsFISH also made it possible to directly compare the organisation of the two sister chromatids within the same cell.

A major focus of the project became the response of chromosome structure to DNA double-strand breaks. scsFISH was applied to six genomic regions in which DNA breaks were induced at defined sites. These experiments revealed both local and larger-scale changes in chromosome conformation around the break. Initial experiments were also carried out in cells in which proteins involved in chromosome cohesion or DNA repair were altered.

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 main advance of MicroChrom is the ability to image and trace the two sister chromatids separately in individual cells. Existing sequencing-based methods can measure how different DNA regions interact across many cells, but they do not directly show the three-dimensional arrangement of the two sister chromatids in a single cell. scsFISH therefore adds a complementary single-cell view with high spatial resolution and the ability to analyse many genomic positions in the same experiment.

The project demonstrated that this approach can be used both to investigate variation in chromosome organisation between individual cells and to follow structural changes associated with DNA damage. This opens new possibilities for studying how chromosome organisation and DNA repair are linked at the level of individual sister chromatids.

Further work will focus on completing the analysis of the generated datasets, improving the computational pipeline and combining the chromosome traces with fluorescent labelling of proteins involved in DNA repair. The scsFISH method and associated analysis tools are planned to be made publicly available together with the corresponding scientific publication.