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

Información del proyecto

MicroChrom

Identificador del acuerdo de subvención: 101103258

DOI

10.3030/101103258

Proyecto cerrado

Fecha de la firma de la CE 17 Abril 2023

Fecha de inicio 1 Agosto 2024

Fecha de finalización 31 Julio 2026

Financiado con arreglo a

• Marie Skłodowska-Curie Actions (MSCA)

Coste total

Sin datos

Aportación de la UE

€ 199 440,96

Coordinado por

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)

Período documentado: 2024-08-01 hasta 2026-07-31

Resumen del contexto y de los objetivos generales del proyecto

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.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

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.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

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.