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Perturb-tracing enables high-content screening of multi-scale 3D genome regulators
Yubao Cheng1, Mengwei Hu1, Bing Yang1
1Department of Genetics, Yale School of Medicine, Yale University, New Haven, CT, USA.
Nature Methods
|April 10, 2025
Summary
We developed Perturb-tracing, a new screening platform to discover regulators of three-dimensional (3D) genome organization. This method identified novel factors influencing chromatin folding and nuclear architecture, advancing our understanding of the 3D genome.
Area of Science:
- Genomics
- Cell Biology
- Epigenetics
Background:
- Three-dimensional (3D) genome organization is crucial for cellular functions and is altered in development, aging, and disease.
- Understanding the factors that regulate chromatin topology is essential, but current technologies lack efficiency in screening for new regulators.
- The multi-scale organization of chromatin influences gene expression and cellular processes.
Purpose of the Study:
- To develop a novel, image-based high-content screening platform for efficient identification of regulators of 3D genome organization.
- To investigate the impact of genetic perturbations on chromatin topology at various length scales.
- To uncover new factors influencing nuclear architecture and their relationship with chromatin compaction.
Main Methods:
- Development of Perturb-tracing, a platform combining pooled CRISPR screens, BARC-FISH (barcode readout), and chromatin tracing.
- Performance of a loss-of-function screen in human cells to analyze 3D chromatin folding conformations.
- Simultaneous visualization of 3D genome alterations and perturbation-paired barcode readout in single cells.
Main Results:
- Discovery of tens of novel regulators of chromatin folding across different scales, including domains, compartments, and chromosome territories.
- Identification of regulators affecting loop extrusion and A-B compartmentalization, as well as those acting through independent mechanisms.
- Uncovering new regulators of nuclear architecture and establishing a link between chromatin compaction and nuclear shape.
Conclusions:
- Perturb-tracing provides a scalable, high-content method for identifying regulators of chromatin and nuclear topology.
- The study expands the knowledge of factors governing the 3D genome, offering new insights into its complex organization.
- Findings pave the way for further research into the functional consequences of altered 3D genome organization in health and disease.
