MFPL: Three-dimensional map shows genome structure of stem cells

New method combines imaging techniques with calculation models

To fit into the nucleus, the roughly two-meter long DNA needs to fold. This effort can only succeed if the DNA passes multiple packaging stages – from the double helix to whole chromosomes. From this folding process, various three-dimensional structures of the genome arise that interact with each other and thus influence cellular processes. For the first time, scientists from Martin Leeb’s group at the Max F. Perutz Laboratories (MFPL), a joint venture of the University of Vienna and the Medical University of Vienna, and the University of Cambridge succeeded in calculating the 3D structures of mammalian genomes in single cells. The results were published in the renowned journal Nature.

Each cell of the human body contains around six billion base pairs – the building blocks that make up the DNA’s double helix. A cell’s nucleus on the other hand ranges from 0.005-0.016 mm in size. To find enough space in the nucleus, the two-meter long DNA molecule thus needs to wrap around protein complexes called “histones”. After having passed multiple complex folding steps in which it forms various 3D structures, the DNA arrives at the chromosome stage.

So far, researchers described the nuclear architecture with microscopic and modern biochemical methods. They separated the genome into two compartments: A (relatively active) and B (relatively inactive). On an even smaller scale they identified “genome-neighborhoods” and DNA-folds. “The structural elements of the genome inside a cell’s nucleus resemble a three-dimensional map”, explains stem cell biologist Martin Leeb. This way, researchers can investigate regulatory DNA-segments and their interactions and draw conclusions about a gene’s activity, which is sometimes influenced by its localization in the nucleus.

In their recent publication, Martin Leeb, group leader at the MFPL, together with colleagues at the University of Cambridge now report the calculation of complete, three-dimensional genome structures of single haploid murine stem cells. Haploid stem cells contain each chromosome only once and not – as is the case for most mammalian cells – twice. Using this technique, the researchers could ensure that the investigated interactions between segments of a chromosome indeed took place on the same chromosome and are not due to sister chromosomes forming structures with each other. “The intention behind the development of haploid stem cells was to establish a simplified genetic model system, mostly for genetic screens. We are delighted, that this simple system also played a crucial role in the clarification of genome structure”, says Martin Leeb, co-author of the study and responsible for the development of the haploid stem cell-technology.

In the first step, the researchers took snapshots of single stem cells using modern imaging techniques. Next, the team of the University of Cambridge determined the same cell’s genome structure and overlaid the image of the cell with its genome structure model – a revolutionary method. “The possibility to overlay calculated genome structures and high-resolution microscopic images does not only allow us to validate the genome models, it also enables us to analyze cellular status and genome structure at the same time. By working at single cell resolution, we could determine, whether there is a correlation between cellular processes and genome structure”, explains Martin Leeb.

The identity of a stem cell is determined by a certain set of genes called pluripotency genes. Over the course of its life, a stem cell receives signals from outside that induce its differentiation into a specialized cell type like skin cells or neurons. This very complex differentiation process is regulated by changes in the network of pluripotency genes and massive changes in the three-dimensional structure of the genome. The structures of the stem cell’s 3D-map are thus literally rebuilt to prepare the cell for its future fate.

“We now have the possibility to investigate changes in the genome structure in dynamic systems such as stem cell differentiation on a single cell level”, says Martin Leeb. This might contribute to a better understanding of the molecular processes that govern the coordinated and exact regulation of stem cell differentiation.  

Publication in Nature:
Tim J. Stevens, David Lando, Srinjan Basu, Liam P. Atkinson, Yang Cao, Steven F. Lee, Martin Leeb, Kai J. Wohlfahrt, Wayne Boucher, Aoife O’Shaughnessy-Kirwan, Julie Cramard, Andre J. Faure, Meryem Ralser, Enrique Blanco, Lluis Morey, Miriam Sansó, Matthieu G. S. Palayret, Ben Lehner, Luciano Di Croce, Anton Wutz, Brian Hendrich, Dave Klenerman and Ernest D. Laue. 3D structure of individual mammalian genomes studied by single cell Hi-C. Nature, DOI: 10.1038/nature21429

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