New microscopy technique reveals unseen molecular processes of gene transcription

Gene transcription, the process of turning genes on or off, is essential for cellular diversity, individual variation, and even health and disease. However, understanding this molecular process has been challenging, as it is not directly visible. Recently, a breakthrough microscopy technique called chromatin expansion microscopy (ChromExM) has enabled scientists to observe previously unseen molecular processes within genetic material, providing valuable insights into gene activation and regulation.

Antonio Giraldez, a professor of Genetics at Yale School of Medicine, focuses on studying DNA codes in the genome and how cells interpret these codes during embryo development. Visualizing the genome is crucial to understanding these processes, but traditional microscopy methods have limitations. To overcome these constraints, Giraldez and his colleagues, including Ph.D. candidate Mark Pownall, collaborated with Joerg Bewersdorf, a professor of Cell Biology and Biomedical Engineering, to develop ChromExM.

In a paper published in Science, they demonstrate the success of ChromExM in significantly increasing the physical volume of zebrafish embryonic cell nuclei by 4,000-fold, leading to a drastic improvement in image resolution. This groundbreaking technique allowed researchers to observe how individual molecules shape gene expression during embryonic development and develop a new model of gene regulation.

Giraldez emphasizes that this research enables them to witness fundamental processes in the nucleus that underlie various biological phenomena, from embryo formation to cancer. By observing these processes directly, they can gain insights beyond mere speculation.

After fertilization, the genome in the fertilized egg is initially “silent.” The egg must undergo a transformation into a transient pluripotent stem cell, capable of developing into different cell types, to form a healthy embryo. Activating the genome in this cell is crucial for its ability to differentiate into various cells.

Giraldez and his team have long studied the process of genome activation, making significant progress in identifying key factors and understanding which genes are activated. However, they had never witnessed this process firsthand. Giraldez highlights the difference between describing how things might occur and actually observing how they unfold.

Unexpanded and expanded nuclei with the DNA fluorescently labelled and imaged with a 10x objective shows how ChromExM dramatically improves resolution. The middle panel shows the unexpanded nucleus at the same physical scale as the expanded nucleus, demonstrating the ~15x physical expansion factor achieved by ChromEx. Credit: M Giraldez Lab

ChromExM helps researchers visualize genome

Previously, Joerg Bewersdorf, co-senior author of the study, developed a technique called pan-ExM, which involved using an expandable gel to anchor cells and visualize cellular features with unprecedented resolution. By expanding the gel, the cells and their proteins were pulled apart while maintaining their spatial organization, resulting in a 64-fold increase in cell volume. In this new study, Giraldez and Bewersdorf collaborated to create ChromExM and applied it to embryos to observe gene regulation.

Using ChromExM, the researchers were able to visualize the molecular machinery of cells with incredible resolution. They compared the process to a toy egg that expands into a dinosaur when placed in water. Initially, the dinosaur’s features are not visible, but as it grows, detailed features emerge. In the case of ChromExM, the expansion occurred at a 4,000-fold scale, allowing the team to witness the fundamental processes of the genome in action.

Based on their observations, the researchers developed a new model of gene regulation, which they named “kiss-and-kick.” This model describes how enhancers, regulatory regions in the DNA, interact transiently with gene promoters to trigger gene expression. The burst of transcription separates the regulatory regions from the gene, pausing expression.

ChromExM has provided researchers with a level of detail that was previously unattainable. It offers new possibilities for examining hypotheses that were once untestable, such as studying how genes are switched on or off, their positioning relative to other genes in the nucleus, and how mutations affect gene positions.

Moreover, ChromExM is a cost-effective technique accessible to most laboratories, making it a valuable tool for research. The team aims to further improve the resolution of the technique to enable the identification of individual genes. This would allow scientists to understand the fundamental principles of gene regulation, mutations, and gene function, contributing to a better understanding of health and disease at the genetic level.

Source: Yale University

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