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The ability to turn genes on or off is fundamental to the diversity we see in cells, in individuals, and even in health and disease. This process, called gene transcription, involves converting the information stored in our DNA into a “carbon copy” called RNA.
Until recently, scientists have relied on vague drawings and indirect experiments to understand this process, as it occurs at the molecular level and cannot be seen directly. However, advanced microscopy techniques now allow researchers to observe previously unseen molecular processes within genetic material, providing valuable insight into how genes are activated and regulated.
Antonio Giraldez, Ph.D., Fergus F. Wallace Professor of Genetics at Yale School of Medicine, studies the DNA codes in the genome and how cells interpret these codes to create an embryo. An important aspect of understanding these processes involves our ability to visualize the genome.
Unfortunately, traditional microscopic methods have limitations. To overcome these limitations, Giraldez and his colleagues, including the first author of the study, Ph.D. Candidate Mark Pownall, in collaboration with Joerg Bewersdorf, Ph.D., Harvey and Kate Cushing Professor of Cell Biology and Professor of Bioengineering and Physics to develop a new technique called chromatin expansion microscopy (ChromExM).
In the document printed online in Science They show their success in increasing the physical volume of the nuclei of cells. Embryonic zebrafish 4,000-fold to improve image resolution significantly. The technique allowed researchers to see for the first time how individual molecules shape gene expression in cells during embryonic development and to come up with new ways of controlling genes.
“Our research helps us see the fundamental processes in the nucleus that underlie everything in life, from embryo formation to cancer,” Giraldez said. “We can see processes that we could only imagine before.”
Giraldez said that after the sperm fertilizes the egg, the genome is initially “silent,” Giraldez said. A fertilized egg must temporarily transform into pluripotent stem cells, or cells that can give rise to many types of cells, to develop into a healthy embryo. Programming this cell’s ability to make other cells requires a jump start in the genome.
For years, Giraldez and his team have studied how the genome becomes activated. They have made great strides, from identifying key players to learning which genes are activated. “But we’ve never seen genome activation for ourselves,” Giraldez said. “There is a difference between describing what might happen and actually witnessing what happened.”
ChromExM allows researchers to visualize the genome
In his previous work, Bewersdorf, who is the senior author of the study, developed a technique called pan-ExM which involves attaching cells to a gel that can be expanded to allow visualization of the characteristics of cells with unprecedented resolution. As the gel expands, it pulls the cells and internal proteins apart while maintaining their spatial organization, until the cells are 64 times larger in volume. Then, with a second gel, the team repeated the process to increase the amount of cells 4,000-fold.
For this new study, the Giraldez and Bewersdorf labs collaborated to create ChromExM and use it with embryos to see how genes are regulated. Now, each cell is about the size of an embryo. “We used a very traditional tool, the confocal microscope, which allowed us to get this amazing resolution of the molecular machinery of the cell when combined with ChromExM,” Giraldez said. “Even the most powerful microscopes cannot see this image.”
The process, he explained, is like a toy egg that expands into a dinosaur when placed in water. When the egg was first dropped into the glass, the dinosaur’s features were not yet visible. But as the toy grows, it transforms from an immortal to a detailed creature. “That dinosaur probably grew two or three times bigger,” Giraldez said. “Now imagine that growing to a 4,000-fold scale.”
Through ChromExM, the team can see the fundamental processes of the genome in action for the first time. This allowed them to develop a new model of gene regulation, which they named “kiss-and-kick” to describe the way regulatory regions in DNA called enhancers interact with the start of a gene (promoters) to activate. Gene expression, and the burst method of transcription then breaks the gene’s regulatory region (or kicks the enhancer) to stop expression.
“It’s like the pixelated black and white cell phone screen of the eighties to the large, high-definition color screen of today,” Giraldez said. “Our technique allowed us to see details that were not possible before.”
With this new method, the team hopes to examine a hypothesis that until recently was untestable. For example, in addition to looking at basic molecular processes, they also hope to find out how different genes are turned on or off, the positioning of genes in the nucleus, and how mutations affect gene positions.
Furthermore, while other microscopy techniques may be prohibitively expensive, ChromExM is accessible to most laboratories. “Our work will democratize the way to see how molecular processes occur inside the nucleus, which will open new areas of research,” said Giraldez.
The team now hopes to further refine its technical solutions. While researchers can now visualize molecules interacting with the genome, they are still unable to identify individual genes. “Imagine you’re in space, and you take a picture of New York City. Before you could see the island, but now you can see the people in the city,” Giraldez explained.
“But we still don’t know who these people are. If you think those people are the genes we want to see, next we need a camera that will help us focus on each individual” This detail will help scientists understand. The basic principles of how genes turn on and off, break, or repair, and how mutations affect their function – all fundamental steps to understanding how genes work in health and disease.
Mark E. Pownall et al, Chromatin magnification microscopy reveals nanoscale organization of transcription and chromatin, Science (2023). DOI: 10.1126/science.ade5308
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