About 2 meters worth of genetic material must fit into the nucleus of each and every one of most of our trillions of cells, coiling and winding to form what looks like a tangled mess. But when genes get used, how they get used, and how often they get used is in fact completely intertwined with that very mess.
“The chromosomes are sort of all squished together in this really tight space in this kind of ‘spaghetti’ configuration,” says David G. Schatz, PhD, chair and Waldemar Von Zedtwitz Professor of Immunobiology at Yale School of Medicine (YSM). “This giant ball of spaghetti has to be organized in very specific ways, and those ways differ from one cell type to another.”
In 2019, Schatz was studying a process through which immune cells optimize the antibodies they produce. At that time, he met Siyuan (Steven) Wang, PhD, associate professor of genetics at YSM, who specializes in the development and application of image-based spatial mapping of genomic organization and cellular environments.
With Wang’s expertise in imaging and Schatz’s expertise in immunobiology, the two were able to map the genetic spaghetti of human tonsil, a tissue that has an important function in the immune system. They then used that map to illuminate the mechanisms that allow immune cells to update their antibodies. Their findings were published July 23rd in Science.
Mapping the 3D genome of the tonsil
The tonsils play an incredibly important role in the immune system. After maturing and differentiating in the bone marrow and thymus, immune cells can migrate to the tonsils where they lie in wait to defend against pathogens. To better understand how the three-dimensional configuration of genetic material—known as the 3D genome—controls and regulates these processes, Wang imaged tonsil cells using state-of-the art techniques developed in his lab.
While other 3D genome mapping techniques sequence individual cells, the technique developed by Wang’s lab, called Multiplexed Imaging of Nucleome Architectures (MINA), uses special microscopes (also developed in Wang’s lab) to image cells in intact ultra-thin slices of tonsil tissue. Rather than simply sequencing the genetic material of single cells out of context, Wang developed fluorescence techniques to trace the path of chromosomes and their products in the context of their local cellular neighborhoods.
“MINA allows one to image DNA, RNA, and proteins all together and that’s crucial for us to build a 3D genome atlas in this complex tissue microenvironment. Because of this, we’re able to distinguish all these different cell type-specific 3D genome changes,” Wang says. “This is actually the first single-cell 3D genome atlas of human tonsil by any technique, and this is also the first image-based 3D genome atlas of any human organ.”
This work, according to Schatz, will serve as an invaluable resource for the scientific community.
“This atlas is an extraordinary tool for future researchers to refer to. If they want to know anything about the structure of chromosomes or gene expression in all these thousands of cells and in an anatomically preserved manner, they can refer to the atlas,” he says.
Making loops and making mutations
With the atlas, Schatz and Wang then explored how changes in the 3D genome are implicated in one of the processes responsible for adaptive immunity.
Immune cells called B cells can produce antibodies that may loosely “match” to an antigen—a molecular structure from a pathogen that the immune system uses to recognize it. Some of those B cells can then undergo a process called somatic hypermutation, in which mutations are randomly introduced into the genes that encode antibodies. This can sometimes lead to a better “match” for that antigen and, therefore, better immunity against it.
As Schatz was studying this process, he discovered that some regions of the genome are more or less likely to undergo somatic hypermutation, which he nicknamed “hot” and “cold” regions. These regions were flanked by areas of self-contact, where the genome folded in on itself in a 3D loop-like structure held together by certain proteins. Thus, Schatz wanted to explore how these 3D loops might be associated with somatic hypermutation.
“The key hypothesis is that the 3D genome organization and the molecular process that sets up and maintains the 3D genome play fundamental roles in somatic hypermutation, and in this paper, we demonstrate that is the case,” Wang says.
When they systematically targeted and rapidly degraded one of the proteins holding the 3D loops together, Schatz and his team found that somatic hypermutation completely stopped. In other words, getting rid of the “loop makers” also got rid of somatic hypermutation. Schatz confirmed these results with Wang’s 3D genome atlas, finding that the genes in the hot regions had more internal loops and the genes in the cold regions had fewer.
But interestingly, while getting rid of the loop makers got rid of somatic hypermutation, it didn’t actually get rid of all the loops, especially some loops previously thought to be important.
“We thought one of the possible outcomes would be that as soon as somatic hypermutation went away, these looping interactions would go away, but that’s not what we found,” says Schatz. “The looping interactions went away more slowly than did the hypermutation, and so that’s not the full explanation. There’s something else happening. There’s still more to be discovered.”
Lymphomas and future questions
Another area of discovery lies in what happens when these processes innately go awry, as is the case in certain B cell lymphomas. While somatic hypermutation is most likely to happen in antibody genes, it can sometimes hit off target regions, introducing unnecessary and potentially cancer-causing mutations.
Schatz and Wang are still working to discover which factors may be disrupted when hypermutation goes haywire, but for Schatz, it all comes back to the genetic spaghetti and the molecular machines that assemble its 3D structure.
“If you don’t organize the spaghetti the right way and don’t have these big molecular machines that are moving the spaghetti around, the whole process that would allow us to make better antibodies just stops,” Schatz says. “That’s the question that my lab is working on in a myriad of other studies.”
As for Wang, imaging and mapping that spaghetti provides the fundamental groundwork for understanding it.
“This is a resource that people can check into regarding the 3D organization of how the DNA is woven together, how the spaghetti is organized,” he says. “Because the 3D genome is linked to so many different processes like gene expression regulation, DNA replication, and now somatic hypermutation, people studying these processes now have this tissue context where they can generate hypotheses that may be linked to their biological process of interest.”