Congenital heart disease is the most common birth defect, affecting about 1 in 100 babies born each year. One of the many causes of this disorder is having only one functional copy of the TBX5 gene, instead of two healthy copies, inherited from the parents.
However, scientists have struggled to understand why a defective copy of this gene, even in the presence of a second healthy copy, can so drastically derail heart development.
Now, researchers at the Gladstone Institutes have shown that TBX5 plays a role in the natural folding of DNA into the architecture that heart cells need to function. In a new study published in Sciencethey found that the loss of even one copy disrupts this DNA organization, with ripple effects on how countless other genes are used by cells.
The study reframes how scientists think about a long-standing puzzle in genetics: why the loss of one copy of a gene, a condition called haploinsufficiency, can cause severe developmental defects.
TBX5 is just one example of a broader class of genes that cause birth defects when only one copy is lost. What’s exciting about our findings is that they suggest that many different birth defects can occur for the same reason: the cell’s 3D instruction manual simply folds the wrong way.”
Benoit Bruneau, PhD, director of the Gladstone Institute of Cardiovascular Disease and senior author of the study
“We developed and used different computational models to analyze results from thousands of individual cells,” says Katie Pollard, PhD, director of the Gladstone Institute for Data Science and Biotechnology and the study’s other senior author. “This allowed us to finally see how the loss of this protein causes the heart’s DNA structure to break down at every level.”
A close look at DNA folding
Placing DNA inside a cell is like taking a mile-long instruction manual and packing it onto the head of a pin. Because each type of cell precisely folds its manual into a unique three-dimensional shape, a heart cell receives different instructions from a brain cell—and therefore functions differently.
The three-dimensional structure of DNA is organized into many layers, including large compartments (like separate textbook binders), domains (like paragraphs), and chromatin loops (like the folding of a page so that two long sentences touch). In cells, these loops bring distant genetic switches known as enhancers into physical contact with genes, allowing the cell to turn on the specific instructions it needs.
Researchers already knew that TBX5 was a master regulator of heart development—a protein that activates many genes that a heart cell needs to form and function properly. Bruneau’s lab has previously shown that losing one copy of TBX5 affects the levels of hundreds of other heart-specific genes. But they didn’t know exactly how.
In the new work, the team investigated whether the way DNA is folded determines how a heart cell works and what role TBX5 might play in the process.
To test this, the scientists combined several advanced techniques to observe how individual cells respond differently to the loss of TBX5. They took human stem cells—either healthy, without one copy of TBX5, or without both copies—to mature into heart muscle cells, and then used high-resolution 3D mapping to examine the DNA loops in unprecedented detail.
Because the study involved millions of data points from thousands of individual cells, they turned to computational models to make sense of the resulting data sets.
“Using the custom computational approaches we developed, we were able to see for the first time how loss of TBX5 triggers the total collapse of the 3D DNA organization of the heart,” says Shuzhen Kuang, PhD, first author of the study and a former bioinformatics fellow in Pollard’s lab. Surprisingly, we discovered that this collapse occurs at every level of genome organization—chromatin compartments, domains, and loops.”
A master architect
The data revealed that as healthy stem cells matured into heart muscle cells, the genome underwent a sweeping reorganization, with large stretches of DNA shifting from inactive to active or vice versa. And, as the team found, TBX5 is the main architect behind these structural changes.
The scientists discovered that TBX5 acts as a GPS, telling a molecular motor known as synesin exactly where to land on the DNA to create the chromatin loops that connect genes to their enhancers. When there isn’t enough TBX5, the DNA folds and fails to form the necessary loops, preventing the activation of vital heart-related genes.
“What was striking was the sheer magnitude of the amount of TBX5,” says Zoe Grant, PhD, first author of the study and a postdoctoral researcher in Bruneau’s lab. “The more TBX5 you remove, the worse the disruption at every level of genome organization we looked at.”
In fact, the study shows that having just half the normal amount of TBX5 is enough to cause the DNA structure to misfold, which directly leads to heart defects.
Furthermore, when the researchers looked more closely at individual cells, they discovered that not all heart cells responded identically to the loss of TBX5. Distinct differences could be seen between two different types of heart cells – atrial and ventricular – and even between individual cells of the same type.
“This could explain why people with the same mutation can have different heart defects,” says Grant.
A New Window on Developmental Disease
While the study sheds new light on why congenital heart disease occurs at the molecular level, the findings could apply to many other developmental disorders.
“We think we’ve discovered a new disease mechanism,” says Bruneau. “We showed that even a small decrease in one protein can cause the DNA blueprint to misfold and lead to disease. Thus, many genetic defects currently attributed to genetic mutations may actually be caused by three-dimensional DNA misfolding.”
The team plans to investigate when during early heart development TBX5 first begins to shape the architecture of the genome and whether the same principles apply to other proteins that cause birth defects.
