SAN FRANCISCO—July 23, 2026—Congenital heart disease is the most common birth defect, affecting approximately 1 in 100 babies born each year. One of the many causes for this disorder is having only one functional copy of the gene TBX5, instead of two healthy copies, inherited from parents.
Yet, scientists have struggled to understand why one faulty copy of this gene, even in the presence of a second healthy copy, can so drastically derail heart development.
Now, researchers at Gladstone Institutes have shown that TBX5 plays a role in physically folding DNA into the architecture that heart cells need to function. In a new study published in Science, they found that even losing one copy dismantles this DNA organization, with ripple effects in how countless other genes are used by the cells.
The study reframes how scientists think about a longstanding puzzle in genetics: why losing one copy of a gene, a condition called haploinsufficiency, can cause serious developmental defects.
"TBX5 is just one example of a broader class of genes that cause birth defects when only one copy is lost," says Benoit Bruneau, PhD, director of the Gladstone Institute of Cardiovascular Disease and a senior author of the study. "What's exciting about our findings is they suggest many different birth defects might happen for the same reason: the cell's 3D instruction manual simply gets folded the wrong way."
"We developed and used different computational models to analyze results from thousands of individual cells," says Katie Pollard, PhD, director of the Gladstone Institute of Data Science and Biotechnology and the other senior author of the study. "That allowed us to finally see how losing this one protein causes the heart's DNA structure to break down on every level."
A Close Look at DNA Folding
Fitting DNA inside a cell is like taking a miles-long instruction manual and packing it into the head of a pin. Because every cell type precisely folds its manual into a unique three-dimensional shape, a heart cell gets different instructions than a brain cell—and, therefore, functions differently.
The DNA's 3D structure is organized into several layers, including large compartments (like separate binders of the manual), domains (like paragraphs), and chromatin loops (like folding a page so two distant 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 switches on many genes a heart cell needs to form and function properly. Bruneau's lab had 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 if the way DNA is folded determines how a heart cell functions, and what role TBX5 may play in the process.
To test this, the scientists combined many advanced techniques to observe how individual cells react differently to the loss of TBX5. They coaxed human stem cells—either healthy, lacking one copy of TBX5, or lacking both copies—to mature into heart muscle cells and then used high-resolution 3D mapping to look at 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 datasets.
"Using the custom computational approaches we developed, we were able to see for the first time how the loss of TBX5 triggers the total collapse of the heart's 3D DNA organization," says Shuzhen Kuang, PhD, a first author of the study and former bioinformatics fellow in Pollard's lab. "Surprisingly, we discovered this collapse happens at every level of genome organization—compartments, domains, and chromatin 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, 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 cohesin, exactly where to land on the DNA to build the chromatin loops that connect genes with their enhancers. When there isn't enough TBX5, the DNA misfolds and fails to form necessary loops, preventing vital heart-related genes from being turned on.
"What was striking was how the amount of TBX5 matters immensely," says Zoe Grant, PhD, a first author of the study and a postdoctoral researcher in Bruneau's lab. "The more TBX5 you removed, the worse the disruption across every level of genome organization we looked at."
In fact, the study proves that having only half the normal amount of TBX5 is enough to cause the DNA structure to misfold, which directly leads to heart defects.
In addition, when the researchers looked more closely at individual cells, they discovered that not all heart cells responded identically to TBX5 loss. Particular differences could be seen between two distinct types of heart cells—atrial and ventricular—and even between individual cells of the same type.
"This could help explain why people with the same mutation can have different heart defects," Grant says.
A New Window Into Developmental Disease
While the study sheds new light on why congenital heart disease occurs at a molecular level, the findings could apply to many other developmental disorders.
"We believe we've uncovered a new mechanism of disease," says Bruneau. "We showed that even a small decrease in a single protein can cause the DNA blueprint to fold incorrectly and lead to disease. So, many birth defects currently attributed to genetic mutations may actually be caused by the 3D misfolding of DNA."
The team plans to investigate when during early cardiac development TBX5 first begins shaping the genome's architecture, and whether the same principles hold for other proteins that cause birth defects.
About the Study
The paper, "Dose-dependent sensitivity of human three-dimensional chromatin to a heart disease–linked transcription factor," was published in the journal Science on July 23, 2026. The authors are Zoe L. Grant, Shuzhen Kuang, Shu Zhang, Abraham J. Horrillo, Zhe Chen, Kavitha S. Rao, Cemre Celen, Vasumathi Kameswaran, Carine Joubran, Deepak Srivastava, Katie Pollard, and Benoit Bruneau of Gladstone; Pik Ki Lau, Keyi Dong, Bing Yang, Weronika M. Bartosik, Nathan R. Zemke, and Bing Ren of UC San Diego; and Irfan S. Kathiriya of UC San Francisco.
The work was supported by the National Institutes of Health (NHLBI U01 HL157989, UM1HG011585, R01 HL155906), the California Institute for Regenerative Medicine, Additional Venture, Gladstone Institutes, the Roddenberry Foundation, the Younger Family Fund, UC San Francisco, the Saving tiny Hearts Society, and the National Science Foundation.
About Gladstone Institutes
Gladstone Institutes is an independent, nonprofit life science research organization that uses visionary science and technology to overcome disease. Established in 1979, it is located in the epicenter of biomedical and technological innovation, in the Mission Bay neighborhood of San Francisco. Gladstone has created a research model that disrupts how science is done, funds big ideas, and attracts the brightest minds.
