Congenital heart disease is the most common birth defect, affecting approximately 1 in 100 babies worldwide. One possible cause involves changes to TBX5, a gene essential for normal heart development. In some children, one of the two inherited copies of TBX5 does not function properly, leaving only a single working copy.
For years, scientists have investigated why losing just one copy of TBX5 can significantly disrupt heart development, even when the remaining copy is functional.
Researchers at the Gladstone Institutes have now found that TBX5 does more than regulate gene activity. The protein also helps organize DNA into the three-dimensional structure required for heart cells to function correctly. In a new study published in Science, the researchers report that losing even one copy of TBX5 can disrupt the genome’s organization and alter the activity of hundreds of genes in heart cells.
The findings offer new insight into haploinsufficiency, a genetic condition in which losing one copy of a gene causes serious developmental problems.
“TBX5 is just one example of a broader group of genes that cause birth defects when just one copy is lost,” says Benoît Bruneau, PhD, director of the Gladstone Institute of Cardiovascular Disease and senior author of the study. “What’s interesting about our findings is that they suggest that many different birth defects can occur for the same reason: the cell’s 3D instruction manual is simply folded the wrong way.”
“We developed a variety of computational models and used them to analyze results from thousands of individual cells,” said Dr. Katie Pollard, director of the Gladstone Institute for Data Science and Biotechnology and another senior author of the study. “This allows us to finally understand how the loss of this one protein disrupts the heart’s DNA structure at all levels.”
How DNA folding helps heart cells function
Fitting DNA inside a cell is an extraordinary biological feat. Although the DNA molecule would stretch for miles if fully extended, it must be tightly packed inside a microscopic nucleus. However, DNA is not folded randomly. Each cell type organizes its genetic material in a unique three-dimensional structure, allowing heart cells to access different genetic instructions than brain or muscle cells.
This 3D genome structure is arranged in several layers, including large compartments, domains, and chromatin loops. These loops bring distant gene switches, known as enhancers, into physical contact with specific genes. These connections help activate the genetic instructions cells need to develop and function.
Scientists already knew that TBX5 is a key regulator of heart development. The protein activates many genes involved in the formation and function of heart cells. Previous research from Bruneau’s lab showed that losing one copy of TBX5 changes the activity of hundreds of heart-specific genes. However, the biological process behind those changes remained unclear.
The research team therefore investigated whether DNA folding influences heart cell development and whether TBX5 helps control the genome’s three-dimensional organization.
Mapping the heart’s 3D genome
To study the effects of different TBX5 levels, the researchers used several advanced techniques to analyze individual cells. They guided human stem cells to develop into heart muscle cells. Some cells had two functioning copies of TBX5, some had only one copy, and others lacked both copies.
The scientists then used high-resolution 3D genome mapping to examine DNA loops and other structural features in detail.
Because the experiments produced millions of data points from thousands of individual cells, the researchers used specialized computational models to analyze the data.
“Using the custom computational approach we developed, we were able to see for the first time how loss of TBX5 causes a complete disruption of the heart’s 3D DNA organization,” said Shuzhen Kuang, PhD, first author of the study and former bioinformatics fellow in the Pollard lab. “Surprisingly, we discovered that this disruption occurs at every level of genome organization: compartments, domains, and chromatin loops.”
TBX5 acts as an architect of heart DNA
As healthy stem cells developed into heart muscle cells, the researchers observed major changes in genome organization. During maturation, large regions of DNA switched between active and inactive states.
TBX5 emerged as a central organizer of these structural changes.
The researchers discovered that TBX5 acts like a GPS for a molecular motor called cohesin. By guiding cohesin to specific locations on DNA, TBX5 helps create chromatin loops that connect genes with their enhancers.
When TBX5 levels are too low, these loops do not form correctly. As a result, DNA can fold incorrectly, preventing important heart-development genes from turning on at the appropriate time.
“What was striking was how extremely important the amount of TBX5 was,” says Zoe Grant, PhD, lead author of the study and a postdoctoral fellow in Bruneau’s lab. “The more you delete TBX5, the worse the disruption at every level of genomic organization we looked at.”
The results showed that reducing TBX5 to half its normal level was enough to disrupt DNA folding and contribute directly to congenital heart defects.
The researchers also found that heart cells did not all respond to TBX5 loss in the same way. Significant differences appeared between the two major types of cardiac cells—atrial cells and ventricular cells. Differences were also observed among individual cells of the same type.
“This could help explain why people with the same mutation suffer from different heart defects,” Grant says.
Implications for developmental disorders
Although the study provides important insight into congenital heart disease, the researchers believe similar mechanisms may contribute to other developmental disorders.
“We believe we have uncovered a new mechanism of disease,” Bruneau says. “We showed that even a small reduction in a single protein can cause the DNA blueprint to misfold and cause disease. Therefore, many birth defects that are currently thought to be caused by genetic mutations may actually be caused by three-dimensional misfolding of DNA.”
The findings suggest that genetic mutations can cause disease in more than one way. In addition to changing individual genetic instructions, mutations may disrupt the physical organization of the genome itself.
Next, the research team plans to determine when TBX5 begins organizing the genome during early heart development. The researchers will also investigate whether other proteins associated with birth defects organize DNA through similar mechanisms.
About the research
The study, titled “Dose-dependent sensitivity of human three-dimensional chromatin to heart disease-related transcription factors,” was published in Science. The authors include Zoe L. Grant, Shuzhen Kuang, Shu Zhang, Abraham J. Horrillo, Zhe Chen, Kavitha S. Rao, Cemre Celen, Vasumathi Kamiswaran, 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 the University of California, San Diego; and Irfan S. Katiriya of the University of California, San Francisco.
This research was supported by the National Institutes of Health (NHLBI U01 HL157989, UM1HG011585, and R01 HL155906), the California Institute for Regenerative Medicine, Additional Ventures, the Gladstone Institutes, the Roddenberry Foundation, the Young Family Foundation, the University of California, San Francisco, the Saving Tiny Hearts Society, and the National Science Foundation.
Source: www.sciencedaily.com