Regulators of DNA folding could be targets for treating cancer

Most cells in the human body each contain about six feet of DNA. Yet the nucleus, where DNA is coiled, is no larger than a single speck of dust. Despite its density, DNA is not a tangled ball of yarn. It is organized into intricate layers of loops that fold and unfold in response to cues from the cell.

Scientists know that the three-dimensional shape of DNA is important. This long helical thread is peppered with genes that are translated into proteins to drive cellular activity. And the structure of the genome—those layers of loops—determines which genes are active at any given time.

How the three-dimensional structure of the genome is maintained, however, is less clear. Structural changes and abnormalities are associated with many diseases, such as cancer and developmental disorders. Identifying what controls genome structure could yield targets for treatment.

In a new study, published in Nature Methods, Yale scientists uncovered 21 regulators of genome structure, 19 of which are associated with diseases. They developed a combination of advanced methods that set a new standard for accuracy and efficiency.

“Through our approach, we were able to create the first screening platform for regulators of multi-scale 3D genome organization,” says Siyuan (Steven) Wang, Ph.D., an associate professor of genetics and cell biology at Yale School of Medicine and senior author of the study. Each of these regulators could be a new drug target for diseases or anti-aging therapies, he adds.

Searching for regulators of genome structure

In the cell nucleus, DNA loops around proteins and together they form a thick fiber called chromatin. Genes at the heart of a tight chromatin coil are hard to reach, and thus, essentially inactive.

Chromatin regulators help moderate gene expression, cell division, and DNA damage repair by unwinding and compacting DNA, changing which genes are exposed. “Genome structure controls many, if not all, genomic functions,” says Wang.

In 2016, Wang led a team that invented the first image-based 3D genomics technology, called chromatin tracing. This approach added more nuance than sequencing-based methods, which deduce structure based on interactions between segments of folded DNA. Chromatin tracing involves labeling DNA with fluorescent tags to track numerous adjacent segments as the strand folds and unfolds.

In this new study, the researchers combined chromatin tracing with pooled CRISPR screening, which inactivates single genes in many different cells, and invented a barcoding system called BARC-FISH that identifies cells based on the inactivated genes. Integrating these methods enabled the researchers to view structural changes resulting from each single gene knockout.

“Essentially, we’re capturing a snapshot of what the folded DNA looks like with each gene perturbation,” says Wang.

The approach can compare many gene candidates at once, unlike previous methods. They tested 137 genes in this study, but, according to Wang, the method is scalable. “We could do 10 times more genes,” he adds.

Disease-related gene regulates genome structure

Because older cells display altered 3D genome structure, the researchers tested genes that are involved in cellular aging. They also targeted genes that encode proteins found in the cell nucleus, which are more likely to regulate genome structure than proteins that exit it.

The study revealed 21 new regulatory genes, each of which codes for proteins that modify chromatin at different ranges. Some interact with nearby segments of DNA while others target distant sequences. Most of these genes have established roles in disease. The gene RB1, for example, can cause retinoblastoma—eye cancer—and was also identified as a chromatin regulator in this study.

One of the genes that jumped out to the researchers, CHD7, causes CHARGE syndrome—a condition that affects numerous organs and structures. According to previous studies, the CHD7 protein regulates gene expression during early human development by unwinding chromatin to make certain genes accessible.

In this study, the researchers observed that CHD7 can also do the opposite.

“We found at a very large length scale, CHD7 proteins actually compact chromatin,” says Wang, indicating that the same gene can modify chromatin in different ways, depending on its activity range. CHD7 targets faraway genes to keep chromatin compacted, which reduces net gene expression.

Depletion of CHD7 can increase total gene expression in the cell, perhaps by too much. “That may explain why CHD7 mutations lead to such complex phenotypes,” says Wang.

CHD7 is also closely related to a gene associated with autism spectrum disorder, which is unrelated to CHARGE syndrome but also characterized by complex and varied symptoms.

Now, Wang and colleagues are preparing to debut the first single-cell 3D genome atlas for cancer in mice, using their flagship chromatin tracing method. A preprint of this study posted on bioRxiv describes DNA structural changes in lung and pancreatic cancer cells tracked throughout disease progression. The genes driving these changes are promising therapeutic targets.

“What if we reverse the 3D genome changes that occur in cancer?” says Wang. “Does it lead to a better outcome? These findings open up a lot of opportunities.”