Mapping ‘dark’ regions of the genome illuminates how cells respond to their environment

Researchers at Duke University used CRISPR technologies to discover previously unannotated stretches of DNA in the ‘dark genome’ that are responsible for controlling how cells sense and respond to the mechanical properties of their local environment.

Understanding how these DNA sequences affect cellular identity and function could give researchers new therapeutic targets for illnesses that involve changes to mechanical properties of tissues, including fibrosis, cancer and stroke, as well as long-term issues such as neurodegeneration and even aging.

This work appears online on September 25 in the journal Science.

The local environment around a cell plays a critical role in determining how that cell functions and the characteristics it develops within different tissues. Advances in epigenetic profiling and CRISPR-based screening tools have enabled researchers to explore how chemical stimuli like hormones, signaling proteins like cytokines, and even pharmacologic medications can shape gene expression. But how mechanical structure, such as tissue stiffness or applied external forces, affects cellular function is not fully understood.

“Mechanical stimuli from a cell’s microenvironment are also potent regulators of many fundamental cell processes, including growth, death, differentiation and migration,” said Charlie Gersbach, the John W. Strohbehm Distinguished Professor of Biomedical Engineering and the Director of the Duke Center for Advanced Genomic Technologies (CAGT). “We know these physical stimuli play key roles in tissue development, regeneration, aging, and disease pathology such as fibrosis and tumor formation, but the precise mechanism through which they act has been difficult to understand.”

Gersbach and his laboratory have spent over a decade developing techniques to use CRISPR to modulate epigenetic activity, which controls how much of our genes are produced. Originally discovered as a bacterial defense system against viruses, CRISPR targets very specific DNA sequences. While the original system carries a protein called Cas9 that slices the targeted viral genomes, the DNA-targeting part of the system can operate independently. One of the methods Gersbach’s team has pioneered involves using a version of CRISPR-Cas9 to explore and modulate genes without cutting them. Instead, it makes changes to the structures that package and store DNA, affecting the activity level of the accompanying genes.

While this CRISPR epigenome engineering technology is useful for modulating genes of known function, through collaboration with genomics experts in the Duke School of Medicine, Gersbach came to appreciate the power of these tools for studying the vast majority of the human genome that we still don’t understand. In particular, Greg Crawford, the Wilburt C. Davison Distinguished Professor of Pediatrics, has spent his career mapping changes to genome structure in many cell types and tissues as well as in response to diverse environments. He has mapped millions of regions of our “dark genome” that become more or less accessible across these settings, but it remains unclear what function they are playing in human cells.

“Only 1-2% of our genome encodes for genes. The other 98% of the genome clearly plays an important role in shaping cell identity, response to the environment, and susceptibility to disease, but until recently we didn’t have the tools to probe the function of this ‘dark’ part of our genome,” said Crawford. “A significant function of the dark genome is to act as ‘enhancers’ of genes – dialing them up and down in different situations. But it’s largely unknown where the enhancers are in our DNA sequence, which genes they are controlling, and how that relates to cell-environment interactions”.

Gersbach and Crawford have been working together closely within the Duke CAGT for the last decade to tackle this challenge. But to understand how cells sense their mechanical environment, their team partnered with Brent Hoffman, an associate professor of biomedical engineering and an expert in mechanobiology. Led by postdoctoral fellow Brian Cosgrove, they made hydrogels that mimic tissues of different stiffness and cultured cells on these gels. The researchers then used sequencing tools to quantify RNA levels and map regions of open chromatin, or accessible DNA, in order to determine changes to gene expression and genome structure in each sample.

“In just 20 hours on the different gels, we observed changes in the levels of thousands of genes and the structure of almost fifty thousand regions of the genome,” shared Hoffman. “This underscores the profound effect of the mechanical microenvironment on cell biology and clarifies how changes in tissue structure can play a significant role in diseases like fibrosis and cancer.”

It was more challenging to determine which changes in the ‘dark’ genome affect cell function by acting as enhancers of specific genes. For this, the team used CRISPR to silence the activity each region of DNA and determined the resulting effects on cell growth and migration. The regions that showed strong functional effects were further profiled by measuring the levels of every gene in response to blocking these enhancers with CRISPR.  The regions that changed structure in response to local mechanical properties, altered cell growth and/or migration, and controlled the levels of specific genes were dubbed ‘mechanoenhancers’ to reflect their role in regulating cell response to environment.

To better understand the function of mechanoenhancers and their role in disease, they teamed up with Yarui Diao and Purushothama Tata, both associate professors of cell biology and members of the Duke CAGT. Diao, an expert in methods for analyzing the physical interactions of enhancers and genes, showed that mechanical environment in fact alters the contact of mechanoenhancers to their gene targets. Tata, who studies lung fibrosis and regeneration, provided samples from lung biopsies that showed activity of mechanoenhancers controlling disease-related genes in idiopathic pulmonary fibrosis (IPF).

“Mapping these mechanoenhancers can improve our mechanistic understanding of diseases that involve changes to tissue mechanical properties, like fibrosis and cancer, and possibly lead to new drug targets or methods for engineering how cells sense pathologic mechanical environments,” said Cosgrove.

The work was primarily supported by the National Institutes of Health’s Consortium for the Impact of Genetic Variation on Function (IGVF), which aims to understand how genomic variation affects genome function. Ongoing and future work by the Duke team includes studying how mechanoenhancers vary across cell types, disease conditions, and age, including how genetic variation within these DNA sequences confers resilience to various environmental stresses. They are also interested in pursuing these regions of the dark genome as direct targets of genetic medicines to treat otherwise intractable conditions.

“The project is a great example of highly effective and unique collaboration across engineering, science, and medicine at Duke with many contributors throughout Duke CAGT,” said Gersbach. “This highlights the type of products that our center is designed to produce by marrying technology with biomedical science, as well as engineering approaches with patient-derived samples.”

This work was supported by the National Institutes of Health (UM1HG009428, UM1HG012053, U01AI146356, RM1HG011123, R01MH125236, R35GM156302, R01HL146557, R01HL160939, R01HL153375), the National Human Genome Research Institute, the NIH Somatic Cell Genome Editing Consortium, the National Institutes of Health PROSPER Grant (T32HL160494), The National Science Foundation Office of Emerging Frontiers and Multidisciplinary Activities (EFMA-1830957), the Allen Distinguished Investigator Award from the Paul G. Allen Frontiers Group (CAG), Open Philanthropy (CAG), the National Science Foundation GRFP (NSF-GRFP DGE – 2139754), and the Regeneration Next Postdoctoral Fellowship.

CITATION: “Mechanosensitive genomic enhancers potentiate the cellular response to matrix stiffness,” Brian Cosgrove, Lexi Bounds, Carson Taylor, Alan Su, Anthony Rizzo, Alejandro Barrera, Tongyu Sun, Alexias Safi, Lingyun Song, Thomas Whitlow, Aleksandra Tata, Nahid Iglesias, Yarui Diao, Purushothama Rao Tata, Brenton Hoffman, Gregory Crawford, and Charles Gersbach. September 25, 2025. DOI: 10.1126/science.adl1988