Epi-PoreC: a nanopore-based method for simultaneous profiling of chromatin conformation, DNA methylation, and chromatin accessibility

The genome is not merely a static library of instructions stored inside a cell; it is a dynamic, three-dimensional structure that folds and twists to control how genes are read. To understand how life works, scientists must look at this structure from three different angles at once. First, they need to see how distant parts of the DNA strand touch and interact, forming loops and bridges that bring specific genes under the control of their regulators. Second, they must map the chemical tags attached to the DNA, which act like switches that turn genes on or off without changing the underlying code. Third, they need to identify which parts of the DNA are open and accessible to the cellular machinery, and which parts are tightly packed and hidden away. For years, researchers have had to study these three features separately, using different experiments on different samples. This approach leaves a gap: because the measurements are taken apart, it is impossible to know exactly how the folding, the chemical tags, and the accessibility of a single piece of DNA relate to one another at the same moment.

A team of researchers at Indiana University has now bridged this gap with a new method called Epi-PoreC. By combining three established techniques into a single workflow, they created a way to capture the shape, the chemical state, and the accessibility of the genome all on the same individual DNA molecule. The method relies on a technology known as nanopore sequencing, which reads DNA by pulling long strands through a tiny protein pore and measuring how the electrical current changes as the molecule passes through. This technology is powerful enough to read very long stretches of DNA, allowing scientists to see how far apart two touching points are and to detect chemical modifications along the way. The researchers tested their new approach on the nuclei of Arabidopsis thaliana, a small flowering plant widely used in biological research. They took plant tissue, isolated the cell nuclei, and then subjected them to a series of chemical steps designed to freeze the DNA in its natural 3D shape, label the open regions, and prepare the strands for reading.

The process begins by crosslinking the DNA to lock its folded structure in place. The researchers then permeabilize the nuclei to allow an enzyme to enter and mark the accessible regions of the genome with a specific chemical tag. This step, borrowed from a technique called Fiber-seq, identifies which parts of the DNA are open and available for use. After this labeling, the DNA is cut into manageable pieces and the ends that were touching in the 3D space are glued back together, creating a physical record of the genome's architecture. This step, known as chromosome conformation capture, ensures that any two DNA segments that were close together in the nucleus will remain connected in the final sample. The resulting DNA strands are then fed into the nanopore sequencer. As the long strands pass through the pores, the machine reads the sequence of letters and simultaneously detects the chemical tags and the junctions where DNA segments were glued together. Because the entire process happens on a single molecule, the researchers can see exactly which chemical tags and which structural connections belong to the same piece of DNA.

The results of this integration were striking. When the team compared the maps generated by their new method against maps created by running the three techniques separately, the data matched with remarkable precision. The patterns of how the chromosomes folded and touched each other were nearly identical to those seen in standard experiments, showing that the extra steps required to label the DNA did not distort the genome's natural shape. Similarly, the chemical maps of DNA accessibility and the locations of natural chemical modifications were indistinguishable from those produced by the standalone methods. The researchers found that the new approach could detect complex interactions, such as those occurring in the dense, repetitive regions near the center of chromosomes, which are often difficult to study with other technologies. While the new method produced slightly shorter DNA reads than the standard version of the structural mapping technique, the lengths were still sufficient to reveal the intricate folding patterns of the genome.

This work demonstrates that it is possible to view the genome as a unified system rather than a collection of separate parts. By capturing the shape, the chemical state, and the accessibility of the DNA all at once, Epi-PoreC provides a more complete picture of how the genome functions. The method is particularly valuable for studying complex or repetitive regions of the genome, where long reads are essential to distinguish one section from another. The researchers noted that while the approach is highly effective, the additional enzymatic steps do introduce a slight reduction in the length of the DNA strands that can be read, which could limit the resolution in the most challenging areas. Nevertheless, the ability to link the physical architecture of the genome directly to its chemical landscape on a single molecule opens new doors for understanding how genes are regulated. This integrated view allows scientists to ask questions that were previously unanswerable, such as how the folding of a specific DNA loop influences the chemical tags on that same loop, or how the accessibility of a region changes when it interacts with a distant part of the chromosome. As the technology continues to improve, this kind of single-molecule analysis promises to deepen our understanding of the complex machinery that drives life.