Northwestern Medicine scientists have discovered that little-studied DNA structures play a central role in organizing the human genome and controlling gene activity, according to a new study published in the Proceedings of the National Academy of Sciences.
The study revealed that G‑quadruplexes (G4s) — four-stranded DNA structures — directly interact with a key genome-organizing protein called CTCF, helping shape how DNA folds inside the cell and how genes are turned on or off.
“This is really a new layer of regulation in the genome,” said the study’s senior author Vipul Shukla, PhD, assistant professor of Cell and Developmental Biology and of Medicine in the Division of Hematology and Oncology. “Normally, when we think about DNA, we think about a sequence of nucleotides. G‑quadruplexes are a layer of regulation in which a linear sequence of the DNA transforms to make alternative structures which are presented as conformational folds.”
While DNA is typically depicted as a double helix, it can also form alternative shapes. G‑quadruplexes arise in regions where DNA folds into stable, stacked structures. Scientists have known about these shapes for over a century, Shukla said, but only recently have they been recognized as important features in living cells.
For Daniela Samaniego-Castruita, PhD, a postdoctoral scholar in the Shukla laboratory and the study’s first author, these structures represent a unique type of epigenetic regulation.
“They’re not necessarily encoded in the genome,” Samaniego-Castruita said. “These secondary structures are located at important regulatory regions in the genome. However, we know very little about the regulation and functions of these G‑quadruplex structures.”
To better understand these structures and how they impact genetic regulation, the team set out to identify which proteins interact with G4s.
Using a large-scale proteomics screen, investigators identified dozens of proteins that bind to G‑quadruplexes. Many of these proteins are involved in fundamental cellular processes, including RNA splicing, gene regulation and chromatin remodeling.
“I think one of the most important findings for this paper is that we identified CTCF as a G‑quadruplex binding protein,” Samaniego-Castruita said.
CTCF is widely known as a master regulator of genome architecture, helping fold DNA into loops and domains and bringing distant regions into contact. Inside the nucleus of a cell, DNA is folded into complex 3D structures. These folds create loops that connect different parts of the genome, enabling precise control of gene expression.
The new study shows that G‑quadruplexes play a key role in this process. About 35 percent of chromatin loops were found to be associated with G4 structures, and roughly a quarter of CTCF-mediated loops depend on G4 interactions.
Shukla said the findings challenge traditional models of genome organization.
“In the loop extrusion model, there is a molecular motor that creates these transient loops,” he said. “You keep creating these loops until this motor gets hit by a roadblock. And more often than not, people describe the roadblock to be CTCF. CTCF bound to a G‑quadruplex is an even stronger roadblock. Once you form loops by G‑quadruplex and CTCF, these loops tend to be much more stable.”
The implications go beyond genome architecture. Because chromatin loops regulate gene activity, the CTCF–G4 interaction directly affects gene expression.
The investigators found hundreds of genes that exhibited altered expression when CTCF was removed and then restored. A subset of these genes appeared to be regulated specifically through G‑quadruplex-dependent looping.
“These loops are an important mode of regulation that you need to turn on or turn off gene expression,” Shukla said.
One of the most surprising findings, Shukla said, was how many different proteins interacted with G4s.
“We weren’t anticipating identifying so many important regulators of genome function,” Shukla said. “This kind of puts G4s at this cross-section of genome biology.”
That suggests G4 structures could influence a wide range of cellular processes: not just gene expression, but also transcriptional control, RNA processing and chromatin accessibility.
Moving forward, Shukla and his collaborators will study how G4-driven changes in genome architecture may contribute to development of cancers and neurodegenerative conditions.
Shukla is also a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University.
The study was supported by the SPARK pilot award, R00 award from the National Cancer Institute (R00CA248835), R01 award from National Institute for Allergy and Infectious Disease (R01AI187142), Research Scholar Grant from American Cancer Society (RSG-25-1432761-01-DMC) and institutional startup funds from Northwestern University and the Lurie Cancer Center.
