Single-Cell Map of Gut Chromatin Reveals Which Cells Drive Crohn’s Disease Risk

Scientists have produced one of the most detailed maps yet of how genes are switched on and off in the human gut, and in doing so they have sharpened our understanding of where the genetic risk of Crohn’s disease actually resides. The new study, published in Nature Genetics, used single-cell sequencing of chromatin accessibility to profile hundreds of thousands of cells from the intestinal mucosa of patients with Crohn’s disease and healthy volunteers. The result is a comprehensive atlas of candidate cis-regulatory elements, the stretches of DNA that act as control switches for genes, resolved at the level of individual cell types across two distinct regions of the bowel.

The research team, led by investigators at the University of Chicago, collected pinch biopsies from the terminal ileum and the ascending colon of 23 patients with Crohn’s disease, some with active inflammation and some without, alongside 16 healthy controls. From 71 biopsies, they generated single-cell assay for transposase-accessible chromatin with sequencing, or scATAC-seq, data. After rigorous quality control, the dataset retained 178,030 individual cells, which the researchers clustered into 29 distinct cell types spanning the epithelial, immune and stromal lineages of the intestinal wall. By annotating these clusters with reference gene-expression data, they built a cell-type-resolved catalog of 557,310 candidate cis-regulatory elements, or cCREs, each representing a region of open, potentially active chromatin.

The scale of the atlas matters because Crohn’s disease is not a disease of a single cell type. It is a chronic inflammatory condition of the gastrointestinal tract that arises from an interplay of host genetics, environmental triggers and the gut microbiota. Genome-wide association studies have linked more than 320 genetic loci to inflammatory bowel disease risk, but most of those risk variants sit in noncoding regions of the genome, far from any obvious gene, and their function has remained opaque. Knowing which cell types open or close which regulatory elements provides exactly the kind of functional annotation needed to interpret those variants.

The cellular composition of the atlas reflects the biology of the gut. Epithelial cells dominated the dataset, accounting for 69.3 percent of all cells, followed by immune cells at 26.3 percent and stromal cells at 4.3 percent. Among the epithelial compartment, the researchers identified mature absorptive cells specific to the ileum, known as enterocytes, and their colonic counterparts, colonocytes, along with goblet cells, Paneth cells, enteroendocrine cells and Tuft cells. The immune compartment included CD4-positive and CD8-positive T cells, gamma-delta T cells, natural killer cells, innate lymphoid cells, plasma cells, several B cell subsets, macrophages, dendritic cells and mast cells. Notably, the team also captured neutrophils, innate immune cells that are notoriously difficult to profile with standard single-cell RNA sequencing and have consequently remained understudied despite their suspected role in inflammatory disease.

Because discrete cell-type clusters cannot fully capture dynamic processes such as inflammation or continuous differentiation, the researchers complemented their cluster analysis with a computational approach called topic modeling, which assigns each cell partial membership in multiple regulatory programs simultaneously. This revealed programs shared across related cell types as well as programs tied to specific contexts. One program, topic 7, stood out as a signature of inflammation in epithelial cells: its activity was high in biopsies taken directly from inflamed regions, moderate in adjacent noninflamed tissue from the same patients, and absent in healthy controls. The regulatory elements within this program were enriched for binding motifs of AP-1 family factors and interferon regulatory factors, implicating interferon and JAK-STAT signaling as central drivers of the epithelial inflammatory response. Putative target genes included well-known inflammatory markers such as LCN2, NOS2, DUOX2 and major histocompatibility complex class II genes.

Inflammation left its mark across the entire mucosa. Comparing inflamed tissue with healthy controls, the researchers identified inflammation-associated differentially accessible regions in 21 cell types, with epithelial cells yielding the largest number. Endothelial cells, which line blood vessels and gate the movement of cells and molecules into inflamed tissue, showed 1,702 such regions enriched for proinflammatory transcription factor motifs. CD8-positive T cells displayed accessibility changes linked to the effector regulator EOMES, plasma cells showed changes involving the maturation factor POU2F2, and macrophages exhibited broad remodeling involving AP-1, IRF and STAT family factors. In goblet cells and Paneth cell-associated programs, the data pointed to plasticity in the secretory lineage, suggesting that these mucus- and antimicrobial-producing cells reprogram their regulatory landscape in response to injury and inflammation.

To connect DNA sequence directly to chromatin state, the team exploited natural genetic variation among their 39 donors. By imputing genotypes from the sequencing data and mapping chromatin accessibility quantitative trait loci, or caQTLs, in 19 cell types, they identified thousands of genetic variants that alter chromatin accessibility in a cell-type-specific manner. Critically, they showed that variants predicted to disrupt transcription factor binding motifs correlate with reduced accessibility at the affected elements. Disruption of TCF3 motifs in plasma cells and CDX2 motifs in colonocytes were among the clearest examples, providing direct evidence that the regulatory logic inferred from motif enrichment reflects real functional relationships between sequence, protein binding and chromatin state.

The atlas then delivered its most consequential finding: a cell-type-resolved view of Crohn’s disease heritability. Using stratified linkage disequilibrium score regression on GWAS summary statistics, the researchers found that 10 of the 29 cell types showed significant enrichment of inflammatory bowel disease genetic risk, and all 10 were immune cells. Adaptive immune cells, particularly T cells, carried the strongest signal, but innate immune cells, including macrophages, dendritic cells and neutrophils, were also enriched for Crohn’s disease risk specifically. Strikingly, ulcerative colitis risk was not enriched in those innate cells, a result consistent with known differences in genetic architecture between the two major forms of inflammatory bowel disease and a demonstration of the specificity of the approach. Epithelial and stromal cells, despite their dramatic inflammatory responses, showed no enrichment for disease risk, and neither did the inflammation-associated regulatory programs themselves.

That separation between genetic risk and inflammatory consequence is one of the study’s most provocative implications. The data suggest that the genetic predisposition to Crohn’s disease operates primarily through immune cells, while the epithelial and stromal changes visible in inflamed tissue are largely downstream effects of the inflammatory process rather than primary causes. The team also linked body mass index heritability to enteroendocrine cells, confirming a previous observation and hinting that part of the genetic contribution to metabolism is mediated through these hormone-producing gut cells.

Finally, the researchers fine-mapped Crohn’s disease GWAS signals and overlaid the resulting variants onto their atlas. Variants with high posterior inclusion probabilities were 3.5-fold enriched within cCREs, and the team prioritized 30 candidate regulatory variants across 22 loci. Most fell in elements active in innate and adaptive immune cells, but several implicated epithelial contexts. Near the key risk gene NOD2, a fully prioritized variant sat in a neutrophil-associated regulatory element. Two variants near CIITA, a co-activator of MHC class II genes, occupied adjacent regulatory elements with strikingly different specificities, one active across immune and endothelial cells and the other strongly associated with epithelial inflammation, suggesting that multiple variants within a single locus can act through different cell types. The authors caution that their moderate cohort size limited power for finer clinical stratification and for detecting caQTLs in rare cell types, and larger cohorts will be needed to dissect disease subtypes and treatment responses. Even so, the atlas stands as a public resource that transforms a list of noncoding risk variants into testable hypotheses about which cells, which switches and which genes set the stage for Crohn’s disease.

Subject of Research: Single-cell chromatin accessibility mapping of the human intestinal mucosa to identify regulatory programs and cell types underlying Crohn's disease genetic risk

Article Title: Single-cell analysis of chromatin accessibility in the human intestine identifies regulatory programs and clarifies genetic associations in Crohn’s disease

Article References: Zhao, Y., Zhou, R., Mu, Z., Carbonetto, P., Zhong, X., Xie, B., Luo, K., Jiang, Z., Liu, J., Cham, C. M., Koval, J., He, X., Dahl, A. W., Liu, X., Chang, E. B., Basu, A., & Pott, S. (2026). Single-cell analysis of chromatin accessibility in the human intestine identifies regulatory programs and clarifies genetic associations in Crohn’s disease. Nature Genetics, 58(10), 2535-2549. https://doi.org/10.1038/s41588-026-02755-z

Image Credits: AI Generated

DOI: 10.1038/s41588-026-02755-z

Keywords: Crohn's disease, single-cell ATAC-seq, chromatin accessibility, regulatory elements, inflammatory bowel disease, GWAS, intestinal mucosa, T cells, neutrophils, epigenomics, fine-mapping, topic modeling