Background

Our body's intestinal mucosa is surrounded by various sentinel cells that maintain immune balance while preventing the invasion of external pathogens. Among these, Type 3 Innate Lymphoid Cells (ILC3) are key cells that play a central role in regulating the intestinal barrier immunity and repairing tissues. However, due to the extremely low abundance of these cells in the body, it has been challenging to investigate their genetic regulatory mechanisms in detail. Conventional three-dimensional genome structure analysis techniques, such as Promoter Capture Hi-C (PCHi-C), require the analysis of millions of cells. Therefore, creating a precise genetic map of ILC3 extracted in small quantities from humans has been considered nearly impossible.

This limitation has hindered the field of genetics from fully understanding the causes of autoimmune diseases, including Crohn's Disease (CD). Genome-Wide Association Studies (GWAS) have identified numerous disease-associated risk variants, but more than 90% of these variants are located in non-coding regions that do not produce proteins. To understand how genetic variations in non-coding regions control distant target genes and induce inflammation, it is necessary to visualize the three-dimensional contact structure of the genome. This highlights the need for analytical techniques that can construct high-resolution genome maps in rare immune cells such as ILC3.

Key Findings

The research team led by Dr. Valeriya Malysheva at the VIB-UAntwerp Center for Molecular Medicine in Belgium developed a 'mini-Capture Hi-C' technique that operates with only 10,000 cells, complementing existing analytical methods. This technique captures three-dimensional chromosomal contact information between promoters that regulate gene expression and distant enhancers with high resolution. Using this technique, the research team successfully created the first three-dimensional interaction map of the human ILC3 genome.

Furthermore, the researchers designed a Bayesian statistics-based, genome-wide fine-mapping framework called multiCOGS and combined it with genome maps and GWAS data. The analysis revealed approximately 100 target genes that interact with CD risk variants within ILC3. More than half of these are novel targets that have not been previously reported in autoimmune disease research.

The most notable target was CLN3, known as the causative gene for Batten Disease, a childhood neurodegenerative disease. The study demonstrated that CD risk variants cause the three-dimensional structure of the genome to fold, bringing the CLN3 promoter, which is located at a distance, into physical contact. The team immediately conducted functional validation. In experiments using a mouse ILC3-like cell line, stimulation with pro-inflammatory cytokines caused a sharp decrease in CLN3 expression. Conversely, artificially increasing CLN3 levels significantly reduced the secretion of interleukin-17 (IL-17), an inflammatory cytokine. These results confirmed that the CLN3 gene acts as a negative regulator that controls the excessive immune response in the intestine.

Significance and Prospects

This research has opened a precise pathway for discovering therapeutic targets for complex autoimmune diseases by elucidating the three-dimensional physical contacts of chromosomes within rare immune cells. The discovery that CLN3 is involved not only in neurodegeneration but also in maintaining immune homeostasis in the digestive tract is expected to be a milestone in multidisciplinary research that explains the connection between the brain and the gut.

In addition, the research team expanded the application of the same genome analysis platform to five autoimmune diseases, including Ulcerative Colitis (UC), Multiple Sclerosis (MS), and psoriasis, and created a catalog of ILC3 target genes for each disease. This list of genes, which has been validated for efficacy through CRISPR interference (CRISPRi) screening, is expected to be widely used for target discovery in the development of new drugs.

However, the fact that this functional validation was mainly performed in mouse cell line models is a challenge that needs to be addressed in the future. Subsequent clinical studies are needed to determine whether the same immune-suppressive mechanism functions reliably in the actual in vivo microenvironment of patients before it can be translated into clinical applications.