A critical CTCF binding site of the Ifng-Il22 locus specifies cytokine expression and finetunes immune response

Abstract

Abstract Precise control of cytokine milieu plays an essential role in homeostasis and diseases, and dysregulated cytokine production leads to undesired inflammation and autoimmunity. Interferon gamma (IFN-γ) and interleukin-22 (IL-22), two key cytokines for against intracellular and extracellular pathogens, respectively, evolutionarily resides in close genomic proximity. Notably, these genes are exclusively expressed in type I and type III lymphoid cells via complex epigenomic regulation that remains largely unknown. Our ATAC-seq, ChIP-seq and PollI ChIA-PET datasets revealed a CTCF binding site 70kb upstream of Ifng setting a boundary of the enhancer landscapes. Therefore, we proposed that this CTCF site segregates Ifng and Il22gene enhancers for lineage-specific gene regulation. To investigate whether this CTCF binding contributes to bifurcated cytokine gene regulation at the Ifng-Il22 locus, we deleted this 17bp Ifng (−70kb)CTCF site in mice using a CRISPR strategy. We found that Ifng (−70kb)CTCFdel CD4+ T cells revealed deficiency in initial Th1 cell differentiation. Using single cell RNA-seq and Toxoplasma gondii infection as a model for type I immune response in vivo, we identified aberrant Th17 differentiation and increased T cells prolifereation in Ifng (−70kb)CTCFdel mice. To further understand the molecular mechanism, we measured CTCF associated loop formation in pre-differentiated Naïve CD4+ T cells and interestingly, this Ifng (−70kb)CTCF site formed a loop with a CTCF-enriched region on the other side of Il22 gene. Our data provide a novel pre-determined mechanism that prevents cross activation for IFN-γ enhancers to target IL-22 prior to differentiation.