Selective requirement of 3D genomic organization for the Mdm1-Il22-Ifng locus regulation in initial Th1 cell lineage specification and differentiation [ATAC-Seq]
Eukaryotic DNA fibers are organized into structured subunits in the nucleus, but the extent to which these 3D chromatin architectures influence cytokine expression remains unclear. Here, we investigate the role of dynamic loop formation within the topologically associating domain (TAD) in regulating the expression of two critical cytokines, interferon-gamma (IFN-?) and interleukin-22 (IL-22). These cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. Using in situ Hi-C, we demonstrate inducible TADs that insulate Ifng and Il22 enhancers during Th1 differentiation. Deleting the boundary of these TADs resulted in imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo. In contrast, this boundary element was dispensable for cytokine regulation in NK cells. Our finding suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes. Overall design: We differentiate naive T cells with TCR signaling CD3 and CD28 and cytokines IL-12 ex vivo to Th1 cells in WT and IfngCTCFdel mice, and performed focused Hi-C for 3-D architecture and chromatin interactions, bulk RNA-seq for transcriptomic analysis, in parellel using ATAC-seq and ChIP-seq (p300, rad21, CTCF, H3K4me1, H3K27ac, H3K4me3) for epigenomic profiling. To understand the immune response change in the WT and IfngCTCFdel mice in vivo, we measured gene expression of cells isolated from mice infected by Toxoplasma gondii for three and seven days, and by LCMV for 4 days and 7 days using single cell RNA-seq. We also used whole genome sequencing to evaluate of our mouse model generated by CRISPR/Cas9.