Journal: iScience
Article Title: The transcription factor IRF4 regulates the homeostasis and function of intestinal ILC3s
doi: 10.1016/j.isci.2025.112800
Figure Lengend Snippet: Cleavage under targets and tagmentation sequencing of IRF4 binding in intestinal ILC3 subsets (A) Heatmap showing the genome-wide distribution of IRF4-binding signals at peak centers in ILC3 subsets sorted from Irf4 f/f and Irf4 f/f Rorc cre mice by CUT&Tag. (B) Occupancy of IRF4 at all gene promoter regions (±5 kb of TSS). (C) Donut chart showing the percentages of IRF4 binding at exon regions, intron regions, or intergenic regions. (D) Venn plot displaying the overlap of the IRF4-regulated genes from pairwise comparisons of NKp46 + ILC3s, NKp46 − CCR6 − ILC3s, and CCR6 + ILC3s. (E) Gene set enrichment analysis (GSEA) of NKp46 + ILC3 signature gene sets enriched in shared IRF4-modified genes. (F and G) IGV visualizes the indicated gene locus containing ATAC-seq and IRF4-binding peaks in ILC3 subsets. IRF4 CUT&Tag sequencing data are from two independent replicates. (H–K) Rescue experiments. Retroviruses were generated by transfection of pMX-IRES-GFP plasmids containing the indicated genes into Plat-E cells using PolyJet. CLPs (Lin − CD127 + c-Kit int Sca-1 int Flt3 + ) were sorted from the bone marrow from Irf4 f/f and Irf4f/f Rorc cre mice and transfected with retroviral supernatants. Retrovirus-transfected CLPs were then collected and adoptively transferred into sublethally irradiated CD45.1 + wild-type recipient mice through intravenous tail vein injection. Transduced cells were transferred with CD45.1 + wild-type bone marrow cells to help the engraftment of the CLPs. After 2 weeks, recipient mice were sacrificed, and organs were collected for analysis. (H) Flow cytometry of CD45.2 + GFP + ILC3 subsets isolated from the siLP in the indicated recipient mice (upper). MHC class II expression of CD45.2 + GFP + ILC3 subsets isolated from the siLP in the indicated recipient mice (lower). (I) Cytokine production in siLP CD45.2 + GFP + ILC3s from the indicated recipient mice. (J) The percentages of the indicated subsets were compared. (K) The percentages of MHC class II + ILC3s, IL-17A + ILC3s, and IL-22 + ILC3s were compared. Bar graphs are presented as mean ± SEM. A two-tailed Student’s t test was performed for comparisons. The data are representative of at least three independent experiments. See also .
Article Snippet: Retroviruses were generated by transfection of pMX-IRES-GFP plasmids containing the indicated genes into Plat-E cells using PolyJet (SignaGen).
Techniques: Sequencing, Binding Assay, Genome Wide, Modification, Generated, Transfection, Retroviral, Irradiation, Injection, Flow Cytometry, Isolation, Expressing, Two Tailed Test
