Fig. 7
From: NFAT primes the human RORC locus for RORγt expression in CD4+ T cells
Functional characterization of RORC2 regulatory regions. a, b p300 and CBP binding to the RORC locus is cyclosporine-sensitive. a p300 and CBP ChIPs were performed in CD3+ T cells from peripheral blood stimulated for 20 h in the shown conditions followed by RT-qPCR of the RORC2 promoter region. Background from an irrelevant IgG was subtracted. Shown are two representative independent ChIP experiments. Additional independent experiments are shown in Supplementary Fig. 3j, k. b p300 ChIP was performed as in A, followed by RT-qPCR of RORC2 enhancer regions. c–g CRISPR-Cas9-mediated deletion of regulatory regions in the RORC locus affects RORC2 expression in primary CD4+ T cells. c Human/murine alignment of the RORC2 promoter and upstream conserved region (CNS-2.5 kb and −1.8 kb). The deleted regions (horizontal bars) and their sizes are represented. d Top panel: PCR amplification of genomic DNA from sorted samples (donor 3) transfected with a ribocomplex of Cas9 protein, crRNA and Atto550-labeled tracrRNA. The left side of the gel shows samples enriched for transfected cells (Atto550 High), the right side samples from Atto550 low cells, carrying wild-type DNA. Asterisks mark fragments carrying the expected deletions. Bottom panel: the proportion of WT and deleted DNA fragments in the Atto550 positive cells was quantified with ImageJ program. e Expression of RORC2 was measured by nCounter technology in CD4+ T cells transfected with a negative control crRNA, or cells carrying a genomic deletion as shown in c, d. Horizontal bars indicate two-tailed Student t-test on samples from 4 different donors. f ANOVA analysis of gene expression Nanostring data in control and CRISPR/Cas9 deleted CD4+ T cell populations from 4 donors (p = 0.002, q = 0.060). Donors 3 and 4 had two independent control transfections each. Green squares indicate control samples, red square samples carrying RORC2 deletions. g Pearson correlation plot of the top differentially expressed variable from the ANOVA analysis in d. Source data are provided as a Source Data file
