Fig. 4: YTHDF2 segregates IKZF1/3 to dictate an active chromatin state in polyfunctional CD8 T cells.
a GO enrichment analysis of downregulated genes in Ythdf2CKO compared with Ythdf2F/F CD8 T cells after priming (anti-CD3/CD28, 5 μg/ml, 24 h). b Proximity ligation assay (PLA) analysis of YTHDF2 associated with IKZF1 or IKZF3 in primed mouse (WT or OT-1) and human CD8 T cells stimulated with anti-CD3/CD28 (5 μg/ml, 24 h) or OVA (10 nM, 24 h). Scale bar, 10 μm. c Coimmunoprecipitation assays of YTHDF2 associated with IKZF1 or IKZF3 in primed WT CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h). d Volcano plot of genes with differential chromatin accessibility between activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h). e Volcano plot of genes with differential chromatin accessibility between Jurkat-shCtrl (Vec) and Jurkat-shYTHDF2 (KD) cells. f, g ChIP-seq datasets for IKZF1 (GSM1296538) and IKZF3 (GSM803106) in mouse T cells were obtained using Cistrome Data Browser. ATAC-seq (f) or H3K4me CUT&RUN (g) profiles of activated Ythdf2F/F and Ythdf2CKO CD8 T cells were represented on IKZF1/3-bound loci or IKZF1/3-associated promoters. h Stat5a (left) and Rasgrp1 (right) mRNA levels detected by RT-qPCR in activated Ythdf2F/F (n = 3 independent samples) and Ythdf2CKO (n = 3 independent samples) CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h). i ATAC-seq and H3K4me CUT&RUN tracks on the gene loci of Stat5a (top) and Rasgrp1 (bottom) in activated Ythdf2F/F and Ythdf2CKO CD8 T cells. j Heatmap showing the relative expression of representative genes (down-regulated in Ythdf2CKO) in activated Ythdf2F/F and Ythdf2CKO CD8 T cells from RNA-seq data. Enhanced chromatin accessibility or putative IKZF1/3 binding is depicted by a filled circle. Error bars, mean ± s.e.m. One-sided Fisher’s exact test with P values adjusted by the Benjamini–Hochberg method (b), two-tailed unpaired Student’s t-test (h).
