Fig. 4: Activity of bZIP and bHLH transcription factors during skin T_reg cell development is associated with binding site hypomethylation.

a, Enrichment of bZIP motifs in DMRs and differential peaks between skin T_reg cells and blood CD45RA⁺ T_reg cells (blood RA⁺ T_reg cells). P values were determined by one-tailed binomial test implemented in homer³¹ (n = 1,742 DMRs, 298,457 DMRs, 5,722 peaks and 3,192 peaks for columns 1–4) with a Benjamini–Hochberg correction. b, Chromatin accessibility in skin T_reg and blood CD45RA⁺ T_reg cells (four donors, ATAC Donors 1, 2, 4 and 5) around genomic sites for a selected bZIP motif. c, Enrichment of bHLH motif sites in DMRs and differential peaks between skin T_reg cells and blood CD45RA⁺ T_reg cells. P values were determined as in a. d, Chromatin accessibility in skin T_reg and blood CD45RA⁺ T_reg cells around genomic sites for a selected bHLH motif. Donors are as in b. e, Transcriptomic footprint of transcription factors (that is, enrichment of target genes) among genes differentially expressed between skin T_reg and blood CD45RA⁺ T_reg cells. Labels correspond to transcription factor gene names. P values were determined by two-tailed permutation test (n = 28,078 genes) with a Benjamini–Hochberg correction. f, Expression of transcription factor genes corresponding to relevant motifs (mean ± s.d.; n = 4 skin T_reg cell donors and 5 blood CD45RA⁺ T_reg cell donors, Tissue Donors 7, 8, 10 and 11 and Blood Donors 3–5, 9 and 10). g, Methylation (top left, Tissue Donors 7, 10 and 11 and Blood Donors 3–5) and chromatin accessibility (middle left, ATAC Donors 1, 2, 4 and 5) in skin T_reg and blood CD45RA⁺ T_reg cells together with c-Myc ChIP–seq signal³² from three cell lines (bottom left) around c-Myc motif sites associated with MLPH and GNA11 and quantitative PCR with reverse transcription showing relative gene expression (mean) of MLPH (guide RNA A or B) and GNA11 in primary blood T_reg cells from six donors (CRISPR Donors 1–6, three donors per guide RNA) after CRISPR-mediated activation originating from regions shown in track plots (right). Highlighted regions correspond to selected DMRs (methylation tracks), scATAC-seq peaks (chromatin accessibility tracks) and c-Myc motif sites (motif ± 100 bases; ChIP–seq and gene tracks). Vertical lines at the bottom of the methylation tracks mark CpG sites. P values were determined by two-tailed paired t-test (n = 3 donors). Data are representative of two or more independent experiments with two or more individual donors.