Figure 1.
Expansion and purification of human CD4+CD25+FOXP3+ Treg cells.
Panel A, Isolation and purity (% indicated) of CD4+CD25+ T cells from human PBMCs. Panel B, Expansion rates of sorted CD4+CD25+ T cells in vitro. Panel C, Evaluation of the suppressive capacity of expanded cells. Panel D, FACS-sorting of CD4+CD25+FOXP3+ regulatory T cells and CD4+CD25+FOXP3− T cells after expansion. The purity of each sub-population is indicated. S:R, Suppressor:Responder ratios
Figure 2.
Distribution and tag density of H3K4me3 enriched peaks. Panel A
, An illustration of the four regions that the human genome was divided into: proximal promoters, exons, introns, and intergenic sequences [39]. The number of islands detected for each sample is listed (Total), followed by the islands among genomic regions with the percentage they comprise listed in the parenthesis. Panel B, Tag density of H3K4me3 enriched peaks in Treg and aTconv cells. Tag density was plotted by splitting upstream (5Kb), downstream(5Kb) and gene body into 40 windows, respectively. Tag density = number of reads in specific region/(reads total number×specific region length).
Figure 3.
Comparison of H3K4me3 enriched regions, proximal promoters or related genes between Treg and aTconv cells. Panel A
, Correlation of H3K4me3 enriched regions between Treg and aTconv cells. Each spot represents a peak. The X- and Y-axis represent the ratio of the total reads of the H3K4me3 enriched peak to the length of the proximal promoter of this gene in Treg or aTconv cells, respectively. Panel B, Overlapping H3K4me3 enriched regions between Treg and aTconv cells. Panel C, Correlation of proximal promoters marked by H3K4me3 between Treg and aTconv cells. Each spot represents an individual peak. The X- and Y-axes are the same as for Panel A. Panel D, The high degree of overlapping proximal promoters marked by H3K4me3 between Treg and aTconv cells. Panel E, Summary of the distribution of overlapping regions enriched by H3K4me3 in Treg and aTconv cells. Panel F, Venn diagram showing the difference of genes associated with H3K4me3 enriched proximal promoters between Treg and aTconv cells.
Figure 4.
H3K4me3 modifications of cell signature genes and common genes in Treg and aTconv cells, as analyzed by the UCSC hg18 Genome Browser.
The following tracks are shown (from top to bottom): Window position; ChIP-Seq tag counts for H3K4me3 modifications in Treg cells; ChIP-seq tag counts for H3K4me3 modifications in aTconv cells; UCSC genes based on RefSeq; mammalian consensus. The red frames indicate the H3K4me3 modifications in proximal promoters. Panel A, H3K4me3 modification on the FOXP3 loci (ChrX:48990000–49010000). The two intragenic H3K4me3 islands, located about 6 kb (ChrX:49001600–49002200) and 4 kb (ChrX:49004100–49005100) downstream of the FOXP3 promoter in Treg cells, are framed in blue. Panel B, H3K4me3 modification on the CCR7 loci (Chr17:35960621–35978190). Panel C, Expression of cell signature genes and common genes. The mRNA expression levels of signature genes and common genes are shown for Treg and aTconv cells, as measured by real-time PCR assays. The 2−ΔΔ CT method is used to quantify the expression relative to the GAPDH housekeeping control. Data are shown as fold-induction relative to Treg cells and are the mean ± standard error of the mean (SEM) from three independent experiments. *, P<0.01, vs Treg cells.
Figure 5.
Genome-wide distribution and comparison of H3K4me1 modifications between Treg and aTconv cells. Panels A and B
, Distribution and tag density of H3K4me1 enriched peaks. (A) Distribution of H3K4me1 enriched peaks. The number of islands for each sample is listed as the total identified islands (Total), followed by the islands among genomic regions with the corresponding percentage listed in the parenthesis. (B) For each gene, uniquely mapped tags were summed in 125 bp windows from 5 kb upstream of the txStart to the txStart, and from the txEnd to 5 kb downstream. Panels C and D, Comparison of H3K4me1 enriched regions between Treg and aTconv cells. (C) Correlation of H3K4me1 enriched regions between Treg and aTconv cells. Each spot represents a single peak. The X- and Y-axis represent the ratio of the total reads of H3K4me1 enriched peak to the length of this gene in Treg or aTconv cells, respectively. (D) Overlapping H3K4me1 enriched regions between Treg and aTconv cells.
Figure 6.
Prediction of enhancers and verification of activity for cell signature genes. Panel A
, UCSC hg18 genome browser views of H3K4me1 modifications of the cell signature gene FOXP3 loci (ChrX:48990000–49010000) in Treg and aTconv cells. The following tracks are shown (from top to bottom): genes' location; ChIP-Seq tag counts for H3K4me1 modifications in Treg cells; ChIP-Seq tag counts for H3K4me1 modifications in aTconv cells; UCSC genes based on RefSeq; mammalian consensus. The blue frames indicate the Treg cell-type specific H3K4me1 enriched regions. Panels B Functional analysis of enhancer candidates specific for Treg cells. These fragments are all enriched by H3K4me1 modifications except the FOXP3 (ChrX:49004128–49005080) fragment, which is H3K4me3 modified and identified as an enhancer in the published literatures. Panel C, Functional analysis of the enhancer candidates common in both Treg and aTconv cells, which are all H3K4me1 modified. All the selected enhancer candidates were cloned into the luciferase vector pGL3-Promoter, respectively. The indicated plasmids were transiently transfected into Jurkat T cells that were stimulated with or without PMA and ionomycin (PI) after transfection. Luciferase activity was normalized against the activity of a co-transfected Renilla construct. Mean values ± SEM are shown relative to the pGL3-promoter alone. * indicates a statistically significant difference between the cloned vs control plasmids (P<0.01; paired Student's t-test).
Figure 7.
Comparison of H3K4me1 and H3K4me3 enriched regions in Treg or aTconv cells. Panel A
, Correlation of H3K4me1 and H3K4me3 enriched regions in Treg cells. Each spot represents a single peak. The X-axis represents the ratio of total reads of the H3K4me3 enriched peak to the length of corresponding gene, while the Y axis represents the ratio of the total reads of the H3K4me1 enriched peak to the length of corresponding gene. Panel B, Regions enriched by H3K4me1 and H3K4me3 in Treg cells. Panel C, Correlation of H3K4me1 and H3K4me3 enriched regions in aTconv cells. Each spot represents a single peak. The X- and Y-axes are the same as for Panel A. Panel D, Regions enriched by H3K4me1 and H3K4me3 in aTconv cells. Panel E, Summary of the distribution of the regions enriched by H3K4me1 and H3K4me3 in Treg or aTconv cells.