| Background and aimsThe CD4~+CD25~+FOXP3~+ regulatory T (Treg) cells are required for proper maintenance of immunological self-tolerance and immune homeostasis. Treg cells develop in the thymus as an independent CD4~+ T cell lineage. It is believed that epigenetic modifications serve as an important regulatory mechanism that regulates type-specific gene expression, especially the Forkhead family transcription factor 3 ( the forkhead / winged-helix protein 3, FOXP3 ) expression in human Treg cells. In mice and humans, distinct regions of the FOXP3 locus have a pattern of DNA methylation and specific histone modifications that differ between Treg cells and conventional T cells(Tconv). The present research mainly concentrated in the regulatory role of DNA methylation, but there is a paucity of information related to the histone modification changes that are implicated in regulating Treg cell type-specific gene expression. Previous studies have suggested that trimethylated histone H3 lysine 4(H3K4me3) is usually associated with active gene promoters and monomethylated histone H3 lysine 4(H4K4me1) at promoter-distal sites is often associated with the presence of an enhancer. In view of the importance of the two histone modifications, we used the ChIP-Seq technology to generate genome-wide mapping of H3K4me3 and H3K4me1 in human CD4~+CD25~+FOXP3~+ Treg cells and CD4~+CD25~+FOXP3- activated (a)Tconv cells, investigating the regulatory role of the two histone methylations during Treg cell type-specific gene expression. Methods1 Expansion and purification of human CD4~+CD25~+FOXP3~+ Treg cells.1.1 Isolation and purity of CD4~+CD25~+ T cells from human peripheral blood mononuclear cells(PBMCs): CD4~+CD25~+ T cells were isolated from PBMCs using the human CD4~+CD25~+ Regulatory T Cell Isolation Kit and the Midi-MACS separation system. The isolated CD4~+CD25~+ T cells were then stained with CD4-FITC and CD25-PE, and their purity was detected with a FACS-Aria high-speed cell sorter.1.2 Expansion and purity of sorted CD4~+CD25~+ T cells in vitro: MACS-purified CD4~+CD25~+ regulatory T cell populations were expanded in vitro using the Dynabeads? Human Treg Expander. The expanded cells were then stained with cell surface anti-CD4-FITC, anti-CD25-PE antibodies and intracellular anti-FOXP3-APC antibody and their purity was detected with a FACS-Aria high-speed cell sorter.1.3 Evaluation of the suppressive capacity of expanded cells: CD4~+CD25- T cells were labeled with the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). CD4~+CD25- T cells were seeded alone or in the presence of expanded CD4~+CD25~+ T cells at different ratios(1:0, 1:1, 2:1, 4:1). Co-cultures were harvested and analyzed on a FACS Calibur flow cytometer.1.4 FACS-sorting of CD4~+CD25~+FOXP3~+ regulatory T cells and CD4~+CD25~+FOXP3- T cells after expansion: The expanded cells were stained with cell surface anti-CD4-FITC, anti-CD25-PE antibodies and intracellular anti-FOXP3-APC antibody, and the fixed cells were separated by fluorescence activated cell sorting(FACS) into batches of CD4~+CD25~+FOXP3- activated Tconv (aTconv) and CD4~+CD25~+FOXP3~+ regulatory T cells.2. The role of the two histone methylations during Treg cell type-specific gene regulation.2.1 chromatin immunoprecipitation(ChIP) and quantitative real-time PCR(QPCR) analysis of known target sites enriched by H3K4me3 and H3K4me1 before and after high-throughput sequencing: The Treg and aTconv cells were used for ChIP analysis and ChIP antibody was anti-H3K4me1, anti-H3K4me3 or anti-rabbit IgG. ChIP samples were tested for the enrichment of positive sites by QPCR.We selected some gene's promoters or H3K4me1 enriched sites as positive sites and these genes were important for Treg cell function and development, such as FOXP3, interleukin 2 receptor alpha (IL2RA), glucocorticoid induced tumor necrosis factor receptor (GITR), cytotoxic T lymphocyte-associated antigen-4(CTLA-4), chemokine (C-Cmotif) receptor 7(CCR7) and Signal Transducer, Activator of Transcription(STAT).2.2 ChIP-Seq and data analysis: ChIP samples were sequenced using the Solexa 1G Genome Analyzer following manufacturer's protocols. ChIP-Seq reads were mapped to the University of California, Santa Cruz (UCSC) human genome (hg18) by SOAP(Short Oligonucleotide Analysis Package), which allowed a uniquely aligned read to have up to two mismatching bases.And then we used a defined analysis model (Model-based Analysis of ChIP-Seq, MACS) with default parameters to find peaks, which were called"peaks"of H3K4me3 and H3K4me1.2.3 Genome-wide maps and comparison of H3K4me3 and H3K4me1 modifications in Treg cells and aTconv cells: The overall profiles of the H3K4me1 and H3K4me3 distribution were generated by dividing the human genome into four regions: proximal promoters (1kb upstream and downstream of the transcription start site (txStart); exons; introns; and intergenic sequences. H3K4me3 peaks and H3K4me1 peaks in the two lineages were compared each other to determine the common and cell-type specific H3K4me3 peaks, H3K4me1 peaks, H3K4me3 proximal promoters or proximal promoters associated genes.2.4 H3K4me3 and H3K4me1 modifications of cell signature genes and common genes in Treg and aTconv cells, as analyzed by the UCSC hg18 Genome Browser: The output of the SOAP analysis data was converted to browser-extensible data (BED) files in order to view the data in the UCSC Genome Browser. Then we examined the H3K4me3 and H3K4me1 pattern for IL2RA, CTLA4, tumor necrosis factor receptor superfamily member 18(TNFRSF18),STATs, CCR7 and FOXP3 genes, each of which was important for Treg cell function and development.The mRNA expression levels of these genes between the two cell types were detected by QPCR.2.5 Functional identification of some common and cell-type specific H3K4me1 enriched regions in Treg and aTconv cells: this type of function can be studied using traditional Dual-Luciferase Reporter Assay System.3.Statistical analysis. The two-tailed Student's t-test was used in the analysis of ChIP-DNA,mRNA expression and of luciferase activity. Significance was defined by a P-value <0.05.Results1. Expansion and purification of human CD4~+CD25~+FOXP3~+ Treg cells.1.1 We purified CD4~+CD25~+ T cells from PBMCs by using the magnetic-activated cell-sorting method (MACS). The purity of products was determined to be >93%.1.2 The CD4~+CD25~+ T cells were exposed to artificial antigen-presenting cells for repeated stimulation via CD3 and CD28 in the presence of high-dose IL-2, which resulted in profound proliferation of up to 1000-fold expansion within an eight to nine week span.During the expansion, the purity of CD4~+CD25~+FOXP3~+Treg was reduced ceaselessly.1.3 Results from the mixed lymphocyte reaction (MLR) assay showed that the proliferation of CD4~+CD25- responder T cells was inhibited, in a dose-dependent manner(0:1, 1:1, 1:2, 1:4), by the expanded CD4~+CD25~+ T cells.1.4 We purified the triple-positive T cells from the expanded CD4~+CD25~+ T cells by the FACS method, and the purity of CD4~+CD25~+FOXP3~+ Treg and activated conventional CD4~+CD25~+FOXP3- T cells (aTconv) reached 99.0% and 99.4%, respectively2. The role of H3K4me3 during Treg cell type-specific gene regulation.2.1 Prior to sequencing, the ChIP samples were confirmed for the target sites in both cell types by regular ChIP-QPCR. Results showed that the promoter of known genes were strongly enriched by H3K4me3 in both Treg cells and aTconv cells, but not in IgG control. In contrast, FOXP3 was marked in its proximal promoter by H3K4me3 in Treg cells, but not in aTconv cells and IgG control.The QPCR results were highly consistent with ChIP-Seq data as expected.2.2 We used the high-throughput ChIP-Seq approach to generate genome-wide H3K4me3 maps of human CD4~+CD25~+FOXP3~+ Treg cells and CD4~+CD25~+FOXP3- aTconv cells.2.3 We compared the H3K4me3 enriched regions between Treg and aTconv cells, and found that the coefficient correlation was 0.92 and that there were 20784 H3K4me3 islands that overlapped in the two cell types.And then we compared the H3K4me3 enriched proximal promoters of Treg and aTconv cells, and determined that the coefficient correlation was 0.83 and that there were 15508 overlapping H3K4me3 enriched proximal promoters.At last, We analyzed the particular genes related to the H3K4me3 enriched proximal promoters, and found that most of the genes were common between the two cell types; only 1220 related genes were Treg cell-type specific.2.4 We examined the H3K4me3 pattern for IL2RA, CTLA4, TNFRSF18, STATs, CCR7 and FOXP3 genes, each of which was important for Treg cell function and development. Results showed that IL2RA, CTLA4, STATs and TNFRSF18 genes were marked in their promoters by H3K4me3 in both Treg and aTconv cells. In contrast, FOXP3 and CCR7 were marked in their proximal promoters by H3K4me3 in Treg cells, but not in aTconv cells.The mRNA expression levels were consistent with the ChIP-Seq data.3. The role of H3K4me1 during Treg cell type-specific gene regulation.3.1 After sequencing, the ChIP samples were confirmed for the target sites in both cell types by regular ChIP-QPCR. Results showed that the known sites were strongly enriched by H3K4me1 in Treg cells or aTconv cells, but not in IgG control. The QPCR results were highly consistent with ChIP-Seq data as expected.3.2 We used the high-throughput ChIP-Seq approach to generate genome-wide H3K4me1 maps of human CD4~+CD25~+FOXP3~+ Treg cells and CD4~+CD25~+FOXP3- aTconv cells.3.3 When comparing the H3K4me1 enriched regions in Treg and aTconv cells, we found that the coefficient correlation was only 0.48 and there were only 8897 overlapping H3K4me1 islands present among the 115391 total regions between both cell types.3.4 We examined the H3K4me1 pattern for IL2RA, CTLA4, TNFRSF18, and FOXP3 genes. Results showed that H3K4me1 exhibited cell-type specific locations in these genes between the two cell types.3.5 As this type of function can be readily studied using traditional reporter gene assays, we selected five Treg cell-specific H3K4me1 regions to evaluate their heterologous enhancer activities. Results showed that only two regions showed enhancer activity. In line with this finding, a H3K4me1 region in intron 1 of the IL2RA gene and a H3K4me1 region upstream of the TNFRSF18 gene, which were enriched in both Treg and aTconv cells, did exhibit enhancer activity in Jurkat cells.3.6 We compared the H3K4me1 and H3K4me3 enriched regions in the same sample, and determined that the coefficient correlation was only 0.16 in Treg cells and 0.19 in aTconv cells. Furthermore, there were only 5030 overlapping H3K4me1 regions and 7063 overlapping H3K4me3 regions.Conclusions:1. We obtained enough and high-purity human CD4~+CD25~+FOXP3~+ Treg cells and aTconv cells by combining in vitro expansion, MACS- and FACS- sorting methods.2. We provide for the first time a comprehensive genome-wide dataset of lineage-specific H3K4me3 patterns in Treg and aTconv cells. We found that most proximal promoters enriched by H3K4me3 were common between the Treg and aTconv cells, suggesting that the related genes of the proximal promoters were co-expressed in the two lineages. But some Treg cell-specific genes such as FOXP3 and CCR7 were enriched by H3K4me3 in their promoters only in Treg cells ,not in aTconv cells. The mRNA expression levels of these genes were consistent with the H3K4me3 status observed for their proximal promoter.These results suggested that Epigenetic control can change the H3K4me3 enrichment status of gene promoters and then regulate Treg cell specific gene expression.In other words, H3K4me3 can regulate gene expression through changing gene promoter activity.3. We provide for the first time a comprehensive genome-wide dataset of lineage-specific H3K4me1 patterns in Treg and aTconv cells. The majority of the H3K4me1 regions differing between Treg and aTconv cells were located at promoter-distal sites, and in vitro reporter gene assays were used to evaluate and identify novel enhancer activity. These results suggested that H3K4me1marked enhancers are the most variable class of transcriptional regulatory element between Treg and aTconv cells, and are probably of primary importance in driving Treg cell-type specific patterns of gene expression. In other words, H3K4me1 can regulate gene expression through changing enhancer activity.4. In addition, we found that most H3K4me1 enriched regions were not enriched by H3K4me3, suggesting that most potential regulatory elements were only enriched by H3K4me1 but lacked H3K4me3 in the whole genome of human Treg and aTconv cells In conclusion, determining the global mapping of H3K4me3 and H3K4me1 modifications in Treg cells and aTconv cells, as related to gene expression status and regulatory regions, such as promoters and enhancers, will provide significant insight into differentiation and lineage commitment processes and Treg-specific function.
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