Genes and Immunity (2010) 11, 554–560 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 www.nature.com/gene ORIGINAL ARTICLE DNA methylome in human CD4 þ T cells identifies transcriptionally repressive and non-repressive methylation peaks T Hughes1,4, R Webb1,4, Y Fei1,2,JDWren1 and AH Sawalha1,2,3 1Arthritis & Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; 2Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA and 3US Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA DNA methylation is an epigenetic mark that is critical in determining chromatin accessibility and regulating gene expression. This epigenetic mechanism has an important role in T-cell function. We used genome-wide methylation profiling to characterize the DNA methylome in primary human CD4 þ T cells. We found that only 5% of CpG islands are methylated in CD4 þ T cells, and that DNA methylation peak density is increased in subtelomeric chromosomal regions. We also found an inverse relationship between methylation peak density and chromosomal length. Our data indicate that DNA methylation in gene promoter regions is not always a repressive epigenetic mark. Indeed, about 27% of methylated genes are actively expressed in CD4 þ T cells. We demonstrate that repressive methylation peaks are located closer to the transcription start site (TSS) compared with functionally non-repressive peaks (À893±110 bp versus À1342±218 bp (mean±s.e.m.), P-value o0.05). We also show that both a larger number and an increased CpG island density in promoter sequences predict transcriptional permissiveness of DNA methylation. TSS in the majority of genes with permissive DNA methylation peaks is in DNase I hypersensitive sites, indicating a failure of DNA methylation to induce chromatin inaccessibility in these loci. Genes and Immunity (2010) 11, 554–560; doi:10.1038/gene.2010.24; published online 13 May 2010 Keywords: CD4 þ T cell; DNA methylation; CpG islands; promoter methylation; methylome Introduction regulation of interferon-g, and interleukin (IL)-4 produc- tion, key cytokines produced by Th1 and Th2 cells, DNA methylation is an epigenetic mark that is critical respectively. DNA demethylation of the FOXP3 locus is in determining chromatin accessibility and regulating pivotal for regulatory T-cell differentiation, and demethyla- gene expression. DNA methylation, which refers to the tion of the IL-2 locus is associated with IL-2 production addition of a methyl group to the fifth carbon in cytosine on T-cell activation.8 Defective T-cell DNA methylation residues within CG dinucleotides, is involved in cell results in T-cell autoreactivity and has an important differentiation, imprinting, X-chromosome inactivation, pathogenic role in both drug-induced and idiopathic and suppression of transcriptional noise and ‘parasitic’ lupus, both in human disease and in animal models.7,9,10 DNA.1–4 Abnormalities in the DNA methylation path- Herein, we characterize the DNA methylome in way are associated with pathological consequences. primary human CD4 þ T cells. We map DNA methyla- For example, mutations in the de novo DNA methyl- tion peaks across the genome, and identify genes with transferase DNMT3B result in a syndrome of Immuno- promoter region methylation in CD4 þ T cells using five deficiency, Centromeric instability and Facial anomalies biological replicates. We further identify distinguishing (ICF syndrome).5 A complete deficiency of the DNA features between transcriptionally repressive and non- methyltransferase DNMT1 is incompatible with life. repressive DNA methylation in CD4 þ T cells. Furthermore, acquired abnormalities in DNA methyla- tion are associated with disease conditions, including cancer and autoimmunity.6,7 Results DNA methylation has a critical role in normal T-cell function such as T helper cell differentiation and the We determined genome-wide DNA methylation peaks in primary human CD4 þ T cells using DNA immuno- precipitation (IP) with an anti-5-methylcytidine antibody Correspondence: Dr AH Sawalha, Arthritis & Immunology Pro- coupled with array hybridization. Both input and IP gram, Oklahoma Medical Research Foundation, 825 NE 13th Street, DNA were labeled and co-hybridized to microarray MS#24, Oklahoma City, OK 73104, USA. chips that included B385 000 probes covering all UCSC- E-mail: [email protected] 4These authors contributed equally to this work. annotated CpG islands and promoter regions for all Received 6 January 2010; revised and accepted 2 March 2010; RefSeq genes (NimbleGen, Reykjavı´k, Iceland). The published online 13 May 2010 experiments were carried out using five biological Human CD4 þ T cell DNA methylome T Hughes et al 555 replicates from five normal healthy female donors (age We identified 2902±187 (mean±s.e.m., n ¼ 5) methy- range from 31 to 48 years). Signal intensity data were lation peaks in CD4 þ T-cell DNA. Further, we identified extracted from the scanned images of each array. Scaled 388 genes that have at least one DNA methylation peak log2-ratios of the IP/input DNA were determined from that appears in the À5to þ 1 kb region relative to the signal intensities, and P-values for methylation enrichment transcription start site (TSS) with its center located within were computed using the one-sided Kolmogorov–Smirnov the À5.5 and þ 1.5 kb region in all the five biological test. Methylation peaks were determined. They represent replicates tested. This stringent requirement that all regions with at least two probes with Àlog10 P-values of at genes should be identified in every sample tested has least 2 within a 500 bp window, and a methylation score of the advantage of adding confidence to the target genes at least 2. The methylation score for each peak is the identified near the methylation peaks. average Àlog10 P-values from probes within that peak. We used gene expression data in normal human Several known methylated genetic loci were included CD4 þ T cells (10 biological replicates) available from in our array for quality control. These included the Gene Expression Omnibus, to determine whether there is regions in the HOXA gene cluster, H19/IGF2/ any correlation between gene expression and methyla- KCNQ1 gene cluster and the IGF2R locus. All were tion status. Expression data were available for 202 genes methylated in all five biological replicates used in this with a methylated promoter region in CD4 þ T cells. study. Figure 1a shows the methylation status of the Only 55 genes (27.2%) had at least one transcript H19/IGF2/KCNQ1 gene cluster in our samples. The expressed in normal human CD4 þ T cells. The majority HOXA gene cluster serves both as a positive and of the methylated genes (72.8%) were not expressed. negative control region, as it contains both known Comparatively, when all annotated genes included in the methylated and hypomethylated regions.11 Our data expression array experiment were analyzed, we found confirm this methylation pattern in all five CD4 þ T-cell that 43.7% of genes (9094 out of 20 828 genes examined) DNA samples (Figure 1b). We further validated the were expressed in normal human CD4 þ T cells (w2 ¼ methylation array data in an independent set of 22.0, Po0.0001) (Figure 2a). These findings are consistent samples from another five normal healthy women (age with DNA methylation being largely a repressive range from 22 to 57 years) using bisulfite DNA epigenetic mark in human CD4 þ T cells. However, sequencing of both methylated and hypomethylated 27.2% of methylated genes are transcriptionally active, regions (Figure 1). indicating that DNA methylation is not always asso- Chromosome 11 1,610,0001,680,000 1,750,000 1,820,000 1,890,000 1,960,000 2,030,000 2,100,0002,170,000 2,240,000 2,310,000 2,380,000 2,450,000 2,520,000 2,590,000 2,660,000 2,730,000 2,800,000 2,870,000 2,940,000 3,010,000 3,080,000 3,150,000 3,220,000 Genes HCCA2 LSP1 TNNT3 H19 IGF2 TSPAN32 CD81 TRPM5 KCNQ1 SLC22A18AS NAP1L4 CARS OSBPL5 8.000 6.400 4.000 Sample 1 2.400 0.000 8.000 6.400 Sample 2 4.000 2.400 0.000 8.000 6.400 4.000 Sample 3 2.400 0.000 8.000 6.400 Sample 4 4.000 2.400 0.000 8.000 6.400 Sample 5 4.000 2.400 0.000 1.000 CpG Islands 0.000 80 85 99 80 84 91 94 87 93 91 91 78 83 91 94 79 83 92 85 79 Chr11: 2,411,956-2,412,208 Methylation (%) 0 255075100 Figure 1 Methylation enrichment signals represented as Àlog10 P-value scores in CD4 þ T-cell DNA from five normal healthy participants in (a) the H19/IGF2/KCNQ1 genetic locus on chromosome 11, and (b) the HOXA gene cluster on chromosome 7. Bisulfite DNA sequencing was used to validate the methylation status in methylated regions within the HOXA3 gene and the KCNQ1/TRPM5 promoter region, and hypomethylated regions in the HOXA1 and HOXA13 promoter regions in an independent set of samples. Genes and Immunity Human CD4 þ T cell DNA methylome T Hughes et al 556 Chromosome 7 27,072,000 27,080,000 27,088,000 27,096,000 27,104,000 27,112,000 27,120,000 27,128,000 27,136,000 27,144,000 27,152,000 27,160,000 27,168,000 27,176,000 27,184,000 27,192,000 27,200,000 27,208,000 27,216,000 27,224,000 27,232,000 27,240,000 27,248,000 27,256,000 HOX Genes A1 A2A3 A4 A5 A6 A7 A9 A10 A11 A13 EVX1 5.500 3.600 Sample 1 2.400 1.200 0.000 5.500 3.600 Sample 2 2.400 1.200 0.000 5.500 3.600 Sample 3 2.400 1.200 0.000 5.500 3.600 Sample 4 2.400 1.200 0.000 5.500 3.600 Sample 5 2.400 1.200 0.000 1.000 CpG Islands 0.000 1002010012211100 79 97 90 78 89 30 88 90 79 90 89 91 14 3 2 13 0 0 17 17 0 2 0 0000000000000000 88 99 95 95 97 85 95 96 90 93 97 92 13 4 10 16 0 15 3 3 0 0 0 0000000000000000 82 94 97 93 95 57 88 94 92 93 95 82 18 8 5 0 15 11 0 9 0 0 0 30000000000000000 85 95 98 84 93 57 88 91 94 93 94 93 504841404800 0000000000000000 74 87 94 86 84 46 91 93 84 94 94 95 14 5 12 18 3 0 9 0 0 0 0 Chr7: 27,101,779-27,102,082 Chr7: 27,129,376-27,129,708 Chr7: 27,206,717-27,207,948 Methylation (%) 0 25 50 75 100 Figure 1 Continued.