DNA methylome in human CD4+ T cells identifies transcriptionally repressive and
non-repressive methylation peaks
Travis Hughes1*, Ryan Webb1*, Yiping Fei1,2, Jonathan D Wren1, Amr H Sawalha1,2,3
1
Arthritis & Immunology Program, Oklahoma Medical Research Foundation, 825 N.E.
13th Street, Oklahoma City, OK 73104, USA;
2 Department
of Medicine, University of Oklahoma Health Sciences Center, 825 N.E.
13th Street, Oklahoma City, OK 73104, USA;
3 US
Department of Veterans Affairs Medical Center, 921 N.E. 13th Street, Oklahoma
City, OK 73104, USA.
Please address correspondence to Amr H. Sawalha MD; 825 N.E. 13th Street, MS#24,
Oklahoma City, Oklahoma 73104. Phone: (405) 271-7977. Fax: (405) 271-4110.
Email: amr-sawalha@omrf.ouhsc.edu
Key words: CD4+ T cell, DNA methylation, CpG islands, promoter methylation,
methylome
Running title: Human CD4+ T cell DNA methylome
* These two authors contributed equally to this work
1
Abstract
DNA methylation is an epigenetic mark that is critical in determining
chromatin accessibility and regulating gene expression. This epigenetic
mechanism plays an important role in T cell function. We used genome-wide
methylation profiling to characterize the DNA methylome in primary human CD4+
T cells. We found that only 5% of CpG islands are methylated in CD4+ 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+ T cells. We demonstrate that
repressive methylation peaks are located closer to the transcription start site
compared to functionally non-repressive peaks (-893±110 bp versus -1342±218 bp
[mean±SEM], p value <0.05). We also show that both a larger number and an
increased CpG island density in promoter sequences predict transcriptional
permissiveness of DNA methylation. The transcription start site in the majority of
genes with permissive DNA methylation peaks are in DNase I hypersensitive
sites, indicating a failure of DNA methylation to induce chromatin inaccessibility
in these loci.
Key words: CD4+ T cell, DNA methylation, CpG islands, promoter methylation,
methylome
2
Introduction
DNA methylation is an epigenetic mark that is critical in determining chromatin
accessibility and regulating gene expression. DNA methylation, which refers to the
addition of a methyl group to the 5th carbon in cytosine residues within CG
dinucleotides, is involved in cell differentiation, imprinting, X-chromosome inactivation,
and suppression of transcriptional noise and ‘parasitic’ DNA
1-4.
Abnormalities in the
DNA methylation pathway are associated with pathological consequences. For
example, mutations in the de novo DNA methyltransferase DNMT3B result in a
syndrome of Immunodeficiency, Centromeric instability, and Facial anomalies (ICF
syndrome) 5. A complete deficiency of the DNA methyltransferase DNMT1 is
incompatible with life. Furthermore, acquired abnormalities in DNA methylation are
associated with disease conditions including cancer and autoimmunity 6; 7.
DNA methylation plays a critical role in normal T cell function such as T helper
cell differentiation and the regulation of IFN, and IL-4 production, key cytokines
produced by Th1 and Th2 cells, respectively. DNA demethylation of the FOXP3 locus is
pivotal for regulatory T cell differentiation, and demethylation of the IL-2 locus is
associated with IL-2 production upon T cell activation 8. Defective T cell DNA
methylation results in T cell autoreactivity and plays an important pathogenic role in
both drug-induced and idiopathic lupus, both in human disease and in animal models
7;
9; 10.
Herein, we characterize the DNA methylome in primary human CD4+ T cells. We
map DNA methylation peaks across the genome, and identify genes with promoter
region methylation in CD4+ T cells using 5 biological replicates. We further identify
3
distinguishing features between transcriptionally repressive and non-repressive DNA
methylation in CD4+ T cells.
Results
We determined genome-wide DNA methylation peaks in primary human CD4+ T
cells using DNA immunoprecipitation (IP) with an anti-5-methylcytidine antibody coupled
with array hybridization. Both input and IP DNA were labeled and co-hybridized to
microarray chips that included ~385,000 probes covering all UCSC-annotated CpG
islands and promoter regions for all RefSeq genes (NimbleGen, Reykjavík, Iceland).
The experiments were performed using 5 biological replicates from 5 normal healthy
female donors. Signal intensity data were extracted from the scanned images of each
array. Scaled log2-ratios of the IP/input DNA were determined from signal intensities,
and p values for methylation enrichment were computed using the one-sided
Kolmogorov-Smirnov (KS) test. Methylation peaks were determined. They represent
regions with at least 2 probes with -log10 p values of at least 2 within a 500bp window,
and a methylation score of at least 2. The methylation score for each peak is the
average -log10 p values from probes within that peak.
Several known methylated genetic loci were included in our array for quality
control. These included the regions in the HOXA gene cluster, H19/IGF2/KCNQ1 gene
cluster, and the IGF2R locus. All were methylated in all 5 biological replicates used in
this study. Fig. 1A shows the methylation status of the H19/IGF2/KCNQ1 gene cluster
in our samples. The HOXA gene cluster serves both as a positive and negative control
region, as it contains both known methylated and hypomethylated regions
4
11.
Our data
confirms this methylation pattern in all 5 CD4+ T cell DNA samples (Fig.1B). We further
validated the methylation array data in an independent set of samples from another 5
normal healthy women using bisulfite DNA sequencing of both methylated and
hypomethylated regions (Fig.1).
We identified 2902±187 (mean±SEM, n=5) methylation peaks in CD4+ T cell
DNA. Further, we identified 388 genes that have at least one DNA methylation peak that
appears in the -5kb to +1kb region relative to the transcription start site (TSS) with its
center located within the -5.5kb and +1.5kb region in all the 5 biological replicates
tested. This stringent requirement that all genes should be identified in every sample
tested has the advantage of adding confidence to the target genes identified near the
methylation peaks.
We used gene expression data in normal human CD4+ T cells (10 biological
replicates) available from Gene Expression Omnibus (GEO), to determine if there is any
correlation between gene expression and methylation status. Expression data were
available for 202 genes with a methylated promoter region in CD4+T cells. Only 55
genes (27.2%) had at least one transcript expressed in normal human CD4+ T cells.
The majority of the methylated genes (72.8%) were not expressed. Comparatively,
when all annotated genes included in the expression array experiment were analyzed,
we found that 43.7% of genes (9,094 genes out of 20,828 genes examined) were
expressed in normal human CD4+ T cells (chi2= 22.0, p <0.0001) (Fig. 2A). These
findings are consistent with DNA methylation being largely a repressive epigenetic mark
in human CD4+ T cells. However, 27.2% of methylated genes are transcriptionally
active, indicating that DNA methylation is not always associated with gene silencing in
5
CD4+ T cells. There was a significant difference in the average distance between the
center of methylation peaks and the transcription start sites of methylated genes that
are expressed compared to non-expressed genes. The center of methylation peaks was
on average 449bp further upstream from the transcription start site in expressed genes
as compared to non-expressed genes (-1342±218 bp versus -893±110 bp
[mean±SEM], p value <0.05). These data suggest that a chromatin distance of 3
nucleosomes (449bp divided by 147bp/nucleosome) is important in determining if DNA
methylation is transcriptionally repressive or permissive in a given genetic locus. There
was no difference in methylation intensities (as measured by -log10 p value methylation
scores) between transcriptionally repressive and permissive methylation peaks
(2.97±0.04 versus 2.98±0.03 [mean±SEM], p value=0.85). We determined the number
of CpG islands and the maximum CG dinucleotide density in CpG islands within the -5.5
to +1.5kb region from the transcription start site of genes that are methylated and
expressed in CD4+ T cells and genes that are methylated but non-expressed. Promoter
regions of expressed genes were more likely to have a CpG island compared to nonexpressed genes (83.3% versus 64.7%, odds ratio= 2.7, Chi2= 6.38, p value= 0.012).
There was a significant difference in the mean CpG island CG dinucleotide densities (as
measured by maximum CG observed/expected ratio) in promoter regions of expressed
compared to non-expressed genes. The mean maximum CG observed/expected ratio
within CpG islands in promoter regions of methylated and expressed genes was
0.95±0.03 compared to 0.88±0.02 in methylated and non-expressed genes
(mean±SEM, p= 0.01).
6
We performed functional analysis of genes that are methylated but expressed in
CD4+ T cells and in genes that are methylated and non-expressed using Ingenuity
Pathway Analysis software (Ingenuity Systems, Redwood City, CA). Interestingly, the
top functional networks identified in genes that are expressed revealed involvement in
basic cell functions such as cellular signaling and interaction, and cellular growth and
proliferation (Fig. 3A). The non-expressed genes showed functional association with
antigen presentation, cell mediated immune response, and humoral immune response
(Fig. 3B). This suggests that the methylation in non-expressed genes is functionally
relevant and that those genes that are involved in immune functions are non-expressed
in primary CD4+ T cells and can demethylate and become transcriptionally active once
T cells are activated and differentiated.
We next analyzed DNase I hypersensitive (HS) sites in primary human CD4+ T
cells, as a marker for chromatin accessibility. This revealed that the TSS is located
within a DNase I HS site in the majority of methylated genes that are expressed but not
in non-expressed genes (85.5% versus 27.9%, chi2=52.45, p value <0.0001). This
further indicates that DNA methylation is functionally relevant in inducing chromatin
inaccessibility at the TSS and transcriptional repression in the latter but not former
group of methylated genes.
Out of all the 27,458 CpG islands examined within the 22 autosomal
chromosomes and the X chromosome, we found that only 1375±65 (mean±SEM) CpG
islands (5% of islands) include methylation peaks in our samples. This indicates that the
vast majority of CpG islands are unmethylated in CD4+ T cells. This is consistent with
recent studies in other cell types 11; 12.
7
The regions in the HOXA gene cluster (Chr7: 26,924,046-27,424,045), the
H19/IGF2/KCNQ1 cluster (Chr11: 1,699,992-3,143,916), and the IGF2R gene region
(Chr6: 160,309,320-160,447,571) were entirely tilted in our arrays. Data from these
regions (~2 Mb titled region) allowed unbiased examination of the DNA methylation
pattern both in CpG islands and in surrounding genetic regions included within these
sequences. We found that most methylation peaks are located outside of CpG islands.
Indeed, in genetic loci close to CpG islands, methylation peaks tend to occur at CpG
‘shores’, just outside of the boundaries of CpG islands. This observation has been
recently reported in other human tissues 13.
We found a higher density of methylation peaks in the subtelomeric regions
compared to the rest of the chromosomes in CD4+ T cells. The mean methylation peak
density in the tilted region within the subtelomeric regions (7Mb regions from the
telomeres) was significantly higher compared to the non-subtelomeric regions in all
chromosomes combined (p<0.0001). This enrichment towards the telomeres is not
explained by tiling as methylation peaks densities were normalized for the size of the
region tiled, and this phenomenon has been recently reported by others
11; 14.
Enrichment of methylation peaks in the subtelomeric regions was evident and
statistically significant in all chromosomes except for chromosome 19 (Table 1).
Unexpectedly, we also found a negative correlation between chromosome lengths and
the density of methylation peaks observed within the tilted regions in our samples
(r2=0.34, p= 0.0035) (Fig. 2B). This is not explained by gene density as there is a
positive correlation between the number of genes on each chromosome and
chromosomal length (r2=0.35, p= 0.003).
8
Discussion
DNA methylation is largely a transcriptionally repressive epigenetic mark that
induces gene silencing and chromatin inaccessibility 15; 16. DNA methylation induces
chromatin inaccessibility and transcriptional repression by several mechanisms. These
include the recruitment of members of the methylcytosine binding domain-containing
proteins, such as MECP2, which is turn recruit histone deacetylases that result in
chromatin condensation 15; 16. In addition, the bulky methyl group on methylcytosine
residues and can prevent the binding of transcription factors to the promoter sequences
of methylated genes 17. Using DNA methylation profiling in primary human CD4+ T
cells, we shed light on transcriptionally permissive DNA methylation marks and find that
~27% of methylated genes in primary human CD4+ T cells are expressed. We find that
most CpG islands are not methylated in CD4+ T cells, consistent with published work in
a number of genetic regions in multiple cell types 18; 19.
We report two characteristics that distinguish transcriptionally repressive from
permissive DNA methylation peaks in promoter gene sequences. A distance between
the transcription start site and the center of methylation peaks of an average of 449 bp
further upstream from the transcription start site prevents DNA methylation from
inducing chromatin inaccessibility and transcriptional silencing. Our data suggest that
methylation peaks that are transcriptionally repressive are on average about 6
nucleosomes away from the transcription start site. Methylation peaks located on
average about 9 nucleosomes away were not functionally repressive. In addition, CpG
islands in the promoter regions of genes that harbor a permissive DNA methylation
9
peak are characterized by an increased maximal CG dinucleotide density compared to
repressive peaks (Fig. 4). Furthermore, we demonstrate that the majority of genes with
a transcription start site located downstream of permissive DNA methylation peaks are
sensitive to DNase I digestion at the transcription start site, as indicated by the
presence of a DNase I HS site, indicating that chromatin is accessible and available for
transcription machinery binding.
We also observe that transcriptionally permissive DNA methylation peaks
correspond to genes involved in cell signaling, growth, and proliferation, while
repressive peaks are more closely associated with genes associated with immune
response and T-cell differentiation. Modulation of repressive DNA methylation patterns
at key regulatory loci plays a critical role in lineage differentiation of certain T helper
subsets. For example, hypomethylation of a single region in the FOXP3 locus reveals
Treg cell lineage commitment and is closely related to the longevity of suppressor
function. 20; 21
Our data indicate that proximity of methylation is directly related to the potential
for methylation-dependent nucleosome remodeling and repression. We suggest a
model whereby failure of DNA methylation to induce inaccessible chromatin
configuration is related to both the distance of the methylation peaks form transcription
start sites and perhaps reduced efficiency to recruit transcription repressor complexes
such as MECP2-SIN3A-HDAC as a result of high CpG island density, leading to an
open chromatin configuration and availability for transcription.
Materials and Methods
10
CD4+ T cell isolation and DNA extraction
Peripheral blood mononuclear cells were isolated from normal healthy donor blood
samples by density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden).
CD4+ T cells were then isolated via magnetic bead separation using direct labeling
(Miltenyi Biotec, Auburn, CA) following the manufacturer’s protocol. DNA was extracted
using the DNeasy Kit (Qiagen, Valencia, CA). The DNA concentration was then
determined using a NanoDrop® spectrophotometer. Our studies were performed using
five biological replicates from five normal healthy participants (age range from 31 to 48).
All participants singed an informed consent. Our studies are approved by our
institutional review boards.
DNA immunoprecipitation and array hybridization
Genomic CD4+ T cell DNA was digested with MseI and purified using Qiagen Quick
PCR Purification kit. Next, we diluted 1.25 μg of MseI digested DNA to a final volume of
300μl in TE buffer (pH7.5), heat denatured for 10 minutes at 95C and then immediately
cooled on ice for 5 minutes. An aliquot of 60μl was then removed and stored at -20C
as the input DNA. Immunoprecipitation (IP) buffer (60μl at 5X) was then added to the
remaining 240μl of the digested and denatured DNA samples. Anti-5-methylcytidine
antibody (Abcam, Cambridge, MA) was added and the samples incubated overnight
with agitation at 4C. Pre-washed Protein A agarose beads were then added and the
samples incubated at 4C with agitation for 3.5-4 hrs to obtain DNA-antibody-bead
complex. This reaction mix was then spun down at 6000 rpm for 2 minutes at 4C and
the pellet washed three times with 1ml 1X IP buffer with 5 minute incubations with
11
agitation in between centrifugation steps. The beads were then resuspended in 250ul of
digestion buffer and 7ul of Proteinase K (10mg/ml) was added and incubated overnight
at 55C with agitation. IP DNA was then purified using ethanol precipitation. We then
performed whole genome amplification (WGA2 kit, Sigma-Aldrich, St Louis, MO) of
input and IP DNA. Input and IP DNA from each participant were then labeled with Cy3
and Cy5 respectively, pooled, denatured, and then co-hybridized to 385K methylation
arrays with tiling that covers all UCSC-annotated CpG islands and promoter regions for
all RefSeq genes (NimbleGen, Reykjavík, Iceland).
Bisulfite DNA sequencing
CD4+ T cell DNA from an independent set of normal healthy controls was isolated and
treated with sodium bisulfite using the EZ DNA Methylation-Gold kit (Zymo Research,
Orange, CA). Sodium bisulfite treatment will convert unmethylated cytosine residues to
thymine, while methylated cytosine residues will remain as cytosines. Sodium bisulfite
treated DNA was amplified and directly sequenced (primer sequences are available
upon request). Percent methylation on each CG site was quantified using the Epigenetic
Sequencing Methylation analysis software (ESME®) 22.
Statistical analysis and methylation peaks identification
Signal intensity data were obtained and analyzed from each scanned array by
NimbleGen. The ratio of IP versus input DNA signals in each probe from each cohybridized sample is determined and the log2-ratio is computed and scaled to center the
ratio data around zero. Scaling is performed by subtracting the bi-weight mean for the
12
log2-ratio values for all features on the array from each log2-ratio value (complete scaled
log2-ratio data for all probes and all samples are available in online supplementary file
1). The -log10 p values are calculated for each probe by placing a fixed-length 750bp
window around each consecutive probe and the one-sided Kolmogorov-Smirnov (KS)
test is used to determine if the probes are drawn from a significantly more positive
distribution of intensity log2-ratios compared to those in the rest of the array. The
resulting score for each probe is the -log10 p value from the windowed KS test around
that probe. Methylation peaks are defined as regions with at least 2 probes with -log10 p
values of at least 2 within 500bp window, and a methylation score of at least 2. Peaks
that are within 500bp apart are merged. The methylation score for each peak represents
the average -log10 p value for the probes within that methylation peak. Complete
methylation peaks data for the autosomal chromosomes and chromosome X are
presented in online supplementary file 2.
Microarray CD4+ T cell expression and DNase I hypersensitivity data
Gene expression profile for human CD4+ T cells obtained from 10 normal healthy
participants were extracted from Gene Expression Omnibus (Lauwerys BR, et al.; GEO
accession: GSE4588). Expression profiling was performed using the Human Genome
U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Genes were considered expressed if
the mean normalized signal value in all 10 samples for at least 1 transcript was above
64. DNase I hypersensitive sites in normal human CD4+ T cells were extracted from the
UCSC Genome Browser and Tables 23.
13
Bioinformatic analysis
The number and density of CpG islands were determined algorithmically using the NCBI
Build 36.1 of the human genome (HG18). CpG islands were defined as a stretch of
DNA of at least 200bp with a C+G content of at least 50% and an observed/expected
CG dinucleotide frequency of at least 0.6.
Online Supplementary information
Supplementary file 1: Scaled log2-ratio data
Supplementary file 2: Methylation peaks data
Acknowledgments: This work was supported by the National Institute of Health Grant
Number P20-RR015577 from the National Center for Research Resources, Grant
Number R03AI076729 from the National Institute of Allergy and Infectious Diseases, the
Oklahoma Rheumatic Disease Research Core Centers; the Department of Veterans
Affairs; the University of Oklahoma Health Sciences Center; the Oklahoma Medical
Research Foundation.
The authors would like to thank Dr. J. Donald Capra, M.D. for his critical review of this
manuscript.
Conflict of Interest: None
14
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16
Figure Legends:
Fig. 1: Methylation enrichment signals represented as -log10 p value scores in CD4+ T
cell DNA from 5 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.
Fig. 2: (A) DNA methylation is significantly association with transcriptional repression in
CD4+ T cells (P<0.0001). About 27% of genes with promoter region methylation escape
transcriptional repression and are actively expressed. (B) Methylation peak density
normalized to tiled regions (peak/Mb) negatively correlates with chromosome length
(r2=0.34).
Fig. 3: Functional network analysis of methylated genes that are expressed in primary
human CD4+ T cells identified involvement in basic cell functions including cellular
signaling and interaction, and cellular growth and proliferation (A). Genes that are
methylated and non-expressed are functionally associated with antigen presentation,
cell mediated immune response, and humoral immune response (B).Gene Key: Solid
lines indicate direct interaction, dotted lines indicate indirect interaction. , an arrow from
17
A to B indicates that A acts on B, a line without an arrowhead indicates binding only,
and a line with a small vertical line at the end from A to B indicates A inhibits B. Grey
indicates genes that are methylated, white indicates genes that are not user specified
but incorporated into the network through relationships with other genes. Node shapes
are: square, cytokine; diamond (vertical), enzyme; diamond (horizontal), peptidase;
dotted rectangular (vertical), ion channel; solid rectangular (vertical), G-protein coupled
receptor; triangle, kinase; oval (horizontal), transcription regulator; oval (vertical),
transmembrane receptor; trapezoid, transporter; circle, other.
Fig. 4: A schematic representation demonstrating distinguishing features between
repressive and permissive DNA methylation. (A) Repressive methylation peaks are on
average 893±110bp upstream of the transcription start site of target genes, which are
characterized by a relatively lower maximum CpG island density. Chromatin is closed at
the transcription start site as indicated by the absence of DNase I HS sites. (B)
Permissive methylation peaks are further upstream from transcription start site
compared to repressive peaks, and are characterized by higher maximum CpG island
densities within promoter sequences of target genes. These methylation peaks fail to
maintain a closed chromatin configuration. This results in accessible chromatin at the
transcription start sites of target genes as evidenced by the presence of DNase I HS
sites, and gene expression. It is possible that these methylation peaks fail to efficiently
recruit transcriptional repressor complexes such as the MECP2-SIN3A-HDAC complex.
MECP2, methyl-CpG-binding protein 2; SIN3A, SIN3 homolog A; HDAC, histone
deacetylase.
18
Table 1: Methylation peak density (peak/Mb) in subtelomeric (up to 7Mb from each
telomere) and non-subtelomeric regions in each chromosome.
Chromosome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Methylation Peak Density
(Peak/Mb)
NonSubtelomeric
subtelomeric
Mean
SEM
Mean
SEM
101.5
6.2
44.3
3.4
175.1
8.3
45.6
3.4
62.1
9.0
31.4
1.6
142.3
4.4
25.3
2.7
120.4
5.8
28.8
2.5
146.3
10.0
36.1
4.1
188.9
9.2
46.7
4.0
167.1
9.1
35.5
0.8
101.2
7.1
63.9
5.1
189.9
10.0
50.2
5.4
121.6
10.2
39.2
2.9
116.7
7.6
35.5
2.4
270.6
15.2
39.2
3.7
87.1
6.5
38.6
2.8
65.9
4.4
38.2
2.4
149.0
6.6
92.0
6.8
113.1
10.3
55.6
3.3
127.3
11.8
52.0
5.3
111.7
8.7
89.8
9.7
106.1
7.4
43.0
4.1
164.6
6.5
74.6
10.0
160.5
12.5
76.4
4.1
132.8
9.9
69.0
7.3
19
P value
1.75E-06
6.83E-09
0.002106
8.37E-11
6.89E-09
2.04E-07
8.62E-09
6.84E-09
0.000393
3.39E-08
2.55E-06
2.06E-07
5.51E-09
7.93E-06
4.86E-05
2.47E-05
6.66E-05
3.10E-05
0.066
3.81E-06
3.35E-06
1.45E-05
8.47E-05