By
From the * Leukocyte Biology Unit and the Cancer Research Unit, The Queensland Institute of
Medical Research, Post Office Royal Brisbane Hospital, Queensland 4029, Australia; and the § Department of Microbiology, University of Queensland, St. Lucia, Queensland 4072, Australia
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Abstract |
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Differential genomic DNA methylation has the potential to influence the development of T
cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon
(IFN)- and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-
and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-
promoter was widespread across >300 basepairs in clones expressing high
levels of IFN-
mRNA, whereas demethylation of the IL-3 promoter was confined to specific
CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-
mRNA exhibited symmetrical methylation of four to six of the IFN-
promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-
gene methylation and mRNA expression in families of clones derived from
resting CD44lowCD8+ T cells or from previously activated CD44highCD8+ T cells. The patterns of IFN-
gene demethylation and mRNA expression were faithfully inherited in all
clones derived from CD44high cells, but variable in clones derived from CD44low cells. Overall,
these findings suggest that differential genomic DNA methylation, including differences among
cytokine genes, among individual T cells, and among T cells with different activation histories,
is an important feature of cytokine gene expression in primary T cells.
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Introduction |
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Differential cytokine gene expression in activated T lymphocytes plays an important part in the diversification of T cell cytokine production patterns and polarization towards type 1, type 2, and other profiles (1). The underlying molecular mechanisms are likely to operate combinatorially, involving a number of pretranscriptional, transcriptional, and posttranscriptional levels of control with multiple interacting factors at each level (5).
At the pretranscriptional/transcriptional level, the involvement of genomic DNA methylation in the regulation of differential cytokine gene expression has not yet been widely investigated. In other systems, methylation of the cytosine residue in CpG dinucleotides can be a negative regulatory influence on the expression of various inducible tissue-specific genes (8). Off-on effects occur when CpG methylation influences chromatin conformation and accessibility (11, 12). In addition, quantitative influences on gene expression are possible since methylation can differentially inhibit the binding of individual transcription factors, directly via the protruding methyl group or indirectly via methyl-CpG-binding proteins (13). Importantly, the methylation status of CpG sites can be heritable, thus contributing to epigenetic regulation of gene expression (16).
Only a few studies have examined the role of DNA methylation in cytokine gene expression in leukocytes. Inhibition of DNA methylation has been used to establish constitutive IL-2 secretion in EL-4 thymoma clones (17).
Reduced CpG methylation in the TNF- promoter has
been described in myeloid leukemia cells and differentiated
monocyte cell lines expressing TNF-
mRNA (18, 19).
Differences in IFN-
expression among long-term T cell clones, T cell lines, and primary human T cell populations
have been found to be associated with differential CpG
methylation in this gene (20). The latter important
findings are supported by data on methylation-mediated alterations in transcription factor-binding to the proximal
IFN-
promoter element (23). Whether heritable methylation patterns in the IFN-
gene can account for the stability of augmented IFN-
expression in memory/effector T
cells, for example, has not yet been tested.
Until recently, analysis of genomic DNA methylation has generally required relatively large amounts of DNA for methylation-dependent restriction endonuclease digestion followed by Southern blotting- or semi-quantitative PCR- based evaluation of restriction patterns. These approaches have been largely superseded by the development of a PCR- and DNA sequencing-based method for positive display of methylated cytosine residues: bisulfite genomic sequencing (24, 25). This method allows evaluation of the methylation status of all cytosine residues on both DNA strands, regardless of their restriction enzyme recognition context. In addition, PCR amplification of bisulfite-modified DNA from small numbers of cells allows clonal analyses (26).
In this investigation, we have applied bisulfite genomic
sequencing to analyze the methylation patterns of the
mouse IFN- and IL-3 genes during development of
short-term CD8+ T cell clones from normal animals. We
have also coupled the methylation studies to quantitative
competitive PCR (QCPCR)1 analyses of IFN-
and IL-3
mRNA expression. To our knowledge, this is the first report of this combination of analyses for any cytokine gene
in any type of leukocyte. The bisulfite sequencing results
extend the previous findings for one CpG site in the IFN-
promoter to cover all of the other IFN-
promoter methylation sites, and provide entirely new information for all of
the methylation sites in the IL-3 promoter.
The results show that, despite similar distributions and
densities of CpG methylation sites, the IFN- and IL-3
promoters exhibit differential demethylation in the same T
cell clone. The coupled methylation QCPCR results show
that demethylation of the IFN-
and IL-3 genes is intimately associated with differential expression of these two
cytokines in activated CD8+ T cell clones. The pattern of
IFN-
promoter demethylation is widespread and compatible with regulatory mechanisms based on altered chromatin conformation. In contrast, the pattern of IL-3 promoter
demethylation is more site specific and focussed on sequence elements and transcription factor-binding sites additional to those currently studied. Thus, differential methylation patterns among cytokine genes and among primary
T cell clones suggest that DNA methylation may be one of
the mechanisms that underpins the generation of diversity
in T cell cytokine profiles during primary immune responses. Furthermore, we show that both IFN-
gene methylation patterns and mRNA expression levels are heritable in the subclones derived from CD44highCD8+ (recently
activated or memory/effector) cells, but variable among the
offspring derived from CD44lowCD8+ (resting or naive)
cells. This indicates that heritable gene methylation patterns
correlate with CD44 and IFN-
expression and thus have
the potential to serve as molecular markers of memory/effector T cells. Together, these results suggest a role for gene
methylation in establishing and/or maintaining differential
cytokine gene expression patterns in primary T cells.
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Materials and Methods |
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Mouse Lymphocyte Preparation.
Specific pathogen-free female C57BL/6 mice (Animal Resources Centre, Murdoch, Western Australia) were used when 6-12 wk old. CD8+ T cells were isolated from pooled brachial, axillary, cervical, inguinal, para-aortic, and mesenteric LNs as previously described (27). In brief, LN cells were stained with PE-conjugated rat anti-mouse CD4 mAb (GK1.5), FITC-conjugated rat anti-mouse CD8 mAb (53.6-7), and 0.5 µg/ml propidium iodide. In some experiments, the staining with anti-CD4 mAb was substituted with biotinylated anti-CD44 mAb (IM7.8.1) followed by PE-conjugated streptavidin. Fluorescence-activated cell sorting was conducted using a FACS® Vantage flow cytometer with Lysis II software (Becton Dickinson, Sunnyvale, CA). Viable CD8+ cells were positively sorted and, upon reanalysis, were >97% pure in all experiments. In some experiments, the CD44low and CD44high subsets of the CD8+ population were also separated by sorting for the lowest or highest 15%, respectively, of the staining distribution. On a four-decade scale, the reanalyzed sorted CD44low cells were contained within the first decade with a mean fluorescence intensity of 4.6, whereas the sorted CD44high cells were within the third decade with a mean intensity of 192. For clonal cultures, single CD8+ T cells were then seeded directly into the wells of prepared (see below) 60-well Terasaki tissue culture plates (Nunc, Roskilde, Denmark) using the automated cell deposition unit of the flow cytometer.Accessory Cell-free CD8+ T Lymphocyte Culture.
Terasaki and 96-well flat-bottomed tissue culture plates were coated with a mixture of hamster anti-mouse CD3Secreted Cytokine Assays.
Supernatants harvested from bulk CD8+ T cell cultures after varying periods of stimulation were assayed for IL-3 by FDC-P1 reporter cell assay as previously described in detail (27). Appropriate controls demonstrated that the presence of 5-azacytidine did not interfere with this bioassay at the dilutions where titration end points were determined (data not shown). Supernatant IFN-Genomic DNA and Cytoplasmic RNA Extraction and cDNA Synthesis.
Nucleic acids were extracted using a combination of published methods (30). After supernatant removal, T cell clones were resuspended in 11 µl of lysis buffer (0.2 × PBS, 20 µg/ml oligo-dT [Boehringer Mannheim, Castle Hill, New South Wales, Australia], 1% NP-40, 4 mM dithiothrietol, 4 U/ml RNasin [Promega Corp., Madison, WI], 100 µg/ml tRNA [Boehringer Mannheim]) and snap frozen atBisulfite Modification of Genomic DNA.
Genomic DNA was bisulfite-treated using a method optimized for small cell numbers (26). In brief, extracted genomic DNA was sheared by pipetting and then denatured in 0.3 N NaOH for 20 min at 75°C. Fresh 4.8 M sodium metabisulfite (pH 5.0) was prepared by adding 4.55 g of Na2S2O5 and 0.4 ml of 10 N NaOH to 8.2 ml H2O and mixing gently. To each 22-µl sample of denatured genomic DNA, 250 µl of 4.8 M Na2S2O5, 14 µl of fresh 10 mM hydroquinone, and paraffin oil were added and the samples were incubated at 55°C, shielded from light, for 4 h. Modified DNA was then purified using Geneclean® kits (BIO 101, La Jolla, CA), and desulfonated in 0.3N NaOH at 37°C for 20 min. Desulfonated DNA was precipitated with ammonium acetate and ethanol, pelleted, washed with 70% ethanol, and resuspended in 20 µl H2O.PCR and Sequencing of Bisulfite-modified Genomic DNA.
Primers flanking CpG sites in the mouse IFN-
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Quantitative Competitive Reverse Transcriptase PCR Assay of Cytokine mRNA Levels.
Competitor plasmids bearing small deletions in the IFN- ![]() |
Results |
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Sequence analysis revealed several, mostly clustered, CpG methylation sites in the mouse IFN- and IL-3
promoters within ~300 bp of the transcription start site
(Fig. 1). Despite the low overall sequence conservation, 1-4
CpG sites ~40-60 bases upstream of the transcription start
site, were present in both genes of all species. Several of the
CpG sites overlapped with the binding sites of transcription
factors shown to be involved in the regulation of human
IFN-
or IL-3 promoter activity. The binding of some of
these factors, including the cyclic AMP response element
binding protein (CREB) and ETS members, has been
shown to be directly affected by cytosine methylation (13,
23, 38). Of all these CpG sites in these two genes, only the
53 site in the IFN-
promoter has hitherto been characterized for its methylation status in T cells (21, 22).
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The cytosine methylation inhibitor
5-azacytidine has previously been shown to increase the
expression of IFN- in long-term T cell clones and lines
(21, 39). Given the similar pattern of potential methylation
sites in the mouse IFN-
and IL-3 promoters (Fig. 1), we
tested the effect of this inhibitor on IFN-
and IL-3 expression in primary mouse CD8+ T cells activated in vitro
in a TCR-dependent, accessory cell-independent, solid-phase mAb system. Treatment with 5-azacytidine increased IFN-
expression up to 25-fold and IL-3 expression up to
14-fold in a dose-dependent manner, with different dose
optima for the two cytokines (Fig. 2). Within the dose
range shown, T cell proliferation was unaffected but 5-azacytidine doses >8 µM resulted in inhibition of proliferation and cell death and consequently cytokine levels below
those of untreated cultures (data not shown). Increased cytokine secretion in the 5-azacytidine-treated cultures was
maximal on day 2 (IFN-
) or 3 (IL-3), with trends towards
plateau levels at day 3 or 4, respectively (Fig. 2). These kinetics represent an acceleration of the expression of IFN-
and IL-3 in this culture system (27, 28). Thus, these results,
in support of the sequence analysis above, suggested a potential role for DNA methylation in regulation of expression of both the IFN-
and IL-3 genes in primary mouse
CD8+ T cells.
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To study comprehensively the potential
cytosine methylation sites in the mouse IFN- and IL-3
promoters, we used bisulfite genomic DNA sequencing to
display the methylation status of all of the cytosines on both
DNA strands of the promoter regions analyzed (Figs. 1 and
3). In both genes, cytosines in a CpG sequence context were virtually the only nonmodified (i.e., methylated) cytosines detected. Methylation at CpNpG sites (40) was encountered at <1% of all potential CpNpG sites and in
<5% of all samples. Consequently, >99% of non-CpG cytosines exhibited cytosine to thymidine conversion, thus internally controlling for the efficiency of the bisulfite modification and for PCR and sequencing artifacts. Repeated
bisulfite modifications, PCR, and direct PCR product sequencing of control mixes of methylated and nonmethylated molecules, similar to experiments reported previously
(25, 41), confirmed that this method reproducibly displays
the predominant methylation pattern of a heterogeneous population of molecules (data not shown). Coincident clear
cytosine and thymidine peaks (Fig. 3), indicative of possible
mixed or transitional CpG methylation status, were seen at
a frequency of <13% of the sites examined in the panel of
clones (see below). The methylation of CpG bps was usually symmetrical with identical patterns on the coding and
noncoding DNA strands in 80% of the sites tested. This result, derived from independent PCR reactions on separate
aliquots of modified genomic DNA from each clone, also
affirmed the reproducibility of the analyses. Symmetrical methylation of all of the predicted CpG sites in the coding
and noncoding strands of the IFN-
and IL-3 promoters
was observed in the control mouse FDC-P1 myelomonocytic cell line that does not express IFN-
or IL-3 (42, 43),
in bulk populations of normal nonstimulated spleen cells,
and in nonstimulated LN CD8+ T cells (Fig. 4 and data not
shown).
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Single CD8+ T cells were cultured with anti-CD3, anti-CD8, and anti-LFA-1 mAbs in
the presence of IL-2 for 4-5 d, until clones of 32-256 cells
had formed. The methylation status of the IFN-
and IL-3
promoters in individual clones was then examined by
bisulfite genomic sequencing. In both the IFN-
and IL-3
promoters, clonal heterogeneity was seen with >30 distinct
patterns recorded in the panel of 40 clones (Fig. 4 and data
not shown). The methylation patterns in the two genes
were not related to each other or to clone size, which is an
approximation of division number. In the IFN-
promoter, the patterns ranged from the two extremes of methylation of nearly all sites on the coding and noncoding
strands (e.g., Fig. 4, clone 1414), to demethylation of all 14 of these cytosines (e.g., Fig. 4, clone 1386). Contrastingly,
in the IL-3 promoter, demethylation of all sites was never
seen. Instead, site-specific differences predominated, ranging from high-frequency demethylation of one or both of
the coding or noncoding strand cytosines at the
164 CpG
site, to low-frequency demethylation of both cytosines at
the
52 site. In both the IFN-
and IL-3 genes, demethylation of the CpG site immediately downstream of the
transcription start site was infrequent. It was concluded that
there was differential methylation of these two cytokine
genes in the same T cell clone, and that the methylation
patterns of the two genes were not closely related to clone
size. Therefore, these results were consistent with a clonally
variable, division-independent demethylation process, affecting these two cytokine genes differentially.
To disclose links between promoter methylation patterns and transcriptional activity, the bisulfite genomic methylation analysis above was coupled to QCPCR measurements of cytokine mRNA levels. In this way, parallel steady state genomic DNA and cytoplasmic mRNA data were derived for each clone at the time of cell lysis.
The QCPCR results for the panel of clones showed that,
by 4-5 d of activation, approximately half of the clones had
detectable levels of IFN- and/or IL-3 mRNA that ranged
over at least two (IL-3) or four (IFN-
) orders of magnitude (Fig. 5). The frequency of IFN-
mRNA expression
after 4-5 d was higher than that of IL-3 mRNA at these
early times. In comparison, clones in the 4-32 cell stage analyzed after 2-3 d of stimulation expressed IFN-
mRNA at a lower frequency, and IL-3 mRNA expression was usually undetectable (data not shown). Correlations between
mRNA levels and clone size (Fig. 5, A and B) were not
significant (r <0.5). However, the probability of detection
of IFN-
or IL-3 mRNA was significantly higher in clones
larger than 256 cells, and clones with detectable IL-3
mRNA almost always coexpressed IFN-
mRNA (Fig. 5 C).
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These data concurred with many of the previous reported findings on the kinetics and relative levels of IFN-
and IL-3 protein expression by primary mouse CD8+ T
cell clones (27, 28, 44, 45), and supported the choice of this
time frame for study of primary CD8+ T cells during an
early phase of in vitro development, when about half the
clones had initiated cytokine mRNA expression.
Comparing the methylation and mRNA data for
individual clones revealed a striking overall association between demethylation of the IFN- promoter and expression of mRNA (Fig. 6). Clones lacking detectable levels of
IFN-
mRNA exhibited relatively dense methylation of
8-13 of the CpG cytosines examined on the two DNA strands of the IFN-
promoter. In contrast, the majority of
IFN-
mRNA-positive clones exhibited multiple and
sometimes uninterrupted demethylation of all CpG sites in
the IFN-
promoter between positions
210 and +1. A
small number of clones (e.g., clones 1417, 1427, and 1408, Fig. 6) expressed relatively low levels of IFN-
mRNA and
retained symmetrical or hemi-methylation of most of the
CpG sites.
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Although these promoter-wide or regional demethylation patterns were pronounced, clone- and site-specific differences across the IFN- promoter were also evident. For
the panel as a whole, symmetrical or hemi-demethylation
of the
45 and
34 CpG sites occurred with the highest
frequency, whereas the site at position +17 exhibited the
lowest frequency of demethylation, yet the status of these
sites was not related to IFN-
mRNA levels. In contrast, the
205,
191, and
53 CpG sites were symmetrically
methylated in most IFN-
mRNA-negative clones, and
their demethylation was closely correlated with expression
of >103 U of IFN-
mRNA. As shown in Fig. 1 A, these
three CpG sites are adjacent to conserved activator protein 1 (AP-1) and activating transcription factor (ATF)/CREB
transcription factor-binding sites.
In the same panel of CD8+ T cell clones, the patterns of
DNA methylation and mRNA expression for IL-3 were
distinctly different from IFN-. Although clones lacking
detectable levels of IL-3 mRNA again exhibited relatively
dense methylation of 8-12 of the CpG cytosines examined
on both strands of the IL-3 promoter from bases
270 to
+30, widespread demethylation of multiple CpG sites across the IL-3 promoter was never seen (Fig. 7). Among
the IL-3 mRNA-negative clones, all of the CpG sites except position
164 exhibited high-frequency methylation.
In the IL-3 mRNA-positive clones, symmetrical demethylation of the
164 CpG site was frequent (10 out of 12 clones, or 83%) and was linked to a slightly higher frequency of symmetrical or hemi-demethylation of the
62
CpG site. However, symmetrical demethylation of the
164 CpG site in the IL-3 promoter also occurred in a
small proportion (6 out of 18, or 33%) of IL-3 mRNA-negative clones. As shown in Fig. 1 B, the
164 CpG site
abuts a GATA transcription factor-binding site that is not
conserved in primates, whereas the
62 site overlaps with
a partially conserved Sp1-binding site.
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We have previously presented evidence for the inheritance of cytokine production
patterns in families of subclones of CD4+ and CD8+ T
cells from normal mice (27, 29). Therefore, we tested
whether the IFN- gene methylation patterns and levels of
mRNA expression described above were heritable characteristics of CD8+ T cell clones, and whether this would
vary for T cells at different stages of in vivo differentiation.
To this end, LN cells from normal mice were sorted into
CD8+CD44high or CD8+CD44low subpopulations to enrich
for previously activated or resting T cells, respectively (46-
48). Single cell cultures were initiated as described above
and, after 4 d, the resultant parent clones were subcloned
by micromanipulation. Parent and progeny subclones were
harvested 2-3 d later, thus allowing clonal expansion for two to six divisions under the stimulation conditions used.
Fig. 8 shows that, in families derived from CD44high
CD8+ T cells, most of the CpG sites in the IFN- promoter were demethylated in all of the parent clones and
their subclones, all of which expressed high levels of IFN-
mRNA. These results corroborated our earlier results on
the close association between regional promoter demethylation and high IFN-
mRNA expression (Fig. 6), and are
consistent with the well-described augmented IFN-
expression status of CD44high T cells (49, 50). In contrast,
more variability in IFN-
gene methylation and expression
was seen among and within the families derived from
CD44low cells. Interestingly, this variability was less marked
for the CpG sites at positions
205,
191, and
53,
whose status was shown (Fig. 6 and see above) to correlate
most closely with IFN-
mRNA expression. As a result,
the previously determined relationships between methylation pattern and mRNA expression (Fig. 6) were largely
maintained in this panel. In addition, partial methylation at
certain sites was perpetuated in some related siblings. This may reflect the maintenance of a balance between methylation and demethylation similar to a previously reported
phenomenon for the adenosine phosphoribosyltransferase
gene in a panel of teratocarcinoma stem cell lines (51).
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These data indicate that demethylation of multiple sites
in the IFN- promoter is different in clones derived from
the CD44high and CD44low subsets of mouse CD8+ T cells,
and that these differences, in conjunction with quantitatively distinct levels of IFN-
expression, can be passed on
to the progeny of individual T cell clones.
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Discussion |
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The above results demonstrate that cytokine gene methylation exhibits many of the features of a mechanism that can regulate two T cell cytokine genes differentially, with heterogeneity between individual T cell clones, and with heritability in differentiated T cells with the CD44high phenotype. Therefore, these findings are germane to our understanding of the diversification of cytokine profiles in primary T cells, the maintenance of established cytokine expression patterns in differentiated T cells, and the molecular mechanisms involved in cytokine gene regulation.
This investigation was initiated upon finding that there
was remarkable similarity in the potential CpG methylation
sites of the mouse IFN- and IL-3 promoters, and that
many of these sites overlapped with binding sites for transcription factors known to be directly affected by CpG methylation (Fig. 1; references 13, 38). These sequence-based
inferences were supported by experiments with the cytosine
methylation inhibitor 5-azacytidine, in which 5-azacytidine increased and accelerated both IL-3 and IFN-
expression in bulk cultures of CD8+ T cells (Fig. 2). This result confirms previously reported results for the effect of
5-azacytidine on IFN-
expression in long-term mouse
CD4+ T cell clones or primary human CD4+ T cells (21,
22), and extends them to both IFN-
and IL-3 in primary
mouse CD8+ T cells.
However, 5-azacytidine is a toxic agent and it is difficult
to distinguish its direct methylation-mediated effects from
other indirect actions, particularly in proliferating polyclonal cell populations. It is also thought that it acts via division-dependent interference in maintenance methylation,
which is unlikely to resemble normal demethylation mechanisms (8, 9, 52). Therefore, we undertook experiments to
characterize IFN- and IL-3 gene methylation, demethylation, and mRNA expression events as they occurred during the in vitro development of cytokine-producing T cells.
Our approach incorporated use of (a) primary mouse
CD8+ T cells analyzed during the early stages of growth
and cytokine expression; (b) solid-phase anti-CD3
, anti-CD8, and anti-LFA-1 mAb to stimulate high frequencies
of T cells from normal mice in a TCR- and accessory molecule-dependent manner; (c) accessory cell-free conditions
to eliminate non-T cell sources of DNA and mRNA; (d)
clonal cultures to disclose the degree of heterogeneity obscured in bulk culture studies, allow visual quantitation of cell proliferation, and permit analyses of heritability by subcloning; (e) bisulfite genomic DNA sequencing to map
comprehensively the sites of cytosine methylation on both
strands of the IFN-
and IL-3 promoters in small numbers
of cells; and (f) sensitive competitive PCR-based quantitation of mRNA levels in small numbers of cells. This approach was thus designed to reveal the full range of cellular and molecular potentialities of the primary developing
CD8+ T cell clones under study.
Clonal heterogeneity, in the levels and combinations of
cytokines produced, is a prominent feature of cytokine expression in primary T cells (2, 4, 53). Broad heterogeneity
in IFN- and IL-3 gene CpG methylation patterns was
seen in the panel of clones examined in this study (Figs. 4,
6, and 7). Indeed, when the methylation patterns of all the
sites in the two genes were considered together (e.g., Fig.
4), nearly every clone in the panel possessed a unique pattern. These results showed that there was not a uniform
demethylation pathway or program for each gene in all
cells, or for two sequences with similar CpG arrays such as
the IFN-
and IL-3 promoters, even when both cytokines were expressed (e.g., Figs. 6 and 7; clones 1383, 1412, 1415, and 1418). Instead, differential demethylation of the
two induced genes occurred in the same T cell clone. Nevertheless, the methylation status of individual CpG sites
studied here was not entirely autonomous or independent
since apparent linkages between the status of neighboring CpG sites in the IFN-
and IL-3 promoters were seen
(Figs. 6 and 7). This may, in part, reflect maintenance methylase activity since this enzyme, while restoring symmetrical methylation to hemi-methylated CpG sites, has also
been found to initiate de novo methylation of neighboring
symmetrically nonmethylated CpG cytosines (54, 55).
Regional or site-specific demethylation of the IFN- or
IL-3 promoters, respectively, was associated with different
levels of the mRNAs derived from these genes. In particular, there was a striking association between promoter-wide
demethylation of the IFN-
gene and expression of high
levels of IFN-
mRNA (Fig. 6). However, large differences in demethylation frequencies of CpG sites in IFN-
mRNA-negative clones also occurred and revealed a subset
of CpG sites, including the previously characterized
53
site, whose methylation was closely associated with the absence of detectable IFN-
mRNA. Similarly, site-specific
effects in the IL-3 promoter were pronounced with most
of the demethylation activity focussed on the
164 CpG
site, which was almost invariably demethylated in IL-3
mRNA-positive clones (Fig. 7). A small proportion of
clones possessing a demethylated
164 CpG site lacked
detectable IL-3 mRNA, suggesting that demethylation at
this site may precede mRNA expression and/or that demethylation of this site alone may be either unnecessary or insufficient for IL-3 gene expression in some T cells.
Together, the above findings justify several levels of studies required to elucidate the role of DNA methylation in the regulation of cytokine gene expression in T cells. The dissection of methylation-mediated regulatory mechanisms is an intensely active field that is currently in a state of flux, with many long-standing views being reconsidered in light of unanticipated new information (10, 15, 52, 55). We believe that similar reconsideration arises from comprehensive bisulfite sequencing methylation analysis of clonal cultures of primary cells.
First, the above mapping in primary T cells of the site-specific CpG differences that correlate with specific levels
of mRNA expression, and with known transcription factor
binding sites, has identified a revised set of sites and transcription factors to be probed for direct methylation-dependence. One study has been conducted for the 53 CpG
site in the human IFN-
promoter where methylation was
shown to affect directly the binding of the factors CREB,
ATF-2, and the c-jun component of AP-1 (23). The results
presented here indicate that similar studies are warranted for the
205 and
191 CpG sites in the mouse IFN-
promoter and the
164 and
62 sites in the mouse IL-3
promoter. Moreover, the transcription factors to be assayed
can be provisionally extended to include two members of
the GATA family, YY1 (which can interact directly with
CREB and ATF members; reference 56) and Sp1. The factors YY1 and Sp1 are reportedly unaffected by CpG methylation in some sequence contexts (13, 38), but, to our
knowledge, the sensitivity of members of the GATA family
has not yet been reported. Conversely, recent description
of the unanticipated creation of an AP-1 factor-binding site
by CpG methylation within a nonconsensus AP-1 sequence (57) underlines the importance of further analyses
at this level.
Second, similar transcription factor-binding studies will
also need to be conducted in the presence or absence of
proteins such as MeCP2 and histone H1 (13, 14). These
well-characterized ubiquitous proteins can bind methylated
cytosines in any sequence context and indirectly inhibit
transcription factor-binding in a competitive manner or via
active repression (15). These effects are methylation density
dependent (58, 59) and compatible with the clustered distribution of the key CpG sites in the IFN- and IL-3 promoters.
The third level of studies requires analyses of the effect of
gene methylation on chromatin structure. Several recent
reports show that a medium to high density of methylated
CpG sites, in the presence of MeCP2 or histones, can promote the formation of a higher-order nucleosome-like
structure that is inaccessible to DNase I and is transcriptionally silent (15, 60, 61). The regional patterns of methylation and promoter-wide demethylation that we have described here for the IFN- promoter are also compatible with the involvement of such a mechanism in the switch
between undetectable and high-level IFN-
mRNA expression.
The results of these three substantial levels of study will determine to what extent DNA methylation-mediated mechanisms are necessary and/or sufficient for regulation of expression of different cytokine genes in T cells. Moreover, the results we have presented here suggest that conclusions on the necessity or sufficiency of individual mechanisms may need to be qualified according to the level of gene expression and the phenotype of the T cell (see below). The potential regulatory diversity arising from this pretranscriptional/transcriptional level is likely to be great given the differences described here between two cytokine genes with similar CpG site densities and distributions, the broad range of promoter CpG methylation patterns in other cytokine genes expressed by T cells, and the possible contributions from methylation in distant enhancer elements (reference 62 and Fitzpatrick, D.R., unpublished data).
Equally important is investigation of the pathways that
regulate methylation and demethylation processes. Two reports have linked activation of the Ras signaling pathway to
DNA methylation or demethylation activities (63, 64), and
a role for the transcription factor NF-B in demethylation
of the immunoglobulin
gene in a B cell line has also been
shown (65). Since the former pathway would tend to link
cytokine gene demethylation to activation of the TCR,
whereas the latter molecular mechanism may be more dependent on costimulatory signals (66), it will be pivotal to
determine which signals are involved in the induction of
cytokine expression in T cells. We have recently shown
that activation of the extracellular signal-regulated kinase
branch of the Ras-activated cascade is required for maximal
TCR-mediated IFN-
and IL-3 expression in primary
mouse CD8+ T cells (67, 68). Although these bulk culture
studies are not directly comparable to the clonal studies described here, it will be interesting to determine whether
this signaling pathway exerts some of its downstream effects
through cytokine gene demethylation.
The importance of the above multi-faceted investigations is emphasized by the experiments herein, which characterize the heritability of IFN- methylation patterns and
mRNA expression levels in T cells at different stages of in
vivo differentiation: previously activated (also referred to as
memory/effector) CD44high cells and resting (or naive)
CD44low cells. In clones derived from CD44high T cells, regional IFN-
promoter demethylation and high-level expression of IFN-
mRNA were inherited characteristics
shared by all parent and progeny clones. In contrast, in
clones derived from CD44low T cells, IFN-
gene methylation and expression patterns were variable both between
and within different families of clones. Therefore, over the
two to six cell division time frame of these studies, the
IFN-
promoter CpG methylation pattern and the fidelity of its vertical transfer were closely associated with both the initial surface marker phenotype of the parental T cells and
with the final IFN-
expression phenotype of both the parent and progeny clones.
This result is germane to four sets of previously published work. First, it confirms and extends the report that
the 53 CpG site in the human IFN-
promoter may
be preferentially demethylated in the recently activated
CD45RAlow/CD45ROhigh subset of primary human CD4+
T cells (22). Second, it corroborates our previous findings on similar kinetic patterns of GM-CSF and IL-3 expression
in the progeny of some CD4+ T cells (27), and on the retention of IFN-
mRNA expression by the progeny of
some CD8+ T cells (29). Third, it compares favorably with
the previously described flexibility of resting or "naive"
CD44low T cells in contrast to the more restricted differentiative potential of previously activated or "memory/effector" CD44high T cells (4, 29, 50). Fourth, it supports earlier
studies on the heritability of methylated CpG sites in long-term cell lines transfected with plasmid or viral DNA (69,
70), and the variable fidelity of this process in some primary
cells (51, 71, 72). Our data add to each of these aspects by
demonstrating differential short-term heritability of multiple contiguous methylation sites in the IFN-
gene, an endogenous induced gene, and linking this difference to the
surface marker phenotypes of two subpopulations of primary CD8+ T lymphocytes.
Thus, differences in cytokine gene methylation represent a molecular correlate of other well-characterized markers of T cell differentiation. Whether these correlations are preserved over longer periods and in vivo, for example during antigen stimulation followed by a return to quiescence (48), merits additional study. Whether DNA methylation can reflect, establish, and/or maintain the commitment or reversibility of T cell cytokine expression patterns, particularly under polarizing priming or restimulation conditions (73), is also worthy of further investigation.
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Footnotes |
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Address correspondence to David R. Fitzpatrick, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, QLD 4029, Australia. Phone: 61-7-3362-0379/0380; Fax: 61-7-3362-0105; E-mail: davidf{at}qimr.edu.au
Received for publication 22 September 1997 and in revised form 23 March 1998.
We thank Grace Chojnowski for expert FACS® assistance; David Sester and Daphne Macaranas for contributions to the early stages of this work; Macky Edmundson for invaluable help with automated DNA sequence analysis; and Dr. Sue Clark for constructive comments on the manuscript.
This work was supported by the National Health and Medical Research Council of Australia, the Queensland Cancer Fund, the Clive and Vera Ramaciotti Foundations, and the Queensland Institute of Medical Research Trust.
Abbreviations used in this paper AP, activator protein; ATF, activating transcription factor; CREB, cyclic AMP response element binding protein; QCPCR, quantitative competitive PCR.
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