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Promoter at CpG and Non-CpG Sites Underlie Differences in IFN- Gene Expression Between Human Neonatal and Adult CD45RO- T Cells1
TVW Telethon Institute for Child Health Research and Centre for Child Health Research, University of Western Australia, Perth, Western Australia, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is a potent pleiotropic Th1 cytokine, the production of
which is tightly regulated during fetal development. Negative control
of fetal/neonatal IFN- production is generally attributed to the
Th1-antagonistic effect of mediators produced by the placenta, but
evidence exists of additional and more direct transcriptional
regulation. We report that neonatal (cord blood)
CD3+/CD45RO- T cells, in particular the
CD4+/CD45RO- subset, are hypermethylated at
CpG and non-CpG (CpA and CpT) sites within and adjacent to the IFN-
promoter. In contrast, CpG methylation patterns in cord blood
IFN--producing CD8+/CD45RO- T cells and
CD56+/CD16+/CD3- NK cells did not
differ significantly from those in their adult counterparts. Consistent
with this finding, IFN- production by stimulated naive cord blood
CD4+ T cells is reduced 5- to 10-fold relative to adult
CD4+ T cells, whereas production levels in neonatal and
adult CD8+ T cells are of a similar order. Evidence of
significant CpA and CpT methylation was not discovered in promoter
sequence from other cytokines (IL-4, TNF-
, or IFN-R
-chain). We additionally demonstrate that overexpression of
DNA methyltransferase 3a in embryonic kidney carcinoma cells is
accompanied by CpA methylation of the IFN-
promoter. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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has a variety of important roles in the maintenance of immunological
homeostasis, in particular in the activation of a range of
Th1-associated cellular immune functions that are central to host
defense against viral and bacterial infections. However, excessive or
prolonged production of IFN- can also contribute to the pathogenesis
of inflammatory diseases as a direct result of its toxic effects on
host tissues or via its role in activation of cytotoxic effector cells
such as macrophages (1).
Prime examples of the dualistic effects of this pleiotropic cytokine
are its activities in relation to fetal development. At one extreme,
IFN- plays a key role in implantation via regulation of arteriolar
remodeling and decidualization (2). In contrast, excessive
production of IFN- at the feto-maternal interface, resulting from
infections or allogeneic interactions, is a major cause of fetal loss,
as a consequence of its deleterious effect on placental integrity
(3, 4). These contradictory findings suggest that fine
control of IFN- gene transcription may be even more important during
fetal life than at later ages.
Consistent with this idea, recent research indicates that biasing of
adaptive immune function in the fetus toward production of
Th1-antagonistic Th2 cytokines is an evolutionary adaptation common to
all mammalian species studied, and furthermore, that this biasing is
orchestrated principally via selective down-regulation of IFN-
production (3, 5). The degree of this down-regulation in
humans is profound, as neonatal lymphocytes produce 10-fold less
IFN- than adult cells when optimally stimulated using PMA and Con A
(6).
The precise mechanism(s) underlying this Th2 polarization of the fetal
response are not fully understood, but production within the placenta
of a range of effector molecules, including IL-4, IL-10,
PGE2, and progesterone, which down-regulate Th1
function via preferential up-regulation of Th1-antagonistic Th2
differentiation (3, 7, 8, 9, 10, 11), is believed to be central in
this process. While these "bystander" mechanisms appear likely to
play a significant role in damping IFN- production at the
feto-maternal interface, it is less plausible to consider that they are
alone responsible for controlling this potentially lethal process.
Instead, given the fundamental importance of this protective mechanism
for fetal survival in the face of the constant danger of Th1-inductive
feto-maternal allogeneic interactions during gestation, it is
reasonable to hypothesize that evolution may have selected for
additional safeguarding mechanisms for control of IFN- gene
expression during this life phase. A prime candidate mechanism in this
context involves methylation of specific CpG sites within the IFN-
gene, in particular within the promoter region.
A wide body of research (reviewed in Refs. 12, 13, 14)
indicates that CpG methylation in mammals is an important mechanism by
which transcription from specific gene loci is regulated in different
cell types. Some recent findings indicate that CpG methylation
contributes to regulation of IFN- gene transcription in the mouse,
and the changes associated with Th1 cell function are also accompanied
by chromatin modification. Firstly, a study of naive and memory
CD8+ T cells demonstrated uncoupling of IFN-
transcriptional control using the methylation inhibitor
5-aza-2'-deoxycytidine (15). Clear evidence presented in
that study showed that CD8+ T cell clones with an
unmethylated IFN- promoter produced 1000-fold more IFN- message
than cells with hypermethylated promoters. In subsequent experiments
(16) long term stable inheritance of an unmethylated
IFN- promoter was demonstrated in murine
CD44high (memory) CD8+ T
cell clones, even after withdrawal of stimulation in culture and return
of IFN- transcription to undetectable levels. 5-Aza-2'-deoxycytidine
treatment has also been demonstrated to restore IFN--production
capacity to murine CD4+ Th2 clones
(17).
In addition to these observations in mice, increased IFN- gene
transcription has been correlated with hypomethylation of the proximal
promoter region in human Th1 cell clones, and hypermethylation has been
demonstrated to inhibit nuclear factor binding to the human IFN-
promoter (17). Of particular interest here are earlier
investigations that used methylation-sensitive restriction mapping to
identify the methylation status of a single SnaBI site in
the proximal IFN- promoter in adult human CD4+
and CD8+ cells (18). In addition,
treatment of human neonatal naive T cells with 5-aza-2'-deoxycytidine
was shown to markedly up-regulate their capacity to produce IFN-
(19). It is also of interest to note that the
down-regulation of IFN- gene expression that follows HIV-1 infection
of human T cells is associated with CpG hypermethylation in the IFN-
promoter, and that treatment of infected cells with an antisense DNA
methyltransferase construct reverses the hypermethylation and markedly
increases IFN- production (20).
The study presented below further investigates the hypothesis that
epigenetic mechanisms involving DNA methylation play an important role
in maintaining the low IFN- phenotype characteristic of human
neonatal T cells. Our results suggest that an intricate relationship
exists between transcription from the human IFN- promoter and the
effects of DNA methyltransferase activity (levels of CpG and non-CpG
methylation), and further, that this relationship differs between
IFN--producing cell populations in neonates. In addition,
experiments employing embryonic kidney cells expressing DNA
methyltransferase 3a
(Dnmt3a)3 demonstrated
patterns of CpA methylation in the IFN- gene that were comparable to
those observed in T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cord blood and adult peripheral blood samples used during this study were collected with prior approval for the study from the Princess Margaret Hospital human ethics committee. PBMC and cord blood mononuclear cells were isolated from whole blood (diluted 50% (v/v) in RPMI medium) over a Lymphoprep gradient at room temperature and 900 relative centrifugal force for 30 min. Purification of cell types of interest was conducted by either immunomagnetic separation employing Dynabeads (Dynal Biotech, Lake Success, NY) or preparative flow cytometry. Magnetic separation using Ab-coated Dynabeads was performed according to the manufacturer’s instructions, except that the buffer employed was 1x PBS, 2% (v/v) FCS, 0.6% (w/v) citrate, and gentamicin (50 µg/ml). In addition, cord blood samples were depleted of nucleated red cells with mAb anti-glycophorin A (Immunotech, Westbrook, ME; clone 11E4B7.6). The final CD45RO- T cells for immediate genomic DNA isolation were prepared by CD45RO depletion (DAKO, Carpenteria, CA; clone UCHL1, CD45RO-FITC; Dynabeads M-450 goat anti-mouse IgG), and CD3 selection (Dynabeads M-450 CD3 pan T). Preparative flow cytometry was performed by standard methodology to >95% purity on cord blood and adult blood mononuclear cells after initial gating on a forward/side scatter profile of medium-sized nongranular lymphocytes.
Bisulfite treatment, PCR, and sequencing
Genomic DNA in the size range of 20–30 kb was prepared from
1–2 x 106 isolated T cells using a QIAamp
DNA Blood Mini Kit (Qiagen, Valencia, CA). The genomic DNA
(
1–2 µg from 1 x 106 cells) was
bisulfite-treated using established procedures (21).
Briefly, 1 µg genomic DNA in 100 µl water was denatured at 37°C
for 15 min with 20 µl freshly prepared 1.8 M sodium hydroxide (NaOH).
Freshly prepared sodium bisulfite (pH 5 with 10 M NaOH) was added
directly (3.1 M final concentration) with hydroquinone (0.5 mm final
concentration), and sulfonation was allowed to proceed at 55°C for
18–24 h under mineral oil. The reaction was recovered and diluted in 1
vol ethanol and 1 vol of water and purified again using a QIAamp DNA
Blood Mini Kit (Qiagen) before alkali desulfonation (0.3 M NaOH) at
37°C for 15 min. Finally, the bisulfite-treated DNA was
ethanol-precipitated as described previously (22) with 20
µg glycogen (Roche, Indianapolis, IN) before resuspension
in 20 µl water for PCR amplification.
Three nested sets of forward and reverse primers were designed against
coding and noncoding strand bisulfite-treated DNA for each gene of
interest. The separate primer sets were tested using the PCR conditions
outlined below, and the most efficient primer sets for each gene were
preserved for later use (Table I
). The
IFN- promoter (GenBank accession no. J00219) noncoding region
primers amplified a 526-bp DNA from nt 21–546, and coding
region primers amplified a 550-bp DNA from nt 21–570. The IL-4
promoter region (GenBank accession no. M23442) noncoding primers
amplified a 600-bp DNA from nt 621-1220, and coding region primers
amplified a 620-bp DNA from nt 611–1,230. The IFN- receptor
-chain (IFNGR1) CpG island promoter region (GenBank accession no.
AL050337) noncoding strand primers and coding strand primers amplified
the same 462-bp DNA from nt 80,872–81,411. The TNF- promoter region
(GenBank accession no. X02910) coding primers amplified a 648-bp DNA
from nt 51–698.
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, IL-4, TNF-, and IFNGR1 promoter
were used in PCR at 300 nM with 1.5 U PLATINUM Taq DNA
polymerase (Invitrogen, Carlsbad, CA), 10–100 ng
bisulfite-treated genomic DNA, 1.5 mM MgCl2, and
200 µM dNTPs. The cycling parameters were 96°C for 3 min and
30 s (1 cycle); 96°C for 15 s, 59°C (decreasing by
1°C/cycle) for 30 s, 72°C for 1 min (9 cycles); 96°C for
15 s, 50°C for 30 s, and 72°C for 1 min (increasing by 5
s/cycle; 30 cycles); and 72°C for 7 min (1 cycle). The PCR product
DNA was cloned directly into pCR2.1 plasmid using an Original TA
Cloning Kit (Invitrogen, San Diego, CA), and plasmid DNA was prepared
for automated sequencing using a QIAprep Spin Miniprep Kit (Qiagen).
Sequencing of 500 ng plasmid DNA was performed with T7 primer (5'-TAA
TAC GAC TCA CTA TAG GG) and BigDye Terminator Ready Reaction Mix (PE
Applied Biosystems, Foster City, CA) according to the manufacturer’s
instructions, except that the cycle-sequencing PCR parameters were
varied to compensate for AT-rich bisulfite-treated DNA using an
annealing temperature of 45°C and extension at 55°C. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-4, TNF-, and IFNGR1 genes
Fig. 1A illustrates the
relative positions of CpG dinucleotides in the human IFN- promoter
compared with the promoters of IL-4, TNF-, and IFNGR1. The
arrangement of symmetrical CpG dinucleotides in the IFN- and IL-4
promoters is sparse compared with the CpG island occurring at the
IFNGR1 promoter and the intermediate frequency of CpG sites in the
promoter of TNF-. Human studies have shown that a hypomethylated
human IFN- promoter results in transcriptional up-regulation
(18, 23), and, conversely, a hypermethylated IFN-
promoter can disrupt nuclear factor binding (17, 24). The
positions of CpG sites that, when methylated, may be potentially
disruptive to nuclear factor binding are identified in Fig. 1B.
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promoter is hypermethylated at CpG and non-CpG
sites in cord blood CD3+/CD45RO- T cells
The IFN- promoter methylation status of
CD3+/CD45RO- T cells was
investigated in neonatal cord blood and adult peripheral blood. Fig. 2A presents a schematic map of
the 526-bp IFN- promoter region that was examined by sequencing
bisulfite-treated and cloned genomic DNA from different randomly
selected individuals. Between 9–12 reverse strand sequences were
scored for their methylation profile at CpG sites -295, -186, -54,
+122, +128, and +171 relative to the start of transcription. During the
scoring of methylated CpGs in the IFN- promoter from cord blood in
particular, a number of apparently methylated cytosines were observed
in the context of CpA (positions -290, -254, -252, -240, -209, and
-2) and CpT (positions -169 and -2) sites.
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promoter of cord blood
CD3+/CD45RO- T cells was
hypermethylated at the three CpG sites 5' to the start of transcription
(-295, -186, and -54) and at an additional three sites downstream
(+122, +128, and +171; Fig. 2
B). Bisulfite sequence analysis
of both DNA strands from the IFN- promoter confirmed the symmetrical
nature of CpG methylation in DNA sequences from cord blood cells (data
not shown). Bisulfite treatment of genomic DNA resulted in DNA strands
that were no longer complementary, and as such, an early decision was
made in this study to primarily analyze data from the noncoding
strand.
Bisulfite sequencing of the IFN- promoter from adult
CD3+/CD45RO- T cells
revealed variable, but consistently lower, levels of methylation at CpG
sites relative to the results for cord blood T cells (Fig. 2C). The measured difference in CpG methylation at the
IFN- promoter of cord blood and adult
CD3+/CD45RO- T cells was
highly significant at positions -295, -186, -54, +122, and +128
(p < 0.01–0.001) by a Fisher’s exact test of
adult/cord vs methylated/unmethylated contingency table for each
CpG site.
The scoring of IFN- promoter non-CpG methylation was almost entirely
restricted to specific CpA sites (positions -290, -254, -252, -240,
-209, and -2) and CpT sites (positions -169 and +42) in the DNA
sequence. There were a number of potential CpA and CpT sites in the
IFN- promoter reverse strand, but clearly, methylation activity
appeared specifically at a limited number of them. We measured a highly
significant differential in IFN- promoter CpA and CpT methylation
between cord blood and adult naive T cells (p
< 0.0001; refer below to Table II), but
found little evidence of previously reported non-CpG methylation at
CpNpG sites in the IFN- promoter (25).
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promoter is hypermethylated at CpG and non-CpG
sites in CD4+/CD45RO- T cells
To further characterize the level of CpG methylation at the
IFN- promoter of adult and neonatal naive cells we purified
CD4+, CD8+ T cells and
CD56+/CD16+ NK cells from
selected individuals by preparative flow cytometry and immunomagnetic
separation. The analysis of IFN- promoter methylation profiles in
these purified IFN--producing cell types revealed that
CD4+ naive T cells were significantly
hypermethylated in cord blood and hypomethylated in adult peripheral
blood (Fig. 3A). In contrast,
there was no significant difference between the IFN- promoter CpG
methylation status of cord blood and that of adult
CD8+ T cells or NK cells (Fig. 3, B
and C). Cytosine methylation at non-CpG sites was also
scored for these cell types, employing the sequences analyzed in Figs. 2 and 3, and is illustrated in Table II. Methylation of IFN-
promoter CpA and CpT sites was observed 1) in the overall naive
CD3+/CD45RO- T cell
population (Fig. 2), and 2) in the purified CD4+
naive T cell population (Fig. 3A). Like CpG methylation,
non-CpG methylation patterns measured at the IFN- promoter were not
significantly different between cord blood and adult
CD8+ T cells and NK cells.
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, 
) among the
three cell types clearly indicates an active process may be affecting
the distribution of methylated CpG sites along an actively transcribing
promoter (Fig. 3, A–C, compare the hypermethylated +122 and
+128 sites proximal to the start of transcription with the demethylated
-186 and -54 sites of known nuclear factor binding). It is also clear
from these results that cord blood CD8+ T cells
and NK cells would be no less inhibited as to their IFN--producing
capacity by CpG methylation than would their adult counterparts (Fig. 3, B and C).
Bisulfite sequence data from the IL-4, TNF-, and IFNGR1 genes
We additionally sought to examine CpG methylation in other
important cytokine promoters, focusing initially on IL-4 promoter
methylation in the cord blood and adult
CD3+/CD45RO- T cell
populations. We sequenced the IL-4 promoter from the same batches of
bisulfite-treated DNA that were used to amplify the IFN- promoter
during the course of our study in Fig. 2. This served two purposes:
first, to confirm whether the disparity in IFN- promoter CpG and
non-CpG methylation observed between cord blood and adult T cells was a
consequence of methyltransferase activity and possibly measurable in
trans at other accessible promoters such as the IL-4
promoter; and second, to quantify the levels of reiterative de novo
methylation at CpA and CpT sites in another promoter of interest.
Methylation at CpA or CpT sites in the IFN- promoter was
approximately 7-fold higher in cord blood
CD3+/CD45RO- T cells than
in adult cells (Table II) and 5-fold higher in purified cord blood
CD4+/CD45RO- T cells than
in adults. In contrast, there was little or no evidence of non-CpG
methylation in IL-4 promoter sequence (data not shown) from cord blood
and adult CD3+/CD45RO- T
cells.
Coding and noncoding strand IFNGR1 CpG-island (GenBank accession no.
AL050337; nt 81,411–80,872) sequences were PCR-amplified and cloned
from the same bisulfite-treated genomic DNA used to isolate IFN- and
IL-4 promoter regions. Of 10 coding strand sequences and nine noncoding
strand sequences, no unconverted cytosines were observed at CpA or CpT
sites. In addition we also sequenced a 648-bp region of the TNF-
promoter from cord CB1
CD3+/CD45RO- T cell
bisulfite-treated genomic DNA and found no evidence of methylated CpA
or CpT sites in a total 5800 bp of sequence.
The possibility exists that the non-CpG methylation we have observed
may have resulted in part from local effects causing resistance to
bisulfite treatment. While we cannot entirely rule out this
possibility, we believe that the contribution of such factors is likely
to be small, for the following reasons. First, the average frequency of
unconverted cytosines measured outside putative non-CpG sites in this
study varied between 4–9 x 10-4/base, and
as such was relatively low and equivalent to the error rate of
Taq polymerase in our system. Second, if a DNA structure
resistant to bisulfite treatment caused unconverted hot spots, such
sites should consistently generate a false signal in all sequences
examined. This was not the case, and CpA/CpT methylation occurred most
commonly in neonatal T cell sequences (Table II). Third, findings
reported for murine embryonic stem cells show that the relative
frequency of non-CpG methylation is CpA>CpT>CpC in genome-wide
analysis (26), which correlates with the frequency of
these sites we have observed at the IFN- promoter, in that most, if
not all, non-CpG methylation appears at CpA dimers, and none appears
above background levels at CpC dimers. Finally, during the course of
bisulfite sequencing we did observe sequences (1 in 200) that were
incompletely bisulfite converted, containing a number of unconverted
cytosines. In contrast to the results reported in Fig. 2, those sites
of incomplete conversion appeared randomly (data not shown).
Non-CpG methylation at specific CpA and CpT residues in the human
IFN- promoter parallels Dnmt3a expression in 293 cells
Reports of non-CpG methylation have been sporadic in the
literature (25, 27, 28, 29) and presumably have only arisen
recently because of the detailed sequence analysis possible using
PCR-amplified gene elements from bisulfite-converted genomic DNA. Two
studies of particular interest used the method of nearest neighbor
analysis to show that in mammals (26), and even insects
(30), CpA and CpT methylation is the most common form of
non-CpG methylation. In another investigation, overexpression of Dnmt3a
in the 293 embryonic kidney carcinoma cell line caused methylation of a
chromosomally integrated copy of the EBV nuclear Ag 1 gene at two
HhaI methylation-sensitive restriction sites
(31). Using DNA from the latter model we
bisulfite-sequenced the IFN- promoter from 293 cells expressing
either Dnmt3a or Dnmt3b (31) to investigate whether the
CpA and CpT methylation patterns in the promoter could be attributable
to the activity of either enzyme. The results demonstrated Dnmt3a
expression in 293 cells was accompanied by the appearance of non-CpG
methylation, specifically at CpA sites in the IFN- promoter
noncoding strand (Fig. 4B).
This effect was not seen in cells that overexpressed Dnmt3b or in
control 293 parent cells (data not shown).
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sequence does not
cause unconverted 5-methylcytosine artifacts in the context of CpA or
CpT sites
To discount the likelihood that the unusual DNA structure of the
IFN- promoter caused incomplete bisulfite treatment hot spots and
unconverted cytosine in the context of CpA and CpT sites, a control
bisulfite experiment was performed using cloned native IFN- (data
not shown). The native IFN- promoter was cloned into pBluescript II
SK+ plasmid, and purified plasmid was
bisulfite-treated essentially as described for the genomic DNA samples.
The PCR-amplified IFN- was then subcloned and sequenced from
bisulfite-treated plasmid, instead of the normal bisulfite-treated
genomic DNA. Little or no unconverted cytosine was discovered in the
context of the CpA or CpT sites that were identified during genomic DNA
bisulfite sequencing. Unconverted cytosines arose from incomplete
bisulfite treatment in the DNA surrounding CpA and CpT sites as a
result of bacterial methylase activity with a similar frequency as it
did at the CpA and CpT sites.
IFN- expression by cord blood and adult CD4+
and CD8+/CD45RO-
T cells
To demonstrate the differential between IFN- production from
cord blood and adult CD4+ and
CD8+ T cells we optimally stimulated
immunomagnetically purified T cells from two neonates and two adults
with PMA/ionomycin using standard methods over a 48-h period (Table III). Under these conditions of short
term stimulation, where little cell division has occurred, the absolute
levels of IFN- are greatest from adult CD8+ T
cells and lowest from cord blood CD4+ T cells.
However, direct comparison of relative levels of IFN- production
within these T cell populations in the two age groups indicates that
while production in cord CD8+ T cells is 50–65%
of that in their adult counterparts, corresponding production levels in
cord CD4+ T cells are reduced 5- to 10-fold
relative to adult levels, as reported previously (6). This
finding parallels our observation of high level IFN- promoter
methylation in cord blood CD4+ T cells compared
with CD8+ T cells (Fig. 3D), in
contrast to the comparable methylation status of respective T cells in
the adult (Fig. 3, A and B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter as an important negative transcriptional regulator of
IFN- production by human T cells. Previous studies using
methylation-sensitive restriction mapping have investigated the status
of the -54 CpG site in human CD4+ and
CD8+ T cells (18, 23). We have
extended these studies by the use of a sensitive bisulfite sequencing
technique that allows measurement of methylated cytosine in any context
over a DNA region of interest. These studies were initially prompted by
an observation that murine CD8+ memory T cell
clones producing large amounts of IFN- message are hypomethylated at
the IFN- promoter, and that this epigenetic characteristic was
preserved upon withdrawal of stimulation and return of message
production to basal levels (15, 16). Our present results
with human cells are consistent with these murine findings, and further
suggest that variations in the degree of CpG methylation in the IFN-
promoter may underlie developmentally related differences in the
capacity to express IFN- responses following stimulation.
In particular, PMA/ionomycin-stimulated
CD4+/CD45RO- neonatal T
cells produce 5- to 10-fold lower levels than their adult
CD4+/CD45RO- counterparts,
and this correlates with methylation in the IFN- promoter of
94–96% vs 62–74% of CpG sites, respectively.
The mechanisms by which CpG methylation regulates gene transcription is
incompletely understood. In particular, there is a paucity of
information on what constitutes the threshold level of CpG methylation
necessary for silencing of individual genes. However, recent findings
implicating chromatin remodeling at both the IFN- and IL-4 loci in
murine T cells during Th1/Th2 differentiation (32) provide
a broad theoretical framework for study of the underlying mechanisms.
Thus, it appears that methylation of CpG dinucleotides in a symmetrical
fashion attracts methyl-binding proteins that may modulate
transcription factor binding by direct steric hindrance or may act in
combination with multiprotein complexes containing histone deacetylase
(HDAC) activity to increase the charge on histone and ultimately cause
chromatin remodeling. The mere association of methyl-binding proteins
to methylated DNA has also been shown to repress transcription without
associated HDAC activity and higher order chromatin formation
(33). Heterochromatin formation is thus an attractive
theoretical mechanism for explaining reduced accessibility and negative
regulation of transcription; however, in practice, proving the link
between methylation and changes to DNA accessibility can be difficult.
Treatment of cells with trichostatin A (HDAC inhibitor) did not, for
example, restore high level transcription from a number of tumor
suppressor genes, even when the cells had been precultured with the
methylation inhibitor 5-aza-2'-deoxycytidine (34, 35).
Our results also suggest the possibility that control of IFN- gene
activation via promoter methylation may be even more complex than
hitherto realized, as non-CpG (notably CpA and CpT) sites may also be
involved. There are few precedents for this possibility in the current
literature, and reports of non-CpG methylation in mammalian gene
sequences have been sporadic and largely restricted to repetitive DNA
elements. Densely methylated CpG islands occurring at the
oriS14 and ori-
origins of replication in
Chinese hamster ovary cells are bilaterally methylated at CpN
dinucleotides (28) and are conserved in humans (36, 37). Bisulfite sequencing of two regions of pBluescript II
SK+ plasmid that had been transfected into mouse
F9 embryonal carcinoma cells and mouse NIH-3T3 fibroblasts revealed
methylated CpNpG sites at different cytosines along both DNA strands
for each of the two regions of the plasmid (25). In a
sequence covering four gene regions, we detected little methylation at
these putative CpNpG sites. In another study examining human embryonic
fibroblasts, a number of non-CpG methylation sites were identified in
the sequence of an L1 retrotransposon (29). The most
recent study of importance here was a genome-wide examination of
non-CpG methylation in murine embryonic stem cells (26),
which showed that methylation occurred at CpA and CpT sites in
wild-type embryonic stem cells and different somatic tissues. The same
group also identified non-CpG methylation in the genome of
Drosophila (30).
We hypothesized that the non-CpG methylation we have observed here may
be an event activated by specific DNA methyltransferase activity in T
cells. Several observations lead us to this suggestion. First, the
proportions of CpA and CpT methylations we observe in the IFN-
promoter of neonatal
CD3+CD45RO- naive T cells
and purified neonatal
CD4+/CD45RO- T cells were
similar, with a bias toward CpA methylation. This observation has also
been made in mouse embryonic stem cells (26). Second, our
IFN- promoter methylation data for CpG sites from adult
CD8+/CD45RO- T cells
demonstrates hypomethylation similar to that observed in mouse
CD44high (memory) CD8+ T
cells (15), which are also capable of generating high
level IFN- production upon activation. Third, in the neonates
studied here the IFN- promoter was hypermethylated in
CD4+/CD45RO- T cells at
all of the six CpG sites, but, conversely, was hypomethylated in
CD8+/CD45RO- T cells,
suggesting that the CD8+ (cytotoxic) T cell
component of neonatal host defenses may be relatively competent
compared with their CD4+ counterparts. This
initial block in CD4+ Th cell function may be a
contributing factor to the poor generation of T cell memory against
viral infection that is characteristic of the neonate
(38).
We have also observed that the differential between IFN- promoter
methylation at CpG and non-CpG sites in neonatal and adult
CD4+/CD45RO- T cells was
not paralleled at the IL-4 promoter. Lastly, we have demonstrated that
Dnmt3a overexpression accompanies IFN- promoter CpA methylation in
the 293 embryonic kidney carcinoma cell line and thus may be the cause
of de novo non-CpG methylation in neonatal
CD3+/CD45RO- and
CD4+/CD45RO- T cells.
However, caution must be exercised in extrapolation of these findings
in 293 cells to primary T cells, as Dnmt3A expression is known to be
significantly higher in the transformed cell line (31),
and additional studies will accordingly be required to resolve this
issue. Nevertheless, our capacity to recapitulate the precise effect
observed in unmodified T cells by supplementation of specific enzyme
activity in a cell line argues strongly that the non-CpG methylation
phenomenon we have observed is not simply a random artifact.
In conclusion, we speculate that the dualistic nature of the functions
of IFN- in fetal development, viz., an obligatory role in
implantation vs potentially catastrophic effects on placental function
if produced locally at significant levels later in gestation, has
dictated the necessity for particularly fine, yet robust, control of
IFN- gene expression during this life phase. We further hypothesize
that the basis for this fine control involves either regulated or
constitutive Dnmt1/3a expression, which, in turn, coordinates a program
of reiterative de novo methylation at specific sites in the IFN-
gene. Current understanding of how this mechanism operates emphasizes
the importance of symmetrical CpG methylation in repressing
transcription via recruitment of methyl-binding proteins. Therefore,
addition of further methyl groups to DNA by reiterative de novo
methylation at CpA and CpT sites, as demonstrated above, could enhance
the efficiency of transcriptional repression either directly by steric
hindrance or via attraction of as yet uncharacterized regulatory
proteins. As such, de novo methylation at CpA and CpT sites may
represent a rapid, yet transient, method that a cell might employ to
modulate epigenetic reprogramming.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patrick G. Holt, Division of Cell Biology, TVW Telethon Institute for Child Health Research, P.O. Box 855, West Perth, Western Australia 6872, Australia. E-mail address: patrick{at}ichr.uwa.edu.au
3 Abbreviations used in this paper: Dnmt3a, DNA methyltransferase 3a; HDAC, histone deacetylase; IFNGR1, IFN-R -chain.
Received for publication July 30, 2001. Accepted for publication January 25, 2002.
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) vs adult (
) T
cells vs cord blood CD8+/CD45RO- (
) T cells
from A and B.