 |
INTRODUCTION |
In mammals, DNA methylation at CpG dinucleotides in the 5' region
of genes is frequently associated with transcriptional silencing (1),
particularly in housekeeping genes on the inactive X chromosome. Numerous studies suggest that this association between promoter hypermethylation and transcriptional repression has a functional basis.
For instance, individual loci on the inactive human X chromosome in
human/hamster hybrid cell lines may be reactivated using
DNA-demethylating agents such as 5-azacytidine (2, 3) and
5-azadeoxycytidine, which inhibit the maintenance methyltransferase and
incorporate into newly synthesized DNA (4), resulting in a failure to
maintain methylation patterns during growth. Likewise, in
vitro methylation of various promoter constructs results in
inhibition of transcription in transient expression assays (5-9).
However, despite significant evidence that DNA methylation represses
transcription, specific mechanisms of this repression are only now
becoming apparent.
Recent reports suggest that DNA methylation mediates transcriptional
repression indirectly, via binding of the methylated DNA-binding
protein MeCP2, which in turn recruits histone deacetylases that modify
the local chromatin structure (10, 11). However, additional mechanisms
may also act to repress transcription by methylation, such
as direct inhibition of transcription factor binding to its cognate
site in DNA. Indeed, methylation of the binding sites of several
transcription factors has been shown to alter the affinity of the
factor for its binding site (reviewed by Tate et al.
(12)).
Whether transcriptional repression relies on methylation at specific
critical CpGs or on the overall level of promoter methylation remains
unclear. Several studies indicate that methylation of CpG dinucleotides
in the vicinity of the transcriptional initiation site(s) of genes is
important for gene silencing (13-17). Therefore, perturbation of the
methylation pattern in this region may identify specific CpG sites
whose methylation is required to maintain silencing and provide insight
into mechanism(s) by which methylation mediates transcriptional repression.
The X-linked human hypoxanthine phosphoribosyltransferase
(HPRT)1 gene exhibits
strong differential methylation in the promoter region on the active
and inactive X chromosomes. High resolution methylation analysis by
ligation-mediated PCR (LMPCR)-assisted genomic sequencing indicates
that the promoter on the active X chromosome is unmethylated, whereas
the promoter on the inactive X chromosome is methylated at most, but
not all, CpG dinucleotides (18). The promoter methylation of the
inactive HPRT allele, like that of other X-linked
housekeeping genes, is unusual in that it occurs in a CpG island, which
is typically unmethylated in autosomes. DNA-demethylating agents such
as 5-azadeoxycytidine (5aCdr) can alter the methylation pattern of the
inactive HPRT allele and can reactivate the gene on the
inactive X chromosome in rodent/human hybrid cell lines (2, 3, 19).
Cis-acting regulatory elements in the HPRT promoter region
have been identified by dimethyl sulfate (DMS) in vivo
footprinting (20).
By examining the altered methylation patterns of the HPRT
promoter in single cell-derived clones from 5aCdr-treated cells, we
have identified three specific CpG dinucleotides in the promoter region
whose methylation is highly correlated with maintaining transcriptional
repression of the HPRT gene on the inactive X chromosome.
Consistent with the requirement for methylation of specific critical
CpG sites, we find no correlation between the level of pre-existing
demethylation and the reactivation frequency of the gene when clonal
lines that have undergone partial demethylation of the promoter are
re-treated with 5aCdr. We also find that the inactive HPRT
allele appears to be insensitive to reactivation by the histone
deacetylase inhibitor trichostatin A (TSA), and we also show that this
resistance to TSA reactivation cannot be overcome by partial
demethylation of the promoter region. Furthermore, we present evidence
for de novo methylation of the HPRT promoter upon
5aCdr treatment, and we discuss its potential relevance to transcriptional silencing by DNA methylation.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
8121 (20) and X8-6T2 (21) are human/hamster
somatic cell hybrids containing an inactive human X chromosome. They
were maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin,
and 1× 6-thioguanine (2-amino-6-mercaptopurine; Sigma). 4.12 (20) is a
human/hamster hybrid containing an active human X chromosome. 4.12 cells were grown in DMEM supplemented with 10% fetal bovine serum, 1%
penicillin/streptomycin, and 1× HAT
(hypoxanthine/aminopterin/thymidine; Life Technologies, Inc.)
supplement. All cells were maintained in culture at 37 °C in 5%
CO2.
5-Azadeoxycytidine (5aCdr) Treatment and Isolation of Single
Cell-derived Clones--
8121 cells were grown to approximately 80%
confluence, switched to medium lacking 6-thioguanine, and treated for
24-48 h with 0.5-2.0 µg/ml 5aCdr. The cells were then washed with
phosphate-buffered saline, trypsinized, counted by hemocytometer, and
serially diluted. Cells were plated at a density of 10-103
cells per 100-mm plate in non-selective medium (without HAT or 6-thioguanine) or 103-105 cells per 100-mm
plate in HAT-supplemented medium. After 2-4 weeks, well isolated
single cell-derived colonies were isolated with cloning rings and
individually expanded in 24-well plates and then in individual culture
flasks. Sixty two clones isolated from 5aCdr-treated cells grown
without selection and four clones isolated from 5aCdr-treated cells
grown under HAT selection were expanded for further analysis.
5-Azadeoxycytidine Reactivation Studies--
Cells were grown to
80% confluence in DMEM with 10% fetal bovine serum and 25 µg/ml
gentamicin and then treated with 1.0 µg/ml 5aCdr for 24 h. The
cells were allowed to recover for 24 h in medium without 5aCdr,
plated in duplicate at a density of 20,000 cells per 150-mm plate in
HAT medium, as well as 1,000 cells per 150-mm plate in non-selective
medium (to normalize for plating efficiency), and incubated at 37 °C
for 10 days. Single cell-derived colonies in each dish were stained
with Coomassie Blue for 5 min and counted. The 5aCdr-induced
reactivation frequency for each cell line was normalized for plating
efficiency by calculating ((average number of HAT-resistant colonies in
each plate)/(average number of colonies on each non-selected plate × 20)). The overall reactivation frequency of all of the clones
together was calculated as ((total number of HAT-resistant
colonies)/(total number of colonies on all non-selected plates × 20)).
Trichostatin A (TSA) Treatment of Cells--
Cells were grown to
50% confluence and then switched to medium supplemented with 300 ng/ml
TSA. Cells were lysed, and RNA was isolated (as described below) after
0, 12, 24, and 48 h of TSA treatment.
Genomic DNA Preparation--
Cells in monolayers were washed
once with phosphate-buffered saline and then lysed and incubated
overnight at room temperature in 10 ml of DNA lysis buffer (150 mM NaCl, 50 mM Tris (pH 8.0), 25 mM
EDTA, 0.5% SDS, 300 µg/ml proteinase K). The lysate was extracted
once with Tris-equilibrated phenol (pH 7.0), twice with phenol/chloroform (50:50), and once with chloroform. The 10-ml aqueous
phase was treated with 10 µl of 10 mg/ml RNase mixture (RNase A (500 units/ml), RNase T1 (2000 units/ml); Ambion) for 1 h at 37 °C
and then extracted once with phenol/chloroform and once with
chloroform. The genomic DNA was precipitated with 0.5 volume of
7.5 M ammonium acetate, spooled out, washed briefly with
70% ethanol, air-dried, and then resuspended in TE (10 mM Tris (pH 8.0), 1 mM EDTA) at approximately 1 µg/µl and
stored at 4 °C.
RNA Preparation--
RNA was prepared using the Trizol reagent
(Life Technologies, Inc.) according to the manufacturer's instructions
and suspended in 100 µl of diethyl pyrocarbonate-treated
H2O. RNA concentration was determined by spectrophotometry
and adjusted to 1 µg/µl in diethyl pyrocarbonate-treated
H2O, and the RNA was stored at
20 °C.
Detection of HPRT mRNA by Reverse Transcriptase-PCR
(RT-PCR)--
HPRT mRNA was assayed using a protocol
modified from Sasaki et al. (19). Briefly, 1 µg of RNA in
6 µl of diethyl pyrocarbonate-treated H2O was denatured
for 5 min at 95 °C and then placed on ice for 2 min. The reverse
transcription (RT) reaction was then performed in a final volume of 20 µl containing 10 units of RNasin (Promega), 7.5 µM
random hexamers, 1 mM dNTPs, 10 mM
dithiothreitol, 50 mM Tris (pH 8.3), 75 mM KCl,
3 mM MgCl2, and 200 units of murine leukemia virus-reverse transcriptase (Promega) and was incubated for 10 min at 20 °C and then 1 h at 37 °C. PCR amplification was performed on 10 µl of the RT products in a final volume of 100 µl
containing 20 mM Tris (pH 8.4), 50 mM KCl, 0.2 mM dNTPs, 1.75 mM MgCl, 60 pmol each of the
HPRT-specific primers HPRT1 (5'-TCCTCCTGAGCAGTCAGC-3') and
HPRT3 (5'-GGCGATGTCAATAGGACTC-3'), 7.5 pmol each of the human MIC2 gene (MIC2)-specific primers XMIC2
(5'-ACCCAGTGCTGGGGATGACTTT-3') and XMIC2R (CTCTCCATGTCCACCTCCCCT-3'),
and 10 units of Taq polymerase (Life Technologies, Inc.).
The PCR cycling conditions were 2 min at 94 °C; 30 cycles of 1 min
at 94 °C, 30 s at 59 °C, and 90 s at 70 °C; 4 min at
72 °C; hold at 4 °C. 10 µl of the PCR reaction was loaded onto
a 1% agarose gel, run at 100 V for 2 h, stained in ethidium
bromide, and visualized by UV fluorescence.
Hydrazine Treatment of DNA for Methylation
Analysis--
Analysis of the methylation status of each CpG
dinucleotide within the 5' region of the HPRT gene was
accomplished using the cytosine-specific Maxam-Gilbert genomic
sequencing reaction (22) followed by gene-specific amplification using
ligation-mediated PCR (LMPCR (23)). 75 µg of genomic DNA were treated
with 60% hydrazine in 1.5 M NaCl for 10 min at 20 °C.
The reaction was stopped with 200 µl of hydrazine stop solution (0.3 M sodium acetate (pH 7.5), 0.1 mM EDTA (pH
8.0)) and then precipitated in ethanol in a dry ice bath. The pellet
was re-precipitated with sodium acetate and washed briefly with 75%
ethanol and dried under vacuum. The sample was resuspended in 90 µl
of H2O, and 10 µl of piperidine was added. Piperidine
cleavage at modified cytosines was performed at 95 °C for 30 min. 1 ml of H2O was added, and the sample was allowed to dry
overnight under vacuum to remove the piperidine. The resulting
chemically cleaved DNA was resuspended at a final concentration of 1 µg/µl in H2O. Fragments representing cleavages at
unmethylated cytosines within the HPRT promoter were
amplified by LMPCR.
LMPCR Amplification of Piperidine-treated DNA--
The LMPCR
amplification of fragments representing cleavages within the
HPRT promoter was performed essentially as described by
Hornstra and Yang (18) except that the PCR step was carried out using
Taq polymerase (20) instead of Vent polymerase. For the
methylation analysis, 2 µg of hydrazine-treated DNA was amplified using the E and M primer sets as described previously (20). The E1 and
E2 primers, 5'-AGCTGCTCACCACGACG-3' and 5'-CCAGGGCTGCGGGTCGCCATAA-3', respectively, were used to examine upper strand methylation, whereas the M1 and M2 primers, 5'-GAATAGGAGACTGAGTTGGG-3' and
5'-GGAGCCTCGGCTTCTTCTGGGAGAA-3', respectively, were used to examine the
lower strand. These two LMPCR primer sets cover the region from
approximately positions
235 to
10 relative to the translation start
site on both strands and include the entire minimal promoter (24).
LMPCR products were size-fractionated on a 5% acrylamide gel DNA
sequencing gel, electrotransferred to a nylon membrane, and hybridized
with an appropriate 32P-labeled probe as described
previously (23).
 |
RESULTS |
Identification of Critical CpG Dinucleotides--
To identify
specific CpG sites within the promoter region whose methylation may be
critical for maintaining transcriptional repression of the
HPRT gene on the inactive X chromosome, we treated 8121 cells (containing an inactive human X chromosome) with the demethylating agent 5aCdr and isolated both HAT-selected (HAT selects
for the HPRT+ phenotype) and non-selected single
cell-derived clones. If critical CpG sites exist within the
HPRT promoter region, these sites should remain methylated
after exposure to 5aCdr in all clones that fail to reactivate the
HPRT gene (and, conversely, undergo demethylation in all
clones that do reactivate). Each of 4 HAT-selected and 62 non-selected
clones were screened for expression of HPRT mRNA by
RT-PCR. The RT-PCR assay was performed on total RNA as a multiplex reaction using both HPRT-specific primers and human
MIC2 (MIC2)-specific primers. Because
MIC2 is an X-linked gene that escapes X inactivation, MIC2 expression serves as an internal positive control for
the RT-PCR. As expected, RT-PCR analysis revealed that all 4 HAT-resistant clones had reactivated the HPRT gene (Fig.
1A). In addition, one of the 62 non-selected clones examined also had reactivated the HPRT
gene following 5aCdr treatment. To determine the sensitivity of the
RT-PCR assay, total RNA from 4.12 cells (which contain an active human
X chromosome and express the HPRT gene) was serially diluted
with total RNA from 8121 cells, and the resulting mixtures were
subjected to RT-PCR. The results (Fig. 1B) indicate that the
RT-PCR assay can readily detect as little as 0.2% of "wild type"
(i.e. 4.12 cells) HPRT mRNA levels. All 5 clones that reactivated the HPRT gene after 5aCdr treatment
exhibited full wild type HPRT mRNA levels, and all 61 clones that failed to reactivate the HPRT gene after 5aCdr
treatment showed no detectable HPRT mRNA; no low or
intermediate HPRT mRNA levels were observed for any of the clones examined in our RT-PCR assays.

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Fig. 1.
RT-PCR analysis of HPRT
mRNA expression. A, representative RT-PCR
analysis of HAT-selected and non-selected single cell-derived clonal
lines from 5aCdr-treated 8121 cells. The lane designations indicate the
name of each clone examined. 8121 is the parental
human/hamster hybrid cell line containing an inactive human X
chromosome. 4.12 is a human/hamster hybrid that contains an
active human X chromosome. HPRT indicates the position of
the expected HPRT-specific RT-PCR product. MIC2
indicates the expected location of the MIC2-specific RT-PCR
product. B, sensitivity of the RT-PCR assay to detect
HPRT mRNA. Total RNA from 4.12 cells (wild type) was
serially diluted with total RNA from 8121 cells, and the RNA mixture
was subjected to RT-PCR analysis. Percentages from 0-100%
indicate the portion of the total RNA that is derived from 4.12 cells.
HPRT and MIC2 represent the expected location of
the respective RT-PCR products.
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All clones assayed for HPRT reactivation by RT-PCR were also
subjected to high resolution DNA methylation analysis by LMPCR genomic
sequencing (Figs. 2 and
3). The specific region of the HPRT promoter examined extends from approximately 140 bp
upstream to 90 bp downstream of the two major transcription initiation sites identified by Kim et al. (25) and encompasses the
entire minimal promoter (26) and all but one of the transcription
factor binding sites previously identified by DMS in vivo
footprinting (20). This region was chosen for analysis because previous
studies suggest the region surrounding transcription initiation sites is crucial for transcriptional repression by DNA methylation (13-17). In the parental 8121 cell line, this region contains 20 symmetrically methylated CpG sites (where paired CpGs on the upper and lower strand
count as a single site) and 12 unmethylated sites (18). Purified
genomic DNA from each clone was subjected to the Maxam-Gilbert cytosine-specific sequencing reaction followed by ligation-mediated PCR
(LMPCR). The cytosine-specific reaction uses hydrazine to modify
cytosine nucleotides in DNA, but this modification is significantly inhibited by methylation at the 5-position of cytosine. The
differential reactivity of hydrazine with cytosine versus
5-methylcytosine allows for examination of the methylation status of
every cytosine (including CpG dinucleotides) within a region of
interest (18, 23, 27). In this high resolution assay, demethylation at
a specific cytosine results in the appearance of a new band (relative to a methylated sample) in the autoradiogram of the cytosine-specific sequencing ladder (Fig. 2). A site was scored as "demethylated" in
5aCdr-treated clones if a new cytosine-specific band was detected that
was previously undetectable (i.e. methylated) at the same position in the cytosine-specific ladder from the 8121 parental cells.
All sites designated as "methylated" showed no detectable band on
the cytosine-specific sequencing ladder above background levels;
therefore, a site in the 5aCdr-treated clones was considered demethylated if the associated band was detectable above
background (Fig. 3).

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Fig. 2.
Representative autoradiogram of genomic
sequencing analysis of the upper strand of the HPRT
gene 5' region. The designation above each lane
indicates the name of the clone examined. HPRT plasmid is a
control sample showing the cytosine-specific sequencing ladder derived
from plasmid DNA containing an unmethylated subclone of this region of
the HPRT gene and reveals the position of every cytosine in
the region. The horizontal bars on the left side
of the figure indicate the positions of each CpG dinucleotide in the
region. Demethylation events in each cell line appear as new bands in
the cytosine-specific ladder at the position of the demethylation
event. The solid circles indicate the position of
demethylation events that have occurred in each clone. All position
numbers are relative to the translation initiation site (26).
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Fig. 3.
Summary of methylation analysis of the
HPRT gene 5' region in unreactivated clones. The
rows of squares immediately above and
below the nucleotide sequence indicate the methylation
pattern of the parental 8121 cell line. Open squares
indicate unmethylated CpG dinucleotides; solid squares
indicate methylated CpG dinucleotides, and half-filled
squares indicate sites methylated in some cells but not others.
? indicates sites for which the methylation status could not
be determined due to technical limitations of the genomic sequencing
methodology. Only those unreactivated clones whose methylation state
differed from the parental pattern at a given CpG site are scored as
open or solid circles. Each open
circle represents one unreactivated clone that exhibited
demethylation at the indicated CpG site. Each solid circle
represents one unreactivated clone that exhibited de novo
methylation at the indicated CpG site. Numbers in
parentheses indicate the number of clones that demonstrated
de novo methylation at a given CpG site. In these cases of
de novo methylation, the number of circles is scaled to half
the number of methylation events at each site. ND indicates
that the methylation status of the CpG site was not determined because
it could not be resolved with the primer set used for that strand. The
large vertical ovals indicate the location of the three
critical methylation sites that remain methylated in all 61 unreactivated clones. Bolded italicized Gs within
the sequence indicate the location of DMS in vivo footprints
in the region (20). Large open rectangles indicate the
location of GC boxes. The arrows indicate the
location of the two major transcription initiation sites determined by
Kim et al. (25), and the dashed line between the
upper and lower strands of the nucleotide sequence indicates the region
of multiple transcription initiation sites described by Patel et
al. (26). All position numbers shown are relative to the
translation start site (26).
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All clones that reactivated the HPRT gene after 5aCdr
treatment demethylated all CpGs in the region around the major
transcription initiation sites of the HPRT gene (data not
shown), a pattern identical to that of the active HPRT
allele on the active X chromosome and consistent with previous 5aCdr
reactivation studies of the HPRT gene (18). In addition, two
HAT-selected 8121-derived clones that had spontaneously reactivated the
human HPRT gene in the absence of 5aCdr treatment also
exhibited complete demethylation of all CpGs in the same region of the
promoter (data not shown). These data suggest that the complete
demethylation of this region of the HPRT promoter in
transcriptionally reactivated clones is not simply an artifact of 5aCdr
or HAT treatment and is a requirement for transcriptional activation of
the gene.
Consistent with these results, clones that failed to reactivate the
HPRT gene following 5aCdr treatment had promoters that remained relatively hypermethylated compared with the unmethylated active HPRT allele (Fig. 2). Among unreactivated clones, the
extent and pattern of 5aCdr-induced demethylation at the
HPRT promoter were quite variable, with as few as 0 and as
many as 11 demethylated CpG sites out of a possible 20 methylated sites
in the parental 8121 cells, with an overall frequency of 7.8%
demethylation/site. Nevertheless, even the clone with 11 demethylated
sites failed to express any detectable HPRT mRNA as
determined by RT-PCR, suggesting that the repressive effect of
methylation on transcription is not simply cumulative nor directly
proportional to the number of methylated CpG sites.
Since any methylation site(s) essential for transcriptional repression
should remain methylated in all unreactivated clones, the methylation
patterns of each of the 61 unreactivated clones were compared (and
summarized in Fig. 3). This analysis identified three CpGs at positions
48,
54, and
97 (relative to the translation initiation site) that
remain methylated on both strands in all 61 unreactivated 5aCdr-treated
clones. The site at position
97 is located immediately upstream of a
major transcription initiation site, and the sites at positions
48
and
54 are located approximately 35-45 bp downstream of the two
major transcription start sites (in the portion of the gene encoding
the 5'-untranslated region). The perfect correlation between
methylation at these three "critical" CpG sites and transcriptional
repression of the HPRT gene in 61 independent clones
suggests that methylation at these sites is necessary for maintaining
repression of HPRT on the inactive X chromosome. In
contrast, all other CpG sites examined in this region exhibited
demethylation in at least one unreactivated clone, indicating that
methylation at each of these sites is not essential for maintaining
transcriptional repression.
Statistical Analysis of the Demethylation Pattern in Unreactivated
Clones--
The 5aCdr-induced demethylation pattern observed in the 61 unreactivated clones was subjected to statistical analysis to determine the likelihood that the three critical CpG sites escaped 5aCdr-induced demethylation simply by chance. To simplify the analysis, statistical analysis was performed using data only from the upper strand (which was
informative at more sites than the lower strand). The null hypothesis
H0 is that the frequency of demethylation in the
m = 20 methylation sites examined in each clone is in
fact uniform, and the pattern observed is based on sampling error. We
reject the null hypothesis if the number of sites that never
demethylate, X, is too large. Let there be r = 61 clones, and at the kth clone nk of
the sites has not been demethylated at least once. Then the
p value to reject the null hypothesis is calculated by the
probability shown in Equation 1,
|
(Eq. 1)
|
where x0 is the observed number of sites
which never demethylated. Probability 1 can be computed recursively by
letting p(k,i) indicate the
probability that there are still i methylated sites never
demethylated after k clones have been observed. Then, under H0 (Equation 2),
|
(Eq. 2)
|
With the initial condition (Equation 3)
|
(Eq. 3)
|
The p value (Equation 1) is shown in Equation 4,
|
(Eq. 4)
|
for m is 20; r is 61; and
nk is 19, 20, 17, ... 18 for k = 1, 2, 3, ... 61. Based on this calculation, the p
value to reject the null hypothesis H0 is
0.00003. Therefore, the distribution of 5aCdr-induced demethylation
events among the 20 methylated CpG sites examined is not uniform or
random. This indicates that failure to demethylate the three critical
methylation sites at
48,
54, and
97 in unreactivated clones is
statistically significant and not due merely to chance. This analysis
supports the notion that methylation of these CpG sites is required to
maintain transcriptional repression of the HPRT gene.
Whether or not these three sites will ever demethylate in a
5aCdr-treated unreactivated clone is unclear. To distinguish between sites that never demethylate and those that have only a very small chance of demethylating would require an impractical volume of additional data. However, if we make the simplifying assumption that
each of the three potential critical sites (at positions
48,
54,
and
97) have the same demethylation probability
p1, we can roughly estimate the maximal
demethylation frequency of these three sites relative to the other
sites in the region. The average probability of demethylation at the 17 sites that did demethylate can be calculated as shown in Equation 5,
|
(Eq. 5)
|
Since the total number of demethylations in the three critical
sites is 0, we cannot estimate a demethylation probability. However if
we take a 95% upper bound for the probability of all three sites
remaining methylated, this probability is p3 =
ln(0.05)/95 = 0.0315. The upper bound probability for each site
then is as shown in Equation 6,
|
(Eq. 6)
|
Therefore, we have a 95% confidence that the demethylation
probability (p1) of each of these three critical
sites is no more than 1/9 the average demethylation probability
(p0) of each of the other 17 methylated sites.
The significant difference in the maximal demethylation probability of
the three critical sites relative to the other sites in the region is
consistent with a functional role for methylation at these sites in
unreactivated clones.
De Novo Methylation of the Human HPRT 5' Region--
An unexpected
finding in these studies is the 5aCdr-induced de novo
methylation of the HPRT promoter just upstream and extending into a cluster of 5 GC boxes (spanning positions
217 to
163; see
Fig. 3) that are normally unmethylated on both the active and inactive
X chromosomes (18). Every unreactivated 5aCdr-treated clone exhibited
some level of de novo methylation in this region, with
nearly 50% of the clones showing de novo methylation at
positions
232,
220,
218, and
212 and occasional de
novo methylation occurring at positions
216 and
191. This
de novo methylation occurs too frequently to arise simply
from a small subpopulation of aberrantly methylated cells in the
parental 8121 cell line. Because 5aCdr is a demethylating agent,
de novo methylation is most likely a secondary rather than
primary effect of 5aCdr treatment. However, since none of the clones
that reactivated the HPRT gene (either spontaneously or as a
result of 5aCdr treatment) show de novo methylation at any
of these sites, the absence of methylation at these sites may be
necessary for transcriptional reactivation. On the other hand,
methylation at these sites is not a requirement for transcriptional
repression, since the parental 8121 cells (where the HPRT
gene is inactive) are not methylated at these sites (18). Recently,
Broday et al. (28) also reported evidence for de
novo methylation of silenced transgenes in cells treated with
5-azacytidine.
Reactivation Frequencies of Partially Demethylated Clones--
To
examine what role the overall level of promoter methylation may play in
the transcriptional silencing of the HPRT gene, we further
analyzed 12 clones that had not reactivated the HPRT gene
after 5aCdr treatment but exhibited partial demethylation of the
promoter to varying degrees. Each clone was subjected to a second round
of 5aCdr treatment, and the reactivation frequency of the
HPRT gene in each clone was determined by selection in HAT
medium (after normalizing for the plating efficiency of each clone in
the absence of HAT). If transcriptional repression of the
HPRT gene is dependent upon a threshold level of overall
promoter methylation, clones with higher levels of pre-existing
demethylation should exhibit a higher 5aCdr-induced reactivation
frequency. However, if methylation of specific critical sites is more
important for maintaining repression, the reactivation frequency should be largely unaffected by the increasing levels of pre-existing demethylation at non-critical sites. The results of this experiment are
shown in Table I.
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Table I
Human HPRT gene reactivation frequencies of clones after a second round
of 5aCdr treatment
Clonal lines were derived from 8121, a human/hamster hybrid cell line
containing an inactive human X chromosome, after treatment with 5aCdr.
The initial number of demethylated sites in the HPRT 5'
region for each clone was determined by genomic sequencing. Each clonal
line was treated with 1.0 µg/ml 5aCdr for 24 h. Plating
efficiency was determined by plating 1000 cells in each of two
duplicate 150-mm plates in nonselective medium and counting colonies
after 10 days. Reactivation frequency was determined by plating 20,000 cells/150-mm plate in duplicate under selection in HAT-supplemented
medium and counting HAT-resistant colonies after 10 days. Normalized
reactivation frequency is normalized for plating efficiency and
calculated as described under "Experimental Procedures." N/A, not
applicable.
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We found no correlation between pre-existing levels of stable
demethylation in the promoter region and the 5aCdr-induced reactivation frequency of the HPRT gene. Most demethylated clones showed
reactivation frequencies similar to that of the parental 8121 cells (no
demethylated clone showed more than a 2-fold increase in reactivation
frequency above 8121 cells), and the clone with the highest degree of
pre-existing demethylation (IIIA4) exhibited a normalized reactivation
frequency slightly lower than 8121 cells. In fact, clones that showed
the highest reactivation frequencies were ones carrying no demethylated sites (NS2A11) and ones carrying only a single demethylated site (IH7),
whereas the clone with the lowest reactivation frequency had 8 demethylated sites (IIH1). Furthermore, the overall reactivation frequency of all partially demethylated clones re-treated with 5aCdr
was 1.3% (calculated by (sum of all HAT-resistant colonies)/(20 × sum of all viable colonies)), a level essentially identical to that
of the 8121 parental cells.
This lack of correlation between overall levels of pre-existing
demethylation and 5aCdr-induced reactivation frequencies is consistent
with the existence of specific critical CpG sites whose methylation is
required to maintain repression of the HPRT gene on the
inactive X chromosome. Since all of the critical sites are methylated
in all of the unreactivated clones, if reactivation requires
demethylation of the critical sites, partial demethylation of
non-critical sites would not be expected to affect subsequent 5aCdr-induced reactivation. On the other hand, these results are not
consistent with models that invoke threshold levels of overall promoter
methylation for maintaining repression of transcription. Since
partially demethylated clones should be closer to the threshold level
of demethylation required for reactivation of the gene, these clones
should undergo reactivation at consistently higher frequencies after
re-treatment with 5aCdr. Because data in Table I show this is not the
case and that partial demethylation of non-critical sites has little
effect on subsequent reactivation frequencies, methylation at
non-critical sites seems to play a secondary role in maintaining
transcriptional repression.
Effects of Trichostatin A Treatment on HPRT
Transcription--
Recent reports (10, 11) have suggested that MECP2
may mediate transcriptional repression at methylated promoters by
recruiting histone deacetylases. Therefore, it is possible that the
three critical methylated sites in the HPRT promoter region
may function on the inactive X chromosome by binding MeCP2 and
recruiting histone deacetylases that, in turn, maintain the repressive
chromatin structure of the promoter. However, in vivo
footprinting studies of both the HPRT gene (20) and the
human PGK-1 gene (PGK-1) on the active and
inactive X chromosomes (29, 30) have not detected any evidence for
stable binding of MeCP2 at methylated CpG dinucleotides on the inactive
X chromosome (i.e. no in vivo footprints have
been detected over methylated CpGs). Despite this caveat, MECP2 may
still play a role in repression of the HPRT locus since the
absence of a footprint could indicate that either MECP2 binding is
transient or that MECP2 simply does not footprint well.
Since TSA has been shown to alleviate MECP2-mediated repression (10,
11), two independent human/hamster hybrid cell lines, 8121 and X8-6T2,
each containing an inactive human X chromosome, were treated for 0-24
h with TSA to assess the role of histone deacetylase-mediated
repression of the HPRT promoter. If HPRT repression by DNA methylation functions primarily via histone deacetylases, TSA treatment should reactivate the HPRT gene
on the inactive X chromosome. Furthermore, because X8-6T2 reactivates at a significantly higher frequency than 8121, both spontaneously and
upon 5aCdr treatment (see Table I), it may be more responsive to
TSA-induced reactivation. Results of this study are shown in Fig.
4A. While TSA treatment did appear
to enhance the expression level of the control MIC2 gene (an
X-linked gene which escapes inactivation), as has been previously
observed in other gene systems (31, 32), it failed to re-activate the
HPRT gene on the inactive X chromosome in both cell lines as
determined by RT-PCR (Fig. 4A). The greater susceptibility
of X8-6T2 cells to 5aCdr-mediated reactivation apparently does not
confer a greater susceptibility to TSA-induced reactivation. These
results are similar to that reported by Riggs et al. (33).
Since these same cell lines can be reactivated by 5aCdr treatment,
these TSA results are inconsistent with repression of the
HPRT gene by the mechanism identified by Nan et
al. (10) and Jones et al. (11). However, we have been unable to find a TSA-responsive gene to act as a direct positive control for TSA reactivation in the human/hamster hybrid cells. Nevertheless, indirect evidence, such as the growth inhibition of the
cells by TSA treatment and increased MIC2 expression over the TSA
treatment time course (Fig. 4A), are consistent with other studies that show that TSA inhibits cell proliferation (34) and
increases the expression of active genes (31, 32), indicating that TSA
is functioning in these cells.

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Fig. 4.
Trichostatin A treatment of cell lines
containing an inactive HPRT allele. A,
time course of trichostatin A treatment of 8121 and X8-6T2 cells.
HPRT gene reactivation in 8121 and X8-6T2 cells, two
human/hamster hybrid cell lines containing an inactive human X
chromosome, was determined over a 24-h TSA treatment time course by
RT-PCR. HPRT indicates the expected position of the
HPRT RT-PCR product. MIC2 indicates the expected
position of the RT-PCR product for human MIC2, a gene that
escapes X inactivation and serves as an internal RT-PCR control. 4.12 is a human/hamster hybrid cell line containing an active human X
chromosome and serves as an external positive RT-PCR control.
B, analysis of HPRT gene reactivation after
48 h of TSA treatment in cell lines, clonally derived from 8121 cells, that failed to reactivate the HPRT gene after 5aCdr
treatment. The bold numbers above the clone designations
indicate the number of sites in the HPRT 5' region that are
demethylated in each clone. Other designations are as above.
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Recently, Cameron et al. (35) have shown that high density
methylation at the promoter may prevent TSA from reactivating genes
even when histone deacetylases are involved in their transcriptional repression. Partial demethylation of the promoter can overcome this
resistance to TSA-mediated reactivation, revealing a role for histone
deacetylases in maintaining repression. To determine if the high
density methylation of the HPRT promoter is preventing reactivation of the HPRT gene by TSA, each of the 12 partially and stably demethylated clonal lines shown in Table I above
were treated with TSA for 0-48 h and assayed for HPRT
expression by RT-PCR. Each of these single cell-derived clones from
8121 cells carried 0-11 CpG sites that were stably demethylated after
5aCdr treatment yet maintained stable repression of the HPRT
gene. As shown in Fig. 4B, regardless of the level of
pre-existing promoter demethylation (i.e. number of
pre-existing demethylated CpGs), TSA treatment failed to reactivate the
HPRT gene in any of the partially demethylated clones. This
was true even for clone IIIA4 which is stably demethylated at 11 of 20 CpGs in the promoter. These results suggest that inhibition of histone
deacetylation is not sufficient to overcome the repressive effects of
DNA methylation, even in the context of reduced levels of promoter
methylation. Because all of the demethylated sites in each of these
TSA-treated clones are non-critical sites, these results are also
consistent with a mechanism of DNA methylation-dependent
repression involving specific critical sites of methylation.
 |
DISCUSSION |
We have examined the high resolution methylation pattern of the
endogenous HPRT promoter region in 61 clonally derived lines that have undergone 5aCdr-induced demethylation but failed to reactivate the HPRT gene. Statistical analysis reveals a
highly significant (p = 0.00003) correlation between
maintenance of transcriptional repression and methylation at three
specific CpG sites. In contrast, the other CpGs in the promoter region
(outside these three critical sites) appear to be able to undergo
demethylation without affecting repression of the gene, even when as
many as 55% of the CpG sites in the promoter are demethylated
(see clone IIIA4 in Table I). Furthermore, prior demethylation of the
promoter region at non-critical CpG sites does not increase the
frequency of HPRT gene reactivation after re-treatment of
cells with a second round of 5aCdr. These data argue that three
critical sites in the promoter region contribute disproportionately to
the repression of the HPRT gene on the inactive X chromosome
and that methylation of critical sites rather than the overall level of
promoter methylation (or a certain threshold level of promoter
methylation) is involved in maintaining transcriptional silencing.
None of the three critical sites of methylation fall within any of the
transcription factor binding sites previously identified by DMS
in vivo footprinting (20), although the critical site at
97 lies just upstream of a DMS footprinted region (see Fig. 3). The
other two critical sites at positions
54 and
48 are located at
least 21 bp from the nearest DMS in vivo footprinted region,
and DNase I in vivo footprinting studies fail to detect footprints at or near these two sites on either the active or inactive
X chromosome.2 Therefore, it is
unlikely that a major mode of action for the three critical CpG sites
involves interference with transcription factor binding upon
methylation of these sites as proposed by Tate and Bird (12).
Furthermore, Rincon Limas et al. (24) have shown that the
region containing the three critical CpG sites is not required for
maximal expression of the HPRT gene in transient expression
assays. Thus, this region may be required more for repression of the
gene on the inactive X chromosome than for activation or expression.
This suggests, at least in part, an indirect mechanism of
methylation-mediated repression of the HPRT gene. Recent
reports have indicated that repression by methylation involves MECP2
recruitment of histone deacetylases (10, 11), a mechanism that was
shown to be TSA-sensitive. However, the HPRT gene on the
inactive X chromosome appears to be resistant to reactivation by TSA,
even when the promoter is partially (and stably) demethylated by 5aCdr treatment. This would suggest that methylation-mediated repression of
the HPRT gene on the inactive X chromosome could act by
either of two possible mechanisms. One possibility is that silencing of
the HPRT gene does not involve a histone deacetylase
complex; however, antibodies against acetylated histone H4 clearly show that the inactive X chromosome is hypoacetylated (36). Alternatively, repression of the HPRT gene by methylation could involve one
or more histone deacetylases that are TSA-resistant, similar to those reported in yeast (37). It is also possible that the dynamic turnover
of deacetylated histones on the inactive X chromosome is so slow, or
histone acetylation occurs at such a low level on the inactive X, that
inhibiting histone deacetylases does not lead to significant
hyperacetylation of the HPRT gene.
Although transcriptional repression of the HPRT gene may
require methylation at specific critical sites, reactivation appears to
require complete demethylation of the promoter. The association between
complete promoter demethylation and transcriptional reactivation is not
specific to 5aCdr-induced reactivation because it also occurs in
spontaneous reactivants. Complete promoter demethylation is also seen
in the X-linked human phosphoglycerate kinase gene (hPGK-1)
upon 5aCdr-mediated reactivation (13). Together with the fact that the
promoters of other X-linked housekeeping genes on the active X
chromosome are also generally unmethylated (16, 27, 30, 38), these
observations argue that the absence of methylation in the promoter
in vivo is essential for transcription of the
HPRT gene and other X-linked housekeeping genes.
Since demethylation of all 20 CpG sites in the promoter region in a
single cell appears to be required for reactivation of the
HPRT gene, the average overall 5aCdr-induced demethylation frequency of 7.8% at each CpG is too low by itself to account for the
observed reactivation frequency of the 8121 parental cells (see Table
I). Therefore, in order to achieve full promoter demethylation and the
HPRT reactivation frequency we observed for 8121 cells, active demethylation of the promoter region must occur following a
crucial initial 5aCdr-induced demethylation event. Such demethylase activities have been reported recently (39, 40). We propose that the
crucial event that triggers active demethylation of the promoter (and
subsequent gene reactivation) is the stable demethylation of one of the
three critical sites. Thus, the reason we never detected one of the
critical sites in an unmethylated state in unreactivated clones is
because demethylation of a single critical site resulted in active
global promoter demethylation and gene reactivation.
Alternatively, the actual 5aCdr-induced demethylation frequency may be
significantly higher than what is observed in the stable cell lines due
to active remethylation of the promoter after 5aCdr is removed. The
high frequency of de novo methylation observed at certain
CpG sites in the HPRT promoter of unreactivated clones suggests that active remethylation of the promoter does occur in these
cells. This remethylation activity could account for the discrepancy
between the overall stable demethylation frequency we observe and the
reactivation frequency of the 8121 cell line. Since the critical
methylation sites are always methylated in cell lines that fail to
reactivate the HPRT gene, these critical sites may either be
relatively resistant to demethylation or have a propensity to
remethylate. This tendency to maintain the methylated status of the
critical sites might act to prevent full promoter demethylation and
reactivation of the HPRT gene.
Although unlikely, we cannot formally exclude the possibility that
methylation-mediated repression does not occur at the promoter at all
and is instead mediated by a distant site. In this case, the critical
sites in the HPRT promoter may remain methylated simply
because they have a strong pre-disposition to de novo
methylate during growth after 5aCdr-induced demethylation. However,
this scenario is extremely unlikely in light of the substantial
evidence that methylation at the promoter mediates transcriptional
repression and that complete promoter demethylation appears to be
required to reactivate the HPRT gene.
Overall, these studies suggest that transcriptional repression by
methylation is a complex process involving non-equivalent methylation
sites that appear to repress transcription by indirect mechanisms. The
complete demethylation apparently necessary for reactivation and the
evidence for de novo methylation further suggest that
methylation itself is dynamic, a product of intrinsic active
methylation and demethylation processes.
Experiments to examine further the function of these three critical CpG
sites include transient expression assays using constructs differentially methylated at the critical and non-critical sites to
determine if methylation of the critical sites is necessary and
sufficient to repress promoter activity. In addition, to assess the
role of chromatin structure in mediating the function of these three
critical sites, these expression constructs could be pre-assembled into
chromatin prior to transfection as described by Buschhausen et al. (5).