The CpG Island Promoter of the Human Proopiomelanocortin Gene Is Methylated in Nonexpressing Normal Tissue and Tumors and Represses Expression
John Newell-Price,
Peter King and
Adrian J. L. Clark
Section of Medicine (J.N.-P.) Sheffield University, Clinical
Sciences Northern General Hospital Sheffield, S5 7AU, United
Kingdom
Departments of Endocrinology (P.K., A.J.L.C.) St
Bartholomews and Royal London School of Medicine and
Dentistry West Smithfield, London, EC1A 7BE, United Kingdom
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ABSTRACT
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Ectopic secretion of ACTH, from sites such
as small cell lung cancer (SCLC), results in severe Cushings
syndrome. ACTH is cleaved from POMC. The syndrome may occur when the
highly tissue-specific promoter of the human POMC gene
(POMC) is activated. The mechanism of activation is not
fully understood. This promoter is embedded within a defined CpG
island, and CpG islands are usually considered to be unmethylated in
all tissues. We demonstrate that much of this CpG island is methylated
in normal nonexpressing tissues, in contrast to somatically expressed
CpG island promoters reported to date, and is specifically unmethylated
in expressing tissues, tumors, and the POMC-expressing
DMS-79 SCLC cell line. A narrow 100-bp region is free of methylation in
all tissues. E2F factors binding to the upstream domain IV region of
the promoter have been shown to be involved in the expression of
POMC in SCLC. We show that these sites are methylated in
normal nonexpressing tissues, which will prevent binding of E2F, but
are unmethylated in expressing tissue. Methylation in vitro
is sufficient for silencing of expression, which is not reversed by
treatment with Trichostatin A, suggesting that inhibition of expression
may be mediated by means other than recruitment of histone deacetylase
activity. The DMS-79 cells lack POMC demethylating
activity, implying that the methylation and expression patterns are
likely to be set early or before neoplastic transformation, and that
targeted de novo methylation might be a potential
therapeutic strategy.
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INTRODUCTION
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POMC is located on the short arm of chromosome 2 and is
normally expressed in the corticotrophs of the anterior pituitary gland
from a highly tissue-specific promoter upstream of exon 1 (1),
producing a transcript of 1,200 nucleotides that encodes the complete
POMC peptide. ACTH is cleaved from POMC. In man, ectopic ACTH secretion
from nonpituitary sites, most notably small cell lung cancer (SCLC) and
carcinoid tumors, leads to the hypercortisolemia of Cushings syndrome
with severe metabolic consequences including hypertension, sepsis,
diabetes mellitus, infertility, psychosis, osteoporosis, and myopathy
(2, 3). In a patient with rapidly advancing SCLC it is likely that this
represents a vastly under diagnosed form of Cushings syndrome with a
high level of comorbidity (4). In the pituitary, the tissue-specific
regulatory elements of the POMC promoter lie approximately -300 bp
upstream from the transcription initiation site (Fig. 1
) and bind the homeobox protein Ptx1 and
NeuroD1 [CE3 and corticotropin upstream transcriptional element
(CUTE), respectively] (5, 6, 7). In a model of nonpituitary expression a
region between 376 and 417 that binds E2F factors has been shown to
be specifically active in DMS-79 cells (SCLC cells) (8) that lack
NeuroD1 (9). The aberrant expression of this promoter seen in the
ectopic ACTH syndrome represents a loss of the normal tight
tissue-specific expression, but the mechanisms by which this occurs are
not fully understood.

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Figure 1. Schematic Representation of Human
POMC
The pituitary promoter is normally expressed in corticotroph cells and
pathologically in the ectopic-ACTH syndrome that causes Cushings
syndrome. Upper panel indicates CpG dinucleotides
represented by short vertical lines and,
above these binding sites for Ptx1, neuroD that binds as
a complex to the corticotroph upstream transcriptional element (CUTE),
and E2F. Lower panel indicates exon structure and
peptide product.
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Cytosine methylation is an epigenetic modification of mammalian DNA and
is associated with condensed chromatin and silencing of gene expression
(10). This is thought to occur mainly through the binding of a family
of methyl binding domain proteins (MBD) such as MeCP1 (11) and MeCP2
(12), and probably less importantly through the direct steric
inhibition of transcription factors binding to their response elements
(13). MBDs recruit reversible histone deacetylase activity and cause
stabilization of condensed chromatin, accounting for much of the gene
silencing (14, 15, 16, 17, 18). Methylation plays a role in tissue-specific gene
expression, with CpG sequences of non-CpG island genes being methylated
in most tissues but unmethylated in the expressing tissue (19).
Tissue-specific methylation patterns are established in
utero and maintained in the adult (20), although these may be
altered in neoplasia with regions of demethylation and de
novo methylation (21). Genome-wide de novo methylation
occurs in the postimplantation embryo and during development various
genes undergo demethylation and transcriptional activation in a
tissue-specific fashion (22).
CpG islands are regions of the genome of between 5002,000 bp in
length in which the G+C content exceeds 50%, and there is an absence
of the normal CpG depletion seen in vertebrate DNA, giving an observed
to expected (O/E) CpG density of more than 0.6 (23). By these
definitions the tissue-specific promoter of POMC lies within
a CpG island that extends to more than 400 bp upstream and 800 bp
downstream of exon 1 (24) (Fig. 1
). With the exception of X chromosome
inactivation (25), parentally imprinted genes (26), and some testes-
specific genes (27, 28), CpG islands are usually considered to be
fully unmethylated in all normal tissues regardless of the state of
gene expression (29). To date, variable methylation of somatically
expressed tissue-specific CpG islands has not been demonstrated,
although this might be a powerful means of ensuring inhibition of
expression in normal nonexpressing tissues. As a result of earlier data
on the 5'- flanking region of this gene, derived from
methylation-sensitive restriction enzyme analysis (30), we hypothesized
that this CpG island promoter might be differentially methylated in
expressing and nonexpressing tissues, contributing to the modulation of
the tissue-specific expression. We have studied the patterns of
methylation of POMC in human normal nonexpressing tissue,
and in expressing and nonexpressing neoplastic tissue, and we have
investigated the influence of methylation on gene expression. Our data
give novel insight into the possible role of tissue-specific CpG
islands and ectopic hormone production.
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RESULTS
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The Tissue-Specific POMC CpG Island Is Methylated in Nonexpressing
Tissues
The tissue-specific 5'-CpG island of human POMC (493
to +98) was studied using the bisulfite conversion technique and direct
sequencing to give a population average of the methylation pattern of a
given tissue (31). This approach allowed study of all CpG sites
regardless of the immediate surrounding sequences. Furthermore, the
tissue-specific region (5, 6, 7) does not lend itself to easy analysis
using methyl-sensitive restriction digestion analysis. Bisulfite
treatment of single-stranded DNA results in the deamination of
nonmethylated cytosines to uracil, whereas methylated cytosines remain
unaltered. After PCR amplification and sequencing, nonmethylated
cytosines appear as Ts in a sequencing gel, whereas only methylated
cytosines appear in the C lane. Using this method there was full
conversion of all non-CpG cytosines to thymidine, indicating
completeness of the bisulfite technique. As expected, the
POMC-expressing human SCLC DMS 79 cell line (32), bronchial
carcinoid tumors and pituitary corticotroph adenoma tissue that had
expressed POMC and synthesized and secreted ACTH in
vivo (all tumors showed positive immunostaining for ACTH, data not
shown), exhibited complete demethylation at all of the 34 CpG sites
examined (no Cs in the C lane; Fig. 2A
, sequences 13, and data not shown). In sharp contrast, in a non-ACTH
secreting pituitary adenoma and an islet cell tumor that secreted
insulin, but not ACTH, methylated cytosines were present at nearly
every site (Fig. 2A
, sequences 46). Interestingly, the CpG island was
heavily methylated in its upstream region in other normal nonexpressing
tissues including pancreas, spleen, kidney, and leukocytes, and cloning
and sequencing of the PCR products from bisulfite-treated DNA revealed
a heterogeneous pattern of methylation of the 5'-CpG island rather than
individual DNA molecules that were either fully methylated or
unmethylated (data not shown). Thus, in nonexpressing normal tissue and
tumors, the tissue-specific region of the CpG island is methylated and
a neuroendocrine or carcinoid cell type per se does not
confer the hypomethylated phenotype. The E2F binding region is
fully unmethylated in expressing tissue (Fig. 2B
). In contrast, this response element is
methylated in normal nonexpressing tissue and nonexpressing tumor
tissue. The pattern of methylation in the normal tissues is, however,
not completely homogenous. One explanation is that this represents
heterogeneity in the tissues. In splenic tissue each of the CpGs is
fully methylated, while in the other tissues there is variability of
the methylation of the CpGs except for the most 5'-CpG site
(lowest arrow), which is fully methylated in all
nonexpressing tissues. This is seen in lung and pancreas, suggesting
that some cells may exhibit an unmethylated cytosine at one or other of
these two sites (Fig. 2
).


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Figure 2. Bisulfite Sequencing Analysis of the Pituitary
Human POMC Promoter
A, Methylated cytosines appear as Cs, whereas nonmethylated cytosines
appear as Ts. Small arrows indicate the position of all
CpG dinucleotides. Full conversion of all non-CpG cytosines is seen (no
Cs in the C lane at non-CpG positions). In ACTH-secreting tissues
(lanes 13) the promoter is fully demethylated, whereas it is
heterogeneously methylated in the nonsecreting tissues (lanes 36).
The positions of the Ptx1 response element, the CUTE and TATA box, are
indicated on the right. Note that there are no CpG sites
within the Ptx1 RE or in the CUTE. B, Analysis of methylation status of
the E2F binding site: In normal nonexpressing tissue and in a
nonfunctioning pituitary adenoma the E2F sites in domain IV are
methylated, whereas in DMS-79 cells and corticotroph tissue this region
is unmethylated.
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The POMC CpG Island Is Unmethylated in a Narrow Central Region
Methylation of this tissue-specific region of the CpG island
in normal tissue prompted us to analyze the remainder of the CpG island
in more detail to assess whether the entire island was methylated in
nonexpressing tissues. We performed sequencing of individual molecules
amplified from 195 to +400 bp across the transcription initiation
site, including 61 CpG sites, which revealed a heterogeneous pattern of
methylation (Fig. 3
). Normal
nonexpressing lung, pancreas, and kidney were methylated at several
sites within this region, which lies in the middle of the CpG island.
Expressing corticotroph tissue exhibited an unmethylated pattern in two
of four molecules sequenced, and of the two methylated cytosines seen,
one was a CpNpG trinucleotide (34). In pituitary tissue, methylation
was apparent at a low density probably from DNA molecules derived from
nonexpressing noncorticotroph cells. A single pattern of methylation of
all molecules for a given tissue was not found. These data indicate
that CpG island methylation is present in normal nonexpressing tissue,
although there is a narrow 100 bp region of protection from methylation
in the center of the CpG island (in exon 1) and another region just
upstream of the TATA box (Fig. 3
). In expressing tissues many molecules
are completely free from methylation, consistent with their state of
activation. Overall, the tissue-specific region of this CpG island is
differentially methylated in expressing and nonexpressing tissues, but
the very center of the island is unmethylated in all tissues.

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Figure 3. The Center of the 5'-POMC CpG Island Is
Unmethylated in All Tissues
Bisulfite sequencing of individual molecules amplified from 195 to
+400 bp from the transcriptional start site of the POMC
pituitary CpG island. The upper panel represents the CpG
island to scale: vertical lines are individual CpG
dinucleotides. Filled gray ellipses represent Sp1
binding sites. Lower panel represents CpG methylation
density to scale: each row represents a single molecule; each
open circle represents an unmethylated CpG site; each
filled oval represents a methylated CpG site.
Filled circles on lines represent position of CpNpG
sites. A 150-bp region in the middle of the island is free of
methylation in all tissues. Nonexpressing normal lung, pancreas, and
kidney have methylated sites, whereas expressing tissues have several
molecules that are fully unmethylated.
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The Tissue-Specific Pattern of Methylation of the POMC Promoter Is
Region Specific
To test whether the differential pattern of methylation of
this CpG island was a region-specific phenomenon, or a more general
effect of neoplastic transformation, we analyzed a CpG-rich region of
18 CpG sites approximately 6 kb downstream in ACTH-secreting and
nonsecreting tumors. In contrast to the tissue-specific promoter, this
region has a methylated pattern in all the tissues studied, with all
the 18 CpG sites examined being methylated (Fig. 4
). Thus, the upstream 5'-CpG island is
specifically unmethylated in ACTH-secreting tissues and DMS-79 cell
line, and methylated in other tissues. This region-specific change
suggests that the process is not merely an effect of random changes in
methylation patterns due to oncogenesis.

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Figure 4. A CpG-Rich Region 6 kb Downstream Is Methylated in
All Tissues
Bisulfite sequencing of a region 6 kb from pituitary promoter in both
ACTH-secreting (lanes 13) and non-ACTH-secreting tumors (lanes 36).
CpG sites are methylated at most positions. A completely homogeneous
pattern is seen in the DMS-79 cells, lane 1.
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DNA Methylation Inhibits Transcription from the Pituitary CpG
Island Promoter in Vitro
To study the effects of methylation on the activity of this
promoter, we conducted transient transfection studies using luciferase
reporter constructs transfected into DMS-79 cells. Before transfection
a variable density of methylation was achieved using site- specific and
generalized methylases (Fig. 5A
) Full
methylation using the generalized methylase SssI resulted in
complete inhibition of expression, as assessed by luciferase activity,
and in comparison with the activity of the mock methylated plasmid,
whereas less dense methylation using FnuD II and
HpaII methylases caused reduction in expression, although
not as great as that seen for SssI (Fig. 6B
). This suggests
that there is a quantitative and context-related effect of cytosine
methylation. The unmethylated 5'-promoter construct was not active in
Hela cells, confirming that the tissue-specific transcriptional
machinery is also necessary for expression (data not shown). Incubation
of the transfected DMS-79 cells for 24 h with the specific
deacetylase inhibitor Trichostatin A (TSA) (35) failed to reverse the
inhibitory effects of methylation, suggesting that inhibition of
expression was not mediated solely by histone deacetylase (Fig. 5C
).
After TSA treatment, cells remained viable, with continued expression
of control reporter constructs. These data suggest that methylation
per se is affecting expression, findings that are consistent
with others (36), and that very little methylation is required to
greatly suppress gene expression.

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Figure 5. Effect of Methylation on the Expression of the POMC
Promoter
Histograms show arbitrary light units corrected for
transfection efficiency by analyzing Renilla luciferase
activity. For each experiment, 2.5 µg of 5'-POMC-LUC
and 150 ng of CMV-Renilla luciferase control vector were
transfected. The data presented are mean ± SD, n
= 3. A, Methylation sites of the human 5'-POMC promoter-luciferase
constructs. Vertical bars indicate CpG dinucleotides (to
scale); filled circles indicate methylated residues
formed by the site-specific methylases FnuDII and
HpaII and the generalized methylase SssI.
The lower panel indicates some of the important
regulatory elements. B, Luciferase activity in DMS-79 cells transfected
with 5'- POMC-LUC construct subjected to variable
methylation with the site-specific methylases FnuDII and
HpaII and either methylation or mock methylation with
the generalized methylase SssI, to introduce methylated
cytosines (as indicated in panel A). C, Luciferase activity of DMS-79
cells transfected with 5'-POMC-LUC with and without
treatment with TSA. In TSA experiments the media were replaced at
24 h with fresh media containing 100 ng ml-1 TSA. D,
Luciferase activity in DMS-79 cells transfected with
5'-POMC-LUC or Ptx1-MUT.
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Figure 6. Lack of Active Demethylation of the POMC Promoter
in DMS-79 Cells
Section of bisulfite sequencing of methylated 5'-POMC construct before
and 120 h after transient transfection into DMS-79 cells. This
promoter remained methylated at 120 h post transfection. The
magnified region indicates two faint bands in the T lane
at two CpG sites (indicated by arrows), which are
present in the right-hand panel after transfection.
These are not present before transfection and suggest a very low level
of demethylation.
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HpaII methylase introduces a single symmetrical methyl
moiety at a CpG site in the tissue-specific enhancer region near the
Ptx-1 response element (Fig. 2A
, arrow with asterisk), with
dramatic effects on expression (Fig. 5B
). The Ptx-1 response element
does not include, but is flanked by, CpG sites, which are methylated in
nonexpressing tissues (e.g., Fig. 2A
, sequence 6).
Site-directed mutagenesis of the Ptx1 binding site greatly inhibited
expression (Fig. 5D
), strongly suggesting that Ptx1, or Ptx1-like
factors, are important for expression in DMS-79 cells and that
methylation near the response element for these factors may inhibit
expression by means other than enhancing histone deacetylation.
POMC Is Not Actively Demethylated in Expressing Cells
No luciferase activity of the SssI-methylated construct
was detected up to 120 h after transfection, even though plasmid
was found to be present using PCR on nuclear extracts, suggesting that
demethylation of the transfected gene construct had not taken place.
Active demethylation has previously been reported to occur within
2 h, and be complete at 72120 h, after transfection of an
-actin gene promoter construct into myoblasts (37). We analyzed the
methylation status of the transfected constructs that had been
methylated in vitro with Sss1 before transfection
into the DMS-79 cells. PCR amplification and sequencing of the
transfected plasmids from bisulfite-converted nuclear extracts made
2120 h following transfection, showed evidence of a tiny degree of
demethylation at one or two CpG sites proximal to the central promoter
at 120 h following transfection (Fig. 6
). However, it in no way resembled the
completely demethylated pattern of the endogenous 5'-CpG island in the
DMS-79 cells (Fig. 2A
, lanes 13). From this we conclude that active
demethylation of this construct was not occurring in the DMS-79 cells,
which is consistent with the lack of luciferase activity observed at
120 h.
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DISCUSSION
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Methylation of the tissue-specific human POMC
CpG island promoter is a powerful means of repressing expression.
Germ-line-expressed genes with CpG island promoters may be methylated
in somatic tissue (27), ensuring maintenance of the repressed state
(28). Apart from the well established examples of genomic imprinting
(38) and X chromosome inactivation (25), evidence is gathering that
while the general maxim holds true that CpG islands are unmethylated in
all tissues, this is not so in every case. In addition to the data
presented here, a CpG island upstream of the chicken
-globin gene
cluster has recently been shown to be methylated in nonerythroid cells,
with repressive effects on expression (39). Here, the CpG island
promoter itself displays differential methylation. Our data may suggest
that in certain CpG islands (possibly those with a TATA box) protection
from methylation is more tightly defined than previously thought, and
that regulatory regions may be differentially methylated in expressing
and nonexpressing tissues. As defined by the percent G+C and O/E CpG,
this CpG island extends over a region of at least 1,200bp (23, 24).
Although the pattern of POMC methylation differs between
individual molecules, taking each tissue as a whole, nonexpressing
tissues are methylated. Methylated CpG dinucleotides may exert
repressive effects over several hundred base pairs (12). Therefore,
even though the transcriptional start site is free of methylation in
several of the molecules analyzed from normal nonexpressing tissues,
expression may be inhibited by methylated CpGs in the nearby sequences.
The patterns seen here, however, are different from those on the
inactive X chromosome in which all CpG dinucleotides of CpG islands are
methylated (25), and in other cancer cells in which methylation of
specific regions may be associated with heterochromatinization of the
transcriptional start site and gene silencing (39). CpG sites at the
edge of CpG islands may be methylated (40). Here, an unmethylated
region of 100 bp lies in the very center of this CpG island in all
tissues, suggesting this is not merely an edge effect. Certainly the
regulatory regions analyzed have a profound influence on expression.
The methylation patterns in the expressing cells do not appear to be
due to a nonspecific demethylation process associated with neoplastic
transformation since the downstream CpG sites are methylated in all the
tissues and tumors studied. In contrast, the pituitary promoter remains
methylated in nonexpressing tumors even if they are of neuroendocrine
origin. Therefore, although the entire CpG island is not fully
methylated in nonexpressing tissues, the level of methylation is far
higher than expected for a CpG island, and this correlates with the
patterns of expression seen for this gene.
Although it was not possible to completely recapitulate the methylation
patterns seen in vivo with our transient transfection
experiments, in most cases the repressive effects of methylation have
been documented to be due to a domain effect involving the density of
methylation rather than critical CpGs (13). A few methylated CpG
dinucleotides in this region appear to substantially diminish
expression. In each of these experiments the complete plasmids were
methylated before transfection. Here, there are 1.8-fold more
HpaII sites than FnuD II in the pGL3 backbone,
but methylation with HpaII methylase results in a 4-fold
greater reduction in expression. This suggests that although some of
the inhibition of expression may be being mediated by vector sequences,
the effects of methylation on the promoter insert itself mediate some
of the effect seen here. This is consistent with data in which distant
methylated sequences have been shown to influence promoter expression
(41). However, the effect of direct methylation of promoters is more
pronounced (15). Interestingly, in the expressing cells and tumors the
methylated 3'-region a few kilobases away does not inhibit the
endogenous POMC promoter, and this may suggest that for this
gene the more distant methylated sequences are not as important an
influence.
From our bisufite sequencing data the E2F-binding domain IV region of
the promoter that has been shown to be specifically active for
expression in DMS-79 cells (8) is methylated in normal nonexpressing
tissue. Since methylation of CpG sites of the response element for E2F
factors inhibits its binding (33), methylation in this region would
prevent activation by these factors. This would fit with the fact that
while there is no documented case of ectopic ACTH arising from splenic
tissue, lung and pancreas are two of the more common sites for ectopic
ACTH expression. Possibly, aberrant POMC expression may
arise in cells from these tissues that possess the necessary
transcription factors for expression and a fully demethylated
phenotype. Moreover, the promoter would need to be unmethylated in
SCLCs before such factors could bind and activate the gene. DMS-79
cells lack NeuroD1 (9), but activation of POMC in these
cells also appears to involve binding of Ptx1, or Ptx1-like proteins,
since a 2-bp mutation of the Ptx1 binding site reduces expression. Ptx1
does not have a CpG in its response element, but methylation nearby may
inhibit expression by other means and the repression may also be
mediated by MBDs. A common theme of repression by MBDs is the
recruitment of corepressors and histone deacetylase activity (15). The
role of MBDs in mediating the repression seen here does not appear to
be due to recruitment of histone deacetylase activity, but it is likely
that they are playing important inhibitory roles, possibly by direct
contact with the basal transcriptional machinery (42). For example, the
mechanism of repression mediated by MBD2 appears to vary depending on
the promoter studied. GAL4-MBD2 represses transcription of a human DNA
polymerase ß and ß-actin promoters through GAL-4 sites, but only
the polymerase ß repression is relieved by TSA (17). Similar
non-TSA-reversible mechanisms may be in play here. An alternative
explanation is that treatment with deacetylase inhibitors may induce
expression of variant histones that are repressive (43).
Active tissue-specific demethylation has been demonstrated for the
-actin gene transfected into myoblasts (37). Similar data exist for
the immunoglobulin
-gene (Ig
) introduced
into B cells and an insulin gene into a hamster insulinoma cell line
(44). In each case gene transfer to nonexpressing cells was not
associated with demethylation. These data support the concept that this
process is occurring through the action of specific
trans-acting factors binding to specific sequences in
cis to direct demethylating activity. Here, we found no such
process even though tissue-specific elements were present in the
transfected construct. One possibility that we cannot exclude is that
distant regions of the gene not present in the transfected plasmid are
necessary for demethylation. These data are consistent with those
showing that embryonal cells are required to demethylate a methylated
CpG island (45) and confirm that tissue-specific CpG island
demethylation or protection from de novo methylation is
likely to occur early in differentiation. CpG islands are thought to be
protected from methylation by the presence of Sp1 (40, 46), but in this
island four Sp1-binding sites (see Fig. 3
) appear incapable of
providing full protection from methylation. An alternative explanation
is that the island may become methylated in tissues where the gene is
not being actively transcribed (47).
Targeted de novo methylation is an attractive
therapeutic proposal since a single hit is likely to silence a specific
gene in subsequent cell generations. Zinc finger protein/Sss
I methyltransferase fusion proteins have been shown to target de
novo methylation in vitro (48). For success in
vivo, this strategy would rely on there being little if any
demethylating activity of the specified sequence in the target cell and
inhibition of expression with the introduction of methylation at only a
few CpG sites in a promoter. Ectopic POMC expression causing
Cushings syndrome is frequently a debilitating state that may fulfill
these criteria and potentially be amenable to such an approach.
Moreover, the mitogenic effects of peptides derived from POMC in SCLCs
may confer a growth advantage to these cells (49), and thus inhibition
of expression may be advantageous even in the absence of ectopic ACTH
secretion. Candidates for directing de novo methylation
include Ptx protein/methylase fusion proteins.
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MATERIALS AND METHODS
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Plasmids and Oligonucleotides
Oligonucleotides were designed to the human POMC
genomic sequence (50) and all primers were obtained from
Genosys, Cambridge, UK. A fragment of 493 to +98 that contains
the previously described positive transcriptional regulatory elements
of the 5'- promoter (5, 31) was amplified by PCR and cloned into the
MluI and BgI II sites of the pGL3-luciferase
plasmid (Promega Corp., Southampton, UK) to form
5'-POMC-LUC; pCMV-Renilla was obtained from
Promega Corp. A 2-bp mutation of the Ptx1 binding site
TGCTAAGCC to TGCTCCGCC was performed using oligonucleotides to the Ptx1
region and a Quikchange site mutagenesis kit
(Stratagene, UK) according to the manufacturers
instructions to form Ptx1-MUT. Orientation and sequence identity was
confirmed in all plasmids by sequencing.
Methylation in Vitro
Before transfection, site-specific methylation was achieved by
incubating plasmid DNA at 37 C for 16 h with FnuDII or
HpaII methylases, or total CpG methylation with the
generalized CpG methylase SssI (all New England Biolabs, Inc., Beverly, MA) using manufacturers recommended
conditions. The mock methylation was performed by omitting
S-adenosylmethionine from the methylation reaction. Plasmids
were phenol-chloroform extracted and ethanol precipitated before
transfection. Completeness of methylation by this process was checked
by bisulfite sequencing and extensive digestion with
HpaII.
DNA Extraction
Written informed consent was obtained from all patients
before the use of tumor/tissue samples in these studies. Samples were
stored at 70 C until extraction of DNA. Tumors studied included 1)
ACTH-secreting: corticotroph adenoma and bronchial carcinoid
tumors associated with Cushings syndrome and cure after excision, and
ACTH immunoreactivity on histology; 2) non-ACTH-secreting:
pancreatic insulinoma and nonfunctioning pituitary adenoma with no
evidence of Cushings syndrome and no ACTH immunoreactivity. Normal
tissue samples were obtained from healthy organs that were not involved
in the pathogenesis of a patients condition but were removed during
operative intervention and included kidney, spleen, whole blood, lung,
and pancreas. Samples were ground on dry ice and DNA extracted using a
Nucleon DNA extraction kit (ScotLabs, Strathclyde, UK),
according to the manufacturers protocol. DNA from whole blood and from
cells grown in culture was prepared using the same kit, according to
the manufacturers protocol.
Bisulfite Modification and Sequencing
Bisulfite modification was performed as described (31).
Briefly, 10 µg of genomic DNA were cut with PstI,
denatured with freshly prepared NaOH at a final concentration of 0.3
M for 15 min at 37 C, and then incubated under
oil at 50 C for 16 h in a final concentration of 3.1
M sodium bisulfite, 0.5 mM
hydroquinone (pH 5.0). Desalting was performed using a Promega Corp. DNA Clean Up System column, and the final desulfonation
was achieved by incubating the eluate with NaOH to a final
concentration of 0.3 M. The solution was then
neutralized with 3 M ammonium acetate (pH 5.2),
and precipitated with four volumes of ethanol. After two 70% ethanol
washes, the converted DNA was resuspended in 100 µl of deionized
water and stored at 20 C. The region of interest was amplified by PCR
using oligonucleotides designed to the bisulfite-modified sequence
(such that non-CpG cytosines are converted to uracils) so as to avoid
any CpG sites, since their methylation status was unknown. For direct
sequencing, each forward primer contained a tail corresponding to the
21 forward M13 sequence for use in dideoxy chain terminator
sequencing. Reverse primers were biotinylated at the 5'-ends:
5'-promoter oligonucleotides corresponding to a fragment 467 to + 30
of the 5'-promoter region: forward:
5'-GTAAAACGACGGCCAGTAGTGGAATAGAGAGAATATG,
3'-reverse: 5'-B-ACTCTTCTTCCCCTCCTT-3'; 3'-region
oligonucleotides: forward:
5'-TGTAAAACGACGGCCAGTGTGTGTTGTAGTTGTTAGTAG-3',
reverse: 5'-B-TCAACTCCCTCTTAAACTCC-3' (converted
genomic sequence in bold, tails in italics).
Direct sequencing was performed on the PCR products after purification
using streptavidin-coated magnetic beads (DynAl, Oslo,
Norway) using the dideoxy chain terminator method and a Sequenase
sequencing kit (Amersham Pharmacia Biotech,
Buckinghamshire, UK). Individual molecules of these products
were cloned into pGEM T and sequenced as above. Individual molecules
from PCR products flanking the transcriptional start site (195 to
+400) were cloned into pGEM 3 using EcoRI and
XbaI restriction tails in the primers and were then
sequenced using Big Dye chain terminators and Taq-FS, and
the products were resolved on a 377 sequencer (all Applied Bio Systems
Ltd., Warrington, UK). For the sequencing of transfected
plasmids, nuclear extracts were prepared as described (37) and
subjected to bisulfite conversion. Using the same 5'-forward PCR primer
and a biotinylated reverse primer designed to the converted pGL3
backbone sequence (+151 to +162), plasmid-specific amplification and
sequencing were performed at 2, 24, 48, 71, 96, and 120 h
following transfection.
Cell Culture and Transient Transfection Assays
DMS-79 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI-1640
(Sigma-Aldrich, Poole, UK) supplemented with 10%
FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml
streptomycin (all Life Technologies, Ltd., Paisley,
UK) under 5% CO2. Cells were passaged 24 h
before transfection. Transfections were performed on
105 cells (seeded per 3-cm well in six-well
plates). 5'-POMC-LUC (2.5 µg) and 150 ng of cytomegalovirus
(CMV)-Renilla control per experiment were incubated for 10
min at room temperature with 15 µl of DOTAP (Roche Diagnostics, Lewes, UK) and used according to recommended
conditions. The cells were then cultured for a further 48 h before
assay. Cell extracts were prepared using Passive Lysis Buffer and
analyzed with the Dual Luciferase kit (both Promega Corp.)
according to the manufacturers instructions. Efficiency of
transfection was controlled for in each experiment by assaying the
Renilla luciferase activity; all results are expressed
corrected in this fashion above background. In the TSA experiments the
media were replaced at 24 h with fresh media containing 100 ng
ml-1 TSA (Sigma).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. John Newell-Price, Section of Medicine, Sheffield University, Clinical Sciences, Northern General Hospital, Herries Road, Sheffield, S5 7AU, United Kingdom. E-mail:
j.newellprice{at}sheffield.ac.uk
This work is supported by the Medical Research Council, UK (J.N.-P.),
The Welcome Trust (P.K.), and The Society for Endocrinology, UK.
Received for publication August 2, 2000.
Revision received October 26, 2000.
Accepted for publication November 15, 2000.
 |
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