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 Bartholomew’s and Royal London School of Medicine and Dentistry West Smithfield, London, EC1A 7BE, United Kingdom


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ectopic secretion of ACTH, from sites such as small cell lung cancer (SCLC), results in severe Cushing’s 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Cushing’s 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 Cushing’s 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. 1Go) 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 Cushing’s 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.

 
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 500–2,000 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. 1Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 T’s 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 C’s in the C lane; Fig. 2AGo, sequences 1–3, 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. 2AGo, sequences 4–6). 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. 2BGo). 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. 2Go).




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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 1–3) the promoter is fully demethylated, whereas it is heterogeneously methylated in the nonsecreting tissues (lanes 3–6). 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.

 
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. 3Go). 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. 3Go). 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.

 
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. 4Go). 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 1–3) and non-ACTH-secreting tumors (lanes 3–6). CpG sites are methylated at most positions. A completely homogeneous pattern is seen in the DMS-79 cells, lane 1.

 
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. 5AGo) 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. 6BGo). 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. 5CGo). 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.

 
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. 2AGo, arrow with asterisk), with dramatic effects on expression (Fig. 5BGo). The Ptx-1 response element does not include, but is flanked by, CpG sites, which are methylated in nonexpressing tissues (e.g., Fig. 2AGo, sequence 6). Site-directed mutagenesis of the Ptx1 binding site greatly inhibited expression (Fig. 5DGo), 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 72–120 h, after transfection of an {alpha}-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 2–120 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. 6Go). However, it in no way resembled the completely demethylated pattern of the endogenous 5'-CpG island in the DMS-79 cells (Fig. 2AGo, lanes 1–3). 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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 {alpha}-actin gene transfected into myoblasts (37). Similar data exist for the immunoglobulin {kappa}-gene (Ig{kappa}) 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. 3Go) 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 Cushing’s 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 manufacturer’s 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 manufacturer’s 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 Cushing’s syndrome and cure after excision, and ACTH immunoreactivity on histology; 2) non-ACTH-secreting: pancreatic insulinoma and nonfunctioning pituitary adenoma with no evidence of Cushing’s syndrome and no ACTH immunoreactivity. Normal tissue samples were obtained from healthy organs that were not involved in the pathogenesis of a patient’s 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 manufacturer’s 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 manufacturer’s 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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