MOLECULAR AND CELLULAR BIOLOGY, July 1996, p. 3245–3254 Vol. 16, No. 70270-7306/96/$04.0010Copyright q 1996, American Society for Microbiology

Site-Specific Methylation of the Rat Prolactin and GrowthHormone Promoters Correlates with Gene Expression

V. NGO,* D. GOURDJI,† AND J.-N. LAVERRIERE‡

Groupe de Biologie de la Cellule Neuroendocrine, Unite de Recherche Associee 1115, Centre Nationalde la Recherche Scientifique, College de France, 75231 Paris Cedex 05, France

Received 25 September 1995/Returned for modification 6 December 1995/Accepted 1 April 1996

The methylation patterns of the rat prolactin (rPRL) (positions 2440 to 220) and growth hormone (rGH)(positions 2360 to 2110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, theanterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilitiesto express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylationof the CpG dinucleotides located at positions 2277 and 297, near or within positive cis-acting regulatoryelements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expressioncoupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNAmethylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase re-porter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in theactivity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of theinhibition was also found to be dependent on the position of methylated sites. Taken together, these datasuggest that site-specific methylation may modulate the action of transcription factors that dictate thetissue-specific expression of the rPRL and rGH genes in vivo.

In mammals, DNA methylation is believed to ensure thesilencing of tissue-specific genes in nonexpressing cells whiledefined combinations of transcription factors dictate the ex-pression of undermethylated genes in specialized cells (14).The coexistence of these two mechanisms raises questionsabout their reciprocal interactions. The rat prolactin (rPRL)and rat growth hormone (rGH) genes belong to the class oftissue-specific genes and, as such, provide models with which toaddress these questions.In the anterior pituitary gland (AP), the restricted pattern of

expression of the rPRL and rGH genes is determined by thepresence of the POU homeodomain transcription factor Pit-1/GHF-1 (2, 42, 45, 56). Multiple cis-acting elements that bindPit-1 in the promoter regions of both genes have been charac-terized (45). Additional factors acting in combination withPit-1 are required for the selective activation of the rPRL andrGH genes in two related cell types, the lactotropes and so-matotropes, respectively. The estrogen receptor, the octamer-binding protein Oct-1, and the recently identified LIM home-odomain protein P-Lim/mLIM-3 are potential cofactors forthe tissue-specific expression of the rPRL gene (1, 13, 29, 62,64, 71). Likewise, the T3 receptor, the zinc finger proteinZn-15, and the ubiquitous transcription factor Sp1 appear tocooperate with Pit-1 with regard to expression of the rGH gene(19, 41, 61; for a review, see reference 69). In addition, Pit-1participates in the protein kinase A (PKA)- and PKC-medi-ated regulation of both the rPRL and the rGH genes (22, 48,53; reviewed in references 20 and 69).

The importance of DNA methylation in the regulation ofrPRL and rGH gene expression has been far less explored.Nonetheless, all studies revealed an inverse coupling betweenCpG methylation and rPRL and rGH gene expression, in APcell lines as well as in normal tissues. This conclusion wasdrawn from investigations that combined DNA methylationanalysis and gene reactivation by the DNA-demethylatingagent 5-azacytidine (10, 36, 72). In all cases, methylation pat-terns were assessed by Southern blot analyses, following actionof methylation-sensitive endonucleases which targeted only asmall proportion of CpG sites. As a result, the methylationstates of the CpG sites lying within or in the vicinity of cis-regulatory elements that bind the transcription factors citedabove are unknown. Evaluation of the methylation states ofsuch sites would be of critical interest, since it is believed thatDNA methylation inhibits gene expression by altering thebinding of transcription factors.By means of genomic sequencing, several groups have in-

deed presented evidence linking site-specific methylation inpromoter regions and transcriptional inactivity (54, 58). How-ever, the great majority of the data collected concerns GC-richand TATA-less promoters (11) and 59 upstream regions ofX-linked housekeeping genes that are associated with CpGislands (7, 26, 50, 51). The implication of promoter methylationin gene regulation is further supported by results from func-tional analyses performed by transfection assays. Reportergene constructs that are artificially methylated and introducedinto cultured cells display an inhibited level of transcription(for a review, see reference 14). Methylation within the preini-tiation domain exhibits the strongest correlation with repres-sion of transcription (38). However, the inhibition of promoteractivity is also proportional to the extent of CpG methylation(9, 27, 32), and modification of coding (33) or vector (32, 52)sequences is effective as well. Hence, it would be useful toanalyze the functional role of promoter methylation regardingtissue-specific gene expression.As in many, but not all, tissue-specific genes, the promoter

regions of the rPRL and rGH genes exhibit rather low GC

* Corresponding author. Present address: Endocrinologie Cellulaireet Moleculaire de la Reproduction, Universite Pierre et Marie Curie,CNRS URA 1449, 4 place Jussieu, Case 244, 75252 Paris Cedex 05,France. Phone: 33-1-44-27-26-60. Fax: 33-1-44-27-26-50.† Present address: Dynamique des Interactions Neuroendocrines,

INSERM U159, Centre Paul Broca, 75014 Paris, France.‡ Present address: Endocrinologie Cellulaire et Moleculaire de la

Reproduction, Universite Pierre et Marie Curie, CNRS URA 1449,75252 Paris Cedex 05, France.

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contents (38% for rPRL and 51% for rGH) and contain aTATA box. Furthermore, the consensus sequence for Pit-1binding is AT rich (16). Therefore, it is not at all self-evidentthat the mechanisms whereby DNA methylation operates onsuch genes are similar to those preventing transcription ofgenes with GC-rich and TATA-less promoters (5). Thus, theaim of this study was to structurally and functionally investigateCpG methylation in relation to the positions of well-charac-terized regulatory elements in the rPRL and rGH promoters.First of all, we analyzed the occurrence of CpG methylation inall four CpG sites of the proximal rPRL promoter, locatedbetween positions 2440 and 220, and in nine CpG sites of therGH promoter, located between positions 2360 and 2110(Fig. 1). This analysis was performed by genomic sequencing ofbisulfite-treated DNA extracted from different rat pituitaryGH3 cell lines (21, 35, 36, 47) according to the method ofFrommer et al. (18). The tumor-derived cell lines are of greatinterest, since they differ in their levels of rPRL and rGHmRNAs by several orders of magnitude while expressingequivalent amounts of the Pit-1 protein. Furthermore, theyalso display similar abilities to express a transfected rPRLpromoter-chloramphenicol acetyltransferase (CAT) construct(47). The same analysis was also performed on DNA originat-ing from the AP and the liver, in which rPRL and rGH geneexpression is high or not detectable, respectively (28). Second,we performed a functional study by selectively methylatingeither the entire rPRL promoter or its distal or its proximalregion before insertion in a CAT expression vector used in atransient-transfection assay.

MATERIALS AND METHODS

Cell lines, culture conditions, and measurement of rPRL and rGH mRNAlevels. The cell lines used all originated from the GH3 tumor-derived cell line(21). They were obtained in this laboratory by subcloning (GH3B6) or selected byalteration of culture conditions (GH3CDL) and action of the demethylatingagent 5-azacytidine (GH3AZA3) (8, 21, 36, 47). Levels of rPRL and rGHmRNAs were measured by dot blot assay, using a 32P-labeled cDNA probe asdescribed by Laverriere et al. (37). The number of mRNA copies per cell wascalculated by assuming 15 pg of total RNA per cell.DNA isolation and bisulfite treatment of genomic DNA. Genomic DNA was

prepared as described by Laverriere et al. (36) and Sambrook et al. (59). Bisulfitetreatment was performed by the method described by Frommer et al. (18), asmodified by Clark et al. and Feil et al. (12, 17). As a control for the full andspecific conversion of cytosines to uracils, we sequenced bisulfite-treated andPCR-amplified pPRL-CAT plasmids, either native or in vitro methylated. Theresults presented here were obtained by analyzing five independent series ofbisulfite treatments, all yielding comparable results.PCR amplifications. Amplifications were performed with 100-ml reaction mix-

tures containing 10 ml of bisulfite-treated DNA, as described by Frommer et al.(18), in the presence of 0.6 U of Taq polymerase (Appligene, Illkirch, France),using a Crocodile II thermocycler (Appligene). The amplification procedureconsisted of 2 min of denaturation at 948C followed by 30 to 45 cycles at 948C (1min), 558C (2 min), and 728C (3 min), ending with 7 min of extension at 728C. Alloligodesoxynucleotides were obtained from Eurogentec (Sart Tilman, Belgium).For amplification of the bisulfite-modified top and bottom strands of the rPRLpromoter, we designed the primer set P1 and P2 and the primer set P3 and P4,respectively, as follows: P1, 59-TGAAGTTAATATATTTTGGTATTTAGTGGA-39; P2, 59-CCACTACTTTCTCATCTACAAACATTAACT-39; P3, 59-TTACATTTTTAAATTAAAATCAACATACCT-39; and P4, 59-AATTATTGTTTTTTTATTTGTAGATATTGA-39.The bisulfite-modified top and bottom strands of the rGH promoter were

amplified with the primer set G1 and G2 and the primer set G3 and G4, re-spectively, as follows: G1, 59-AAATTAAGAGGAATAAGATATTATGGGGA-39; G2, 59-CATAACTAAAACCACTAACAACTTATACTA-39; G3, 59-TTGGAGTTATTGATAGTTTGTGTTGATGGA-39; and G4, 59-AATTACCTTCTAATCCCTACATATACACAC-39. None of the original sequences correspond-ing to the primers contained CpG dinucleotides.Cloning of PCR products and sequencing. Amplified DNA was purified by gel

electrophoresis and cloned into a pGEM-T vector (Promega, Charbonniere,France). Sequences were obtained from double-stranded plasmid templates byusing the thermal cycle sequencing method with the same primers used for PCR(65). The amplified fragments were resolved on 6 or 7% Hydrolink Long Rangerdenaturing gels as recommended by the supplier (Bioprobe Systems, Montreuil,France). Vacuum-dried gels were exposed to Kodak X-Omat AR films without

an intensifying screen for 4 to 24 h at room temperature. Nine to 34 clones weresequenced per DNA strand, corresponding to a total of 462 clones.Plasmid constructions and in vitro DNA methylation. The p0.4kbPRL-CAT

plasmid has been described previously (46) and contains the proximal rPRLpromoter (positions 2427 to 136) inserted between the HindIII and XbaIsites of the multiple cloning site of the pCAT-Basic vector (Promega). ThepGEMPRL-CAT expression vector contains the 2-kb rPRL promoter andwas designed specifically for selective methylation of promoter sequences. TheHindIII site located at position2427 in the 2-kb upstream sequence of the rPRLpromoter was replaced by an NheI site and inserted into the pGEM3Zf(2)vector between the HindIII and BamHI sites located in the polylinker regiontogether with the CAT-simian virus 40 region originating from the pCAT-Basicvector. The resulting construct contained three unique restriction sites (HindIII,NheI, and XbaI) delimiting the distal region (positions 21973 to 2427) andproximal region (positions 2427 to 136) of the rPRL promoter. It displayed thesame activity in a transient-transfection assay as the pPRL-CAT expressionvector kindly provided by C. Bancroft (47).In the first set of experiments, the whole constructs were methylated by using

either HpaII methylase (M z HpaII), HhaI methylase (M z HhaI), or SssI meth-ylase (M z SssI) (2 U/mg of DNA, 2 h at 378C) (New England Biolabs). Com-pletion of methylation was checked by HpaII or HhaI digestion. Mock-methyl-ated constructs were obtained by treating plasmids in the absence of methylase.In a second series of experiments, the pGEMPRL-CAT or the p0.4kbPRL-CATconstruct was regionally methylated. DNA fragments corresponding to the entire(positions 21973 to 136), distal (positions 21973 to 2427), or proximal (posi-tions 2427 to 136) rPRL promoter region were excised by restriction enzymedigestions. DNA fragments were then purified by gel electrophoresis and meth-ylated at all CpG sites with M z SssI (10 U/ml, 2 U/mg of DNA, 2 h at 378C), ormock methylated. M z SssI was then inactivated at 658C for 15 min, and meth-ylation in the entire or distal rPRL promoter was checked by HhaI digestion.pGEMPRL-CAT and p0.4kbPRL-CAT were then reconstituted by ligation ofthe promoter with the unmethylated plasmid DNA fragment and purified byphenol-chloroform extraction and ethanol precipitation. The plasmid concentra-tions were determined by measuring the A260.Transfection. GH3AZA3 cells were transiently transfected by electroporation

as previously described (47), using 10 to 30 mg of plasmid DNA per 12 3 106

cells. The pCMV-bgal expression vector kindly provided by J. A. Martial (Liege,Belgium) was used as an internal control for transfection efficiency when indi-cated.Statistics. Statistical comparisons between methylation patterns obtained by

genomic sequencing were performed by using the two-sided test of exact prob-abilities in 23 2 contingency tables (66). The confidence levels used were 95 and99%, indicated by P , 0.05 and P , 0.01, respectively. For transfection exper-iments, statistical analyses were performed by one-way analysis of variance fol-lowed by Fisher’s test.

RESULTS

The characteristics of the GH3 cell lines used have beendescribed previously (35, 36, 47). The constitutive levels ofrPRL and rGH mRNAs were nevertheless reevaluated herein two independent experiments by dot blotting analysis, us-ing cells grown under serum-free culture conditions. TheGH3CDL cells contained limited amounts of both rPRL andrGH mRNAs ('50 copies per cell). The GH3AZA3 cell vari-ant (47) expressed increased contents of rPRL mRNA ('400copies per cell) and rGH mRNA ('1,700 copies per cell).Finally, the GH3B6 cells produced considerable amountsof rPRL mRNA ('7,000 copies per cell) and rGH mRNA('6,000 copies per cell).Genomic sequencing revealed that the CpG located at po-

sition 236 in the rPRL promoter sequence of the Sprague-Dawley rat strain was replaced by a CpT in that of the Wistar-Furth strain from which cell lines and adult tissues originated.Because of this genetic polymorphism, the rPRL promoteranalyses thus concerned only four CpG sites, located at posi-tions 2373, 2277, 2252, and 297.Methylation of the proximal rPRL promoter in GH3 cell

lines. The analysis of GH3B6 DNA showed a virtual absence ofunconverted cytosine at all four CpG sites, in a total of 12 and21 clones analyzed for the top and bottom strand, respectively(data not shown). This observation indicated that the proximalrPRL promoter was extensively unmethylated in GH3B6 cells,which express the rPRL gene at a high level. By contrast, analy-sis of genomic DNA extracted from GH3CDL and GH3AZA3

3246 NGO ET AL. MOL. CELL. BIOL.

cells revealed that their promoter sequences were both meth-ylated. Unexpectedly, methylation patterns were found to behighly heterogeneous, as illustrated by the three examplesshown in Fig. 2 (lanes C1 to C3). Furthermore, the 16 (24)theoretical patterns generated by the combination of methyl-ated and unmethylated CpG sites were actually observed

among the 235 clones analyzed, which included those derivedfrom normal tissues (not illustrated). Facing this diversity, weperformed statistical comparisons between the top and thebottom strand to determine whether the methylation of CpGsites was symmetrical in each type of DNA. In GH3CDL cells,the top strand was less methylated than the bottom strand at

FIG. 1. Genomic sequencing strategy for CpG sites and regulatory elements in the rPRL (A) and rGH (B) promoters. CpG dinucleotides and their locations(vertical lines on top and bottom strands with numbers) and the positions of the four sets of primers used for PCR amplification following bisulfite modifications ofDNA (arrows) are indicated. The coding, or sense, strands of the rPRL and rGH promoters were amplified with primer set P1 and P2 and primer set G1 and G2,respectively, and referred to as the top strand in the text. Alternatively, the noncoding, or antisense, strands were amplified with primer set P3 and P4 and primer setG3 and G4, respectively, and referred to as the bottom strand in the text. Boxes symbolize the locations of regulatory elements indicated below. cis-acting elementsare indicated above or below each box with their upstream and downstream boundaries and the names of related transcription factors in parentheses. 1P through 4P,Pit-1 or GHF1 binding sites 1 through 4; FP1, footprint 1; LSF-1, lactotrope specific factor 1; BTF, basal transcription factor; RRE, Ras response element (6); TRE,thyroid hormone response element; and NF1, nuclear factor 1.

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position 2252 (P , 0.05). No statistical difference was ob-served for the other positions in the three DNAs analyzed (11cases). We could thus conclude that methylation of the rPRLpromoter occurred rather symmetrically in GH3 cell lines.However, despite the heterogeneity of methylation patterns

within a given cell line, there were significant differences be-tween cell lines (Fig. 3A). Statistical comparisons betweenDNAs were performed with the top and bottom strands con-sidered together because they present overall symmetricalmethylation patterns. In GH3CDL cells, which express therPRL gene at a very low level, sites2373,2277, and297 weremore methylated than in GH3B6 cells (P , 0.01) (Fig. 3A).The average level of methylation per individual rPRL pro-moter molecule increased from 0 in GH3B6 cells to 1.7 (i.e.,42.5%) methylated sites in GH3CDL cells. The GH3AZA3DNA displayed a significantly lower methylation level thanGH3CDL DNA for the sites at positions 2373 (P , 0.01),2277 (P , 0.01), and 297 (P , 0.05) (Fig. 3A), with anaverage of 1.1 (27.5%) methylated sites per individual mole-cule. Comparison of GH3B6 and GH3AZA3 rPRL promotermethylation patterns revealed that sites at positions 2277,2252, and 297 were significantly more methylated inGH3AZA3 than in GH3B6 DNA (P, ,0.01, ,0.05, and ,0.01,respectively) (Fig. 3A). These analyses thus demonstrated aninverse correlation between gene expression and promotermethylation in GH3 cell lines.

Methylation of the proximal rPRL promoter in adult tis-sues. In contrast to what was observed with the cell lines, aclear-cut asymmetry of methylation patterns was obvious be-tween the top and bottom strands from the AP and the liver,except at site 2373 in the latter (Fig. 3B). The bottom strandsfrom the AP and liver rPRL promoters were found to besimilarly unmethylated while their top counterparts differed, asshown by statistical analysis. The average level of methylationwas 2.1 (53%) methylated sites per molecule of rPRL pro-moter in the liver compared with 1.2 (30%) in the AP, whilethe percentage of fully unmethylated rPRL promoter mole-cules was increased from 14% in the liver to 54% in the AP.Levels of methylation at positions 2277 (P , 0.05) and 297(P , 0.01) were significantly lower in the AP than in the liver(Fig. 3B). The methylation status of these CpGs was thusinversely correlated with the level of rPRL gene expression.Methylation of the rGH promoter in GH3 cell lines. As

already observed for the rPRL promoter, the methylation pat-tern of the rGH promoter was obviously heterogeneous in eachof the three cell lines. Six clones derived from the rGH pro-moter of GH3B6 cells displayed six different methylation pat-terns. Similarly, five clones derived from GH3CDL cellsshowed five distinct methylation patterns (Fig. 4). With nineCpG sites, there were 512 (29) theoretical methylation pat-terns. Among the 227 clones analyzed, including those derivedfrom normal tissues, 63 different methylation patterns wereactually observed. Nevertheless, statistical comparisons indi-cated that the methylation patterns of top and bottom strandswere symmetrical in DNAs from GH3B6 and GH3AZA3 cells.

FIG. 3. Percentage of DNA methylation at individual CpG sites in the prox-imal rPRL promoter. Data obtained from genomic sequencing of bisulfite-treated DNA were compiled by individual CpG dinucleotide and expressed asthe ratio of methylC to the number of clones. (A) Cell lines. The followingnumbers of clones were analyzed for the top and bottom strands, respectively: forGH3CDL cells (open bars), 20 and 22; for GH3AZA3 cells (hatched bars), 24 and14; and for GH3B6 cells (black bars), 12 and 21. (B) Tissues. The followingnumbers of clones were analyzed for the top and bottom strands, respectively: forliver tissue (open bars), 21 and 21; and for AP tissue (black bars), 39 and 34.

FIG. 2. Genomic sequencing and methylation analysis of clones derived fromthe rPRL proximal promoter. The positions of CpG sites are indicated (arrow-heads). (A) Methylation analyses of clones derived from the top strand of theproximal rPRL promoter of GH3CDL cells. Unmethylated C’s were converted toT’s, whereas 5-methylC remained as C. (B) Methylation analyses of clonesderived from the bottom strand of the proximal rPRL promoter of GH3AZA3cells. Conversion of C to T is shown by complementary G-to-A replacements.

3248 NGO ET AL. MOL. CELL. BIOL.

This contrasted with the asymmetry existing between the topand bottom strands in DNA from GH3CDL cells at sites 2200(P , 0.01), 2197 (P , 0.01), 2174 (P , 0.05), 2149 (P ,0.05), and 2147 (P , 0.01). Despite this diversity and similarlyto what was observed for the rPRL promoter, significant dif-ferences between cell lines were detected at individual CpGsites. The analysis of the rGH promoter from GH3B6 cells, themost active of the three cell lines, revealed an elevated averagemethylation level of 4.8 (53.3%) methylated sites per molecule.This value was unexpectedly the highest observed and wasclose to that found for DNA from GH3CDL cells (4.1 [45.5%]methylated sites per molecule), the cell line with the lowestlevel of expression. The GH3AZA3 rGH promoter exhibitedthe lowest methylation level, with an average of 2.4 (26.7%)methylated sites per molecule. The whole analysis is summa-rized in Fig. 5A. As for the rPRL promoter, statistical com-parisons between DNAs were performed by considering thetop and bottom strands together. The three sites located atpositions2335,2319, and2231 were found significantly moremethylated in GH3B6 than in GH3CDL DNA (P, ,0.01,,0.05, and ,0.01, respectively). This result revealed that hy-permethylation of these CpG sites was compatible with a highlevel of rGH expression. Conversely, the comparison ofGH3CDL and GH3AZA3 DNAs showed that lower levels ofmethylation at positions 2319, 2231, 2200, 2197, and 2149(P, ,0.01, ,0.05, ,0.01, ,0.05, and ,0.01, respectively) co-incided with higher levels of rGH gene expression. Further-more, the proportion of fully unmethylated rGH promotermolecules increased from 7 of 52 in GH3CDL DNA to 17 of 52in GH3AZA3 DNA.Methylation of the rGH promoter in adult tissues. Unlike

the rPRL promoter, the top and bottom DNA strands of therGH promoter displayed symmetrical methylation patterns inthe two normal tissues analyzed (Fig. 5B). As expected fromthe lack of rGH gene expression, the liver promoter was almost

completely methylated, with an average of 7.4 (82.2%) meth-ylated sites per molecule. This value contrasted with the aver-age level of methylation found in AP DNA of 3.2 (36%) meth-ylated sites per molecule (Fig. 5B). In addition, statisticalcomparisons of liver and AP methylation patterns, performedby considering the top and bottom strands together, indicatedthat all nine CpG sites were significantly less methylated in APDNA (P , 0.01). Furthermore, the proportion of fully unmethy-lated molecules increased from 3% in the liver to 36% in the AP.Altogether, these results and those regarding the rPRL gene

have established an inverse relation between methylation ofpromoter sequences and levels of gene expression in five of thesix situations analyzed.Regional methylation of the rPRL-CAT reporter gene. We

then performed experiments to evaluate the functional role ofpromoter methylation in gene expression, limiting our analysisto the rPRL gene. For this, we designed the expression vectorpGEMPRL-CAT (see Materials and Methods and Fig. 6). Theinfluence of methylation was monitored by transfecting con-structs into GH3AZA3 cells thereafter cultured for 48 h undercontrol conditions or treated with TF (1 mM forskolin and 16nM tetradecanoyl phorbol acetate [TPA]).Methylation of specified regions of the promoter resulted in

various levels of reporter gene expression (Fig. 7, constructs athrough d). The greatest inhibition was observed with con-struct d, which contained the 2-kb rPRL promoter bearing 14methylated CpG sites. The inhibition levels in untreated cellsand TF-treated cells were equivalent, reaching (57 6 7)% and(61 6 5)%, respectively. Compared with that obtained withconstruct d, methylation of the nine CpG sites located in thedistal rPRL promoter region led to significantly (P , 0.05)weaker inhibition in untreated as well as in TF-treated cells[construct c, (23 6 4)% and (28 6 2)%]. The decrease in geneexpression caused by the methylation of the five CpG siteswithin the proximal promoter (a and b constructs) was thelowest observed in untreated cells, reaching (17 6 1)% and(166 7)%, respectively. By contrast, in TF-treated cells, meth-ylation of the same five CpGs resulted in a strong inhibitoryaction that reached (61 6 4)% and (41 6 12)% for the a andb constructs, respectively. Such inhibitions were significantlydifferent from those observed in untreated cells (P , 0.05).The responsiveness of the proximal promoter thus differedfrom that of the distal or entire promoter, in which methylationaltered in parallel constitutive and TF-stimulated expression.To determine whether methylation in the region surround-

ing the rPRL promoter might also be efficient in regulatinggene expression, we methylated the pGEMPRL-CAT plasmid,using either M z HpaII or M z HhaI. The resulting constructscontained CpG sites lying in vector sequences (Fig. 7A, con-structs e and f), except for one site located in the distal pro-moter (construct f). The number of methylated sites (i.e., 16and 18) was of the same order as in the entire promoter (14sites). After methylation with M z HpaII, we observed a smallinhibition of CAT expression in untreated cells [(16 6 3)%]and no significant inhibition in TF-treated cells (Fig. 7B). Bycontrast, HhaI-methylated sites yielded a potent inhibition ofCAT activity, which reached (396 18)% in untreated cells and(44 6 9)% in TF-treated cells. Finally, after full in vitro meth-ylation by M z SssI, CAT expression was totally abolished (Fig.7B, construct g) (see also reference 47).Taken together, these results indicated that methylation in

promoter sequences and in surrounding regions repressedgene expression. Furthermore, the extent of inhibition ap-peared to be dependent on both the number of methylatedCpG sites and their locations.

FIG. 4. Genomic sequencing and methylation analysis of clones derived fromthe rGH proximal promoter. Experiments were carried out as described for Fig.3. The positions of the nine CpG sites analyzed are indicated (arrowheads).Clones are derived from GH3B6 DNA (lanes B6 to B11) and GH3CDL DNA(lanes C5 to C9).

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DISCUSSION

To determine the relationship between expression andmethylation in the rPRL and rGH genes, we have analyzed themethylation state of several CpG sites within their promoterregions by using bisulfite genomic sequencing. This procedure

provides high-resolution data which allow a detailed map ofmethylated sites to be drawn. Three subclones of pituitarytumor-derived cell lines and two normal tissues, the AP andthe liver, which differ in their ability to express the rPRL andrGH genes, were examined. Comparative analyses establish an

FIG. 5. Percentage of DNA methylation at individual CpG sites in the rGH promoter. Calculations were performed and expressed as reported for Fig. 3. (A) Celllines. The following numbers of clones were analyzed for the top and bottom strands, respectively: for GH3CDL cells (open bars), 29 and 23; for GH3AZA3 cells(hatched bars), 27 and 25; and for GH3B6 cells (black bars), 29 and 23. (B) Tissues. The following numbers of clones were analyzed for the top and bottom strands,respectively: for liver tissue (open bars), 27 and 12; and for AP tissue (black bars), 23 and 9.

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inverse correlation between the extent of promoter methyl-ation and the level of gene expression in all but one case tested.In addition, we have shown that targeted methylation of rPRLpromoter regions yields significant inhibition of reporter genesin transient-transfection assays. Altogether, data obtained bytwo independent and complementary approaches argue for akey role of CpG methylation in rPRL and rGH promoteractivities.The results obtained from genomic sequencing on CpG sites

lying in promoter regions are consistent with previous analysesusing HpaII and MspI restriction enzymes in conjunction withSouthern blotting (36). By this method, we observed that themethylation status of three sites in the rPRL gene and of fivesites in the rGH gene was correlated with the level of geneexpression. Likewise, the methylation status of the rGH genein the GH3B6 cell line had already appeared as an exception,with the gene being hypermethylated despite a high level ofexpression. However, a major advance is made here because itbecomes possible to correlate the impact of CpG methylatedsites with their positions on cis-acting elements crucial forcell-specific and regulated gene expression.As concerns the rPRL gene, all the cis-acting elements char-

acterized in the proximal region of the promoter are involvedin positive regulation. If DNA methylation down-regulatesrPRL gene transcription, hypomethylation is expected to cor-relate with a high level of gene expression. In keeping with thishypothesis, it is significant that specific hypomethylation ofCpG sites 2277 and 297 coincides with a high promoter ac-tivity in both GH3 tumor cells and the normal AP. Further-more, the increase in rPRL gene expression in GH3AZA3 cellscompared with that in GH3CDL cells is additionally linked tothe demethylation of CpG site 2373. Of note, CpG site 297

FIG. 6. Methylation strategy. (A) Graphic map of the pGEMPRL-CAT ex-pression vector designed for regional methylation. (B) Map of CpG sites in the2-kb rPRL promoter of the pGEMPRL-CAT vector. The rPRL sequence, fromthe Sprague-Dawley rat strain, contains a CpG at position 236. The locations ofPit-1 binding sites lying in the 2-kb region (black boxes) and the positions ofCpG-methylatable sites (CpG) are indicated.

FIG. 7. Effect of regional methylation on reporter gene expression. (A) Linear representation of regionally methylated constructs. The positions (vertical lines) andthe numbers of methylated CpG sites are indicated. The CAT coding sequence (dotted boxes), the vector sequences (open boxes), and the proximal (black boxes) anddistal (grey boxes) promoter regions are shown. Construct a, p0.4kbPRL-CAT; constructs b to f, pGEMPRL-CAT; construct g, pPRL-CAT (45). (B) Reporter geneexpression. The modified constructs were transfected in GH3AZA3 cells left untreated (black bars) or treated with TF (hatched bars) (see Materials and Methods).The activities of methylated and mock-methylated constructs obtained by ligation (constructs a through d) were quantified as CAT activity per well. Activities of HpaII-,HhaI-, and SssI-methylated and mock-methylated constructs (e to g) were quantified as CAT/b-galactosidase activity ratios. Inhibition was calculated as the ratio of(U2M)/U, where U is the activity of the mock-methylated construct andM is the activity of its corresponding methylated counterpart. Results are the means6 standarddeviations for four (constructs b, c, d, and f) or three (construct e) independent transfections and the means 6 ranges of two independent experiments (constructs aand g), with each experiment performed in triplicate.

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occupies a strategic position: it is located near regulatory ele-ments, including Pit-1, pLim, Oct-1 (positions 262 to 238),the LSF-1 binding site (positions 265 to 245), and the basaltranscription element (BTE) (positions 2117 to 285) (1, 23,29, 45, 62, 71). In addition, this particular CpG lies in the cyclicAMP (cAMP)-like response element (CLE) (positions 2100to 285), which mediates not only cAMP-stimulated but alsoTPA-stimulated regulation (40). Significantly, a CpG is presentat the same position in the PRL promoter sequences of twoother rat strains (Sprague-Dawley and Hooded) and of bovineand human species. Altogether, these data underline the prob-able importance of this CpG site in the regulation of PRL geneexpression.Similar to the rPRL promoter, the rGH promoter contains

cis-acting elements implicated in positive regulation. However,it also possesses negative regulatory elements, such as silencer1 and the proximal repressive element (PRE), which serverepressive functions (34, 49, 57). In this context, hypometh-ylation of positive regulatory sequences as well as hypermeth-ylation of negative regulatory sequences may result in a highlevel of rGH gene expression. In GH3AZA3 cells, comparedwith GH3CDL cells, a higher level of gene expression wascorrelated with hypomethylation of five CpG sites. Four ofthese sites, located at positions 2231, 2200, 2197, and 2149,are situated inside or near cis-acting elements which bind pos-itive regulatory factors, such as growth hormone factor 3(GHF3), T3 receptor, Pit-1, GHF2, and SP1 (19, 45, 61). Con-versely, the site located at position 2319 lies inside the nega-tive regulatory element termed silencer 1. In the AP, all sitesare hypomethylated compared with those in the liver. Conse-quently, the high level of rGH gene expression appears to becorrelated with the hypomethylation of four additional sitesthat lie inside or in the vicinity of positive (CpG sites2174 and2147) or negative (CpG sites 2335 and 2168) regulatorysequences. The relatively high level of gene expression inGH3B6 cells correlates with hypermethylation of two sites lo-cated near or inside silencer 1 at positions2335 and2319 andof one site located on the GHF3 binding site at position 2231.Thus, no simple correlation can be established between highlevels of rGH promoter activity and the methylation of positiveand/or negative cis-acting elements. Additional parameters,such as unequal sensitivity of transcription factors regardingDNA methylation and/or differential distribution of transcrip-tion factors among cell lines, should be considered to account forthe present observation.However, data obtained by genomic sequencing do not pro-

vide information regarding the functional relevance of differ-ential methylation at CpG sites. It cannot be excluded that thedifferent methylation patterns are linked to phenotypic mod-ulations unrelated to gene expression. For example, alterationsin the activity of factors associated with de novo modification(or demodification), such as DNA methyltransferase, may beresponsible for changes in DNA methylation, especially in celllines (67). In this respect, regional methylation experimentsprovide important information. Methylation of the rPRL pro-moter, which involves only 14 CpG sites dispersed within 2 kb,strongly represses CAT gene expression in transient-transfec-tion experiments. This methylation-induced inhibition affectsequally basal and regulated transcription, suggesting that thefactors involved in tissue-specific and in PKA- and PKC-stim-ulated expression, such as Pit-1, are sensitive to CpG methyl-ation. Methylation of the distal part of the rPRL promoterinduced a weaker but similarly balanced effect. In contrast,methylation of CpGs lying in the proximal region preferentiallydecreased the stimulation induced by the activation of PKA-and PKC-dependent pathways. These experiments thus re-

vealed a dual feature for DNA methylation. On the one hand,the modification of a limited domain, such as in the case of theproximal 0.4-kb region, may inhibit specialized functions of theDNA sequence by preventing directly the binding of particulartranscription factors, such as those involved in PKA- and PKC-mediated regulation of the rPRL gene. This hypothesis is sup-ported by in vitro experiments demonstrating that CpG meth-ylation decreases the binding of transcription factors such asCREB/ATF and AP2 (15). Of note, the consensus cis-actingsequences of these two factors present homology with therPRL promoter sequence lying between 1P and 2P bindingsites and containing the CpG site 297. On the other hand, themethylation of extended regions of DNA, such as the 2-kbpromoter or its 1.5-kb distal region, may operate by alteringoverall chromatin structure, thereby influencing gene accessi-bility. In support of such an indirect mechanism, several pro-teins that bind preferentially to methylated DNA have beendescribed, namely, methyl-CpG-binding proteins, methylatedDNA-binding proteins, and histones belonging to the H1 fam-ily (4, 30, 31, 39, 43, 60; for a review, see reference 68). Thisinterpretation is also consistent with the fall in CAT activityobserved in HhaI-methylated constructs. With the exception ofone site located within the rPRL promoter at position 21231,all HhaI-methylated CpG sites are located in the plasmid se-quence. In accordance with the findings of Kass et al. (32), themultiple sites located in the vicinity of the rPRL promotermight provide a focus of methylation able to induce the for-mation of inactive chromatin, which in turn may spread to thecontiguous unmethylated rPRL promoter. The HhaI site lo-cated in the rPRL distal region may provide an additionalseeding point for propagating an inactive chromatin structure.Such a spreading was observed 24 to 48 h following transfec-tion, i.e., as under our experimental conditions (32). WhyHpaII methylation seems to be relatively inefficient at inducinga similar phenomenon remains to be elucidated.A secondary and unexpected finding resulting from genomic

sequencing analysis was the heterogeneous nature of the rPRLand rGH promoter methylation profiles in cell lines as well asin normal tissues. This mosaicism may result from unfaithfulmaintenance of DNA methylation patterns, such as is typicallyobserved in cultured and continuously replicating cells (25, 55).Mosaic methylation patterns were also observed in clonal, his-tologically homogeneous tumoral tissue (63). Moreover, all ofthe few studies performed by genomic sequencing underlinethe heterogeneity of the methylation profiles found in tissuesand clonal populations of animal cells, as well as in plants (18,44, 50). However, the great diversity of patterns observed inthe course of the present analysis raises questions about themechanisms whereby DNA methylation is propagated in cul-tured cells and in normal tissues.On the other hand, and as surprising, is the asymmetry

observed in the methylation patterns of top and bottom strandsof the rPRL promoter in normal tissues. Hemimethylated siteshave been described previously, and some of them appear tobe maintained during several rounds of cell division (58, 70).However, it is generally accepted that hemimethylated CpGsites are efficiently methylated by the maintenance methylasefollowing DNA replication (reviewed in reference 24). More-over, the rGH promoter displays symmetric methylation pat-terns in DNAs from the same origin. This discrepancy betweenthe rPRL and the rGH promoters might be connected to thefindings of Borrelli et al. (3), who demonstrated that PRLexpression and lactotrope differentiation are postmitotic events,in contrast to GH expression and somatotrope differentiation.In keeping with this idea, the hypothetical setting of a rPRL

3252 NGO ET AL. MOL. CELL. BIOL.

promoter methylation pattern might be a postmitotic phenom-enon in normal tissues, in which cell division occurs unfre-quently compared with that in tumor cells. This might explainwhy the rPRL promoter is not hemimethylated in GH3 celllines, in which the doubling time is '40 h. According to thesame hypothesis, the establishment of an rGH promoter meth-ylation pattern in differentiating cells would occur before entryof the cells into the postmitotic phase, leading to symmetricalmodification of both strands, as actually observed in normaltissues as well as in tumor cells.In keeping with this idea, it would be crucial to characterize

the methylation patterns of the rGH and rPRL promotersfrom early to late stages of pituitary ontogenesis. Such knowl-edge should provide important clues in the understanding ofthe interplay between DNA methylation and the complex net-work of transcription factors suspected to govern lactotropeand somatotrope cell differentiation or to be involved in themaintenance of their differentiated state.

ACKNOWLEDGMENTS

We thank J. Martial (Liege, Belgium) for the pCMV-bgal expres-sion vector and S. Magre (Paris, France), who kindly provided us withadult Wistar-Furth rats. We are indebted to J.-P. Jost (Basel, Switzer-land) for critical reading of the manuscript. We are also grateful to E.Etienne for his expert photographic work and to S. N. Thornton forassistance with the English.This work was supported by the Centre National de la Recherche

Scientifique (URA 1115). V.N. was supported by a fellowship from theMinistere de la Recherche et de l’Enseignement Superieur.

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