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a Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208
ABSTRACT
The mouse lactate dehydrogenase c gene (mldhc) is transcribed only in cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-base pair fragment was able to drive testis-specific transcription in vitro and in transgenic mice. Several testis-specific genes are believed to be regulated at least in part through differential methylation of CpG dinucleotides. We investigated the possibility that transcriptional repression of the mldhc gene is mediated in somatic tissues by hypermethylation of CpG dinucleotides. The CpG dinucleotides within a fragment of the mldhc promoter containing a GC box and tandem activating transcription factor/cAMP-responsive element binding sites are hypermethylated in somatic tissues and hypomethylated in testis. Methylation of the activating transcription factor/cAMP-responsive elements altered the protein binding pattern observed in electrophoretic mobility shift assays using mouse liver but not testis nuclear extract. Furthermore, methylation of an extended mldhc promoter fragment driving lac Z silenced transcription from the promoter in a transient transfection assay. These data suggest that tissue-specific differential methylation plays a role in mldhc silencing in somatic tissues.
cyclic adenosine monophosphate, gene regulation, spermatogenesis, testis
INTRODUCTION
The ordered regulation of gene expression during spermatogenesis makes the testis an excellent tissue in which to study transcription. Silencing of testis-specific genes in nonexpressing somatic tissues is critical, and DNA methylation is emerging as a general mechanism through which this task is accomplished. The transcription of several testis-specific genes is believed to be regulated at least in part by CpG hypomethylation within the testis and hypermethylation in somatic tissues [1–8].
Transcription of the testis-specific mouse lactate dehydrogenase c gene (mldhc) is limited to cells of the germinal epithelium. Cloning, sequencing, and analysis of the mldhc promoter revealed a 100-base pair (bp) core region, defined by its ability to direct testis-specific expression of a reporter gene in in vitro transcription assays [9]. This result was confirmed when the same sequence was found responsible for testis-specific expression of a reporter gene in transgenic mice [10]. Analysis of the sequence residing within the core promoter revealed several CpG dinucleotides that are potential sites for methylation. Three of the CpGs were located within putative transcription factor binding sites including a GC box located at -70 bp 5' to the transcription start site and tandem near-consensus activating transcription factor/cAMP-responsive elements (ATF/CREs) at -53 bp and -39 bp. The location of these elements relative to each other and to the transcription start site is of particular interest in light of the recent finding by Iannello et al. [4] that somatic repression of the testis-specific Pdha-2 gene occurs through targeting of an ATF/CRE. The ATF/CRE site within the Pdha-2 promoter is located at -62 bp, similar to the placement of the ATF/CREs in the mldhc promoter. In addition, both the Pdha-2 and mldhc promoters contain a GC box, a consensus binding site for transcription factor Sp1, upstream of their ATF/CREs, separated by 12 bp and 11 bp, respectively.
The present study was undertaken to determine whether CpG dinucleotides within the mldhc core promoter are differentially methylated in somatic tissue vs. testis and whether these differences affect in vitro binding to GC box and ATF/CRE binding sites within the core promoter. We also sought to assay the effect of CpG methylation on transcription from the mldhc promoter. Our results indicated that the mldhc core promoter is hypomethylated in testis and, except for the 5' ATF/CRE, is hypermethylated in all somatic tissues tested. Methylation of CpGs within either the 3' ATF/CRE or both ATF/CREs affects transcription factor binding activity present in liver nuclear extract but not in testis nuclear extract. Also, methylation of the mldhc promoter silences its ability to drive reporter expression in transiently transfected mouse L cells.
MATERIALS AND METHODS
Restriction Endonuclease/Polymerase Chain Reaction Assay
A modification of the method first described by Singer-Sam et al [11] was employed. Mouse (CD-1) testis and liver genomic DNA was isolated by standard methods [12]. Ten micrograms of genomic DNA was digested overnight with 20 units of either AciI (for GC box) or TaiI (for ATF/CRE) followed by phenol:chloroform extraction and ethanol precipitation. Digested genomic DNA (1 µg) was used as template in a polymerase chain reaction (PCR) containing 1 µM of each primer, 200 µM dNTP mix, and 1x PCR buffer (New England Biolabs, Beverly, MA). Primers used to amplify the region surrounding the GC box were sense mouse ldhc sequence (MC) -311 to -291 (5'-CCTACACACAGATGTAAGGGC-3') and antisense MC +28 to +8 (5'-CACAGGTAAGAAACCAGGAT-3'). Primers used to amplify the region surrounding the ATF/CREs were sense MC -311 to -291 and antisense MC +128 to +109 (5'-GCTCACAGGCCTACAATGGC-3'). Internal positive control reaction included with the GC box reaction used oligonucleotides sense MC +8 to +30 (5'-ATCCTGGTTTCTTACCTGTGC-3') and antisense MC +128 to +109. Internal positive control included with the ATF/CRE reaction used oligonucleotides sense MC +372 to +392 (5'-TAGACTACATATTGACCTCCT-3') and antisense MC +561 to +542 (5'-CTAGGTTCTGAATCAGCTGC-3'). PCR conditions were as follows: 94°C for 5 min; 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min; 72°C for 10 min; and soak at 4°C. Template for PCR positive controls was plasmid pNAssß (Clontech, Palo Alto, CA) containing a fragment of the mldhc promoter extending from -425 to +10 bp 5' to the transcription start site but not mldhc intron I. Amplimers were electrophoresed on 1.5% agarose gels and photographed under ultraviolet illumination.
Bisulfite Sequencing
Bisulfite sequencing was performed as previously described [13–15] with modifications. Genomic DNA from CD-1 mouse testis, liver, kidney, heart, and skeletal muscle was isolated using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) according to manufacturer's instructions. Genomic DNA (5 µg) was digested overnight with EcoRI, extracted with phenol:chloroform, and ethanol precipitated. The DNA pellet was resuspended in 25 µl of 0.3 M NaOH and incubated at 37°C for 20 min. Then 250 µl of freshly made metabisulfite solution (2 M sodium metabisulfite and 0.55 mM hydroquinone), pH 5.0, was added, and the solution was mixed, covered with a layer of mineral oil, and incubated at 55°C for 12–16 h. DNA was purified using the Wizard DNA Clean-up System (Promega) and eluted in two 50-µl aliquots, which were combined. NaOH was added to a final concentration of 0.3 M, and the solution was incubated for 20 min at 37°C. The solution was neutralized by addition of 7.5 M ammonium acetate, pH 7.0, to 3 M final concentration. The DNA was ethanol precipitated and resuspended in 100 µl Tris-EDTA buffer.
PCR was used to amplify the sense strand of the bisulfite-converted DNA. The first reaction used oligonucleotides sense -426 to -399 Meta (5'-GGTTTATAGAGTTTTAGGATGGTTAGGG-3') and antisense +134 to +110 Meta (5'-CCCCAACTCACAAACCTACAATAAC-3'). Bisulfite-converted DNA (50 ng) was used as template, and MgCl2 concentration was adjusted to 2 mM final in 1x PCR buffer (MBI Fermentas, Hanover, MD). Cycles were as follows: 94°C for 5 min; 5 cycles of 94°C for 1 min, 53°C for 2 min, and 72°C for 2 min; 30 cycles of 94°C for 30 sec, 53°C for 2 min, and 72°C for 1.5 min; 72°C for 6 min; and soak at 4° C. PCR product was purified using the Wizard DNA Clean-Up System and eluted in 50 µl H2O. One microliter of eluted DNA was used as template in a second PCR. Nested oligonucleotides sense -203 to -176 Meta (5'-TTGGTTGTTATTTTTTGTGTGGTTTTAGGG-3') and antisense +56 to +30 Meta (5'-AACACACACCTTACTACTAACTCCRCAACAC-3') were used as primers. MgCl2 concentration was reduced to 1.5 mM final. Cycles were identical to those listed above except that the annealing temperature was changed to 58°C. Amplimers were gel purified and sequenced on an ABI PRISM 310 automated sequencer (Applied Biosystems, Foster City, CA). Bisulfite sequencing of each tissue was performed three times with tissue isolated from three individual mice.
Nuclear Extract Preparation
Nuclear extracts were prepared from CD-1 mouse tissues by a modification of previously reported methods [9, 16, 17]. All manipulations were performed on ice in prechilled labware using prechilled solutions. Liver from 10–15 adult mice or testes from 50 adult mice were harvested and placed on ice. Aggregate weight of each tissue was approximately 15 g. Testes were decapsulated. Tissue was minced fine with a razor blade and then added to a 30-ml Teflon-glass homogenizer (Wheaton, Milville, NJ). The homogenizer was filled approximately two-thirds full with homogenization buffer (2 M sucrose, 10 mM Hepes, pH 7.6, 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 10% glycerol, 0.5 mM dithiothreitol [DTT]) containing protease inhibitors (0.33 µg/ml aprotinin, 0.5 mM benzamidine, 1.14 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 0.1 mM PMSF). Tissue was homogenized by five strokes of a Teflon pestle attached to a drill. Tissue suspension was brought to 85 ml with additional homogenization buffer and then layered over three 10-ml cushions of homogenization buffer in 40-ml polyallomar centrifuge tubes (Beckman, Palo Alto, CA). Samples were centrifuged for 30 min at 24 000 rpm and 1°C in an SW 28 rotor (Beckman). Nuclei sank as sediment to the bottom of the tube, and cell debris remained suspended in the buffer. The buffer was carefully aspirated, and the pellet was resuspended in 10 ml of a 9:1 mixture of homogenization buffer:glycerol. Nuclei were transferred to a 15-ml Teflon-glass homogenizer (Wheaton) and gently homogenized two strokes by hand. The suspension was brought to 50 ml with additional 9:1 homogenization buffer:glycerol and layered over two 10-ml cushions of buffer (without additional glycerol) in 40-ml tubes. Nuclei were centrifuged for 30 min at 24 000 rpm and 1°C, and the supernatant was aspirated as above. Nuclei were resuspended in 10 ml of buffer (10 mM Hepes, pH 7.9, 10% glycerol, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, and 0.1 mM PMSF) and gently homogenized two strokes by hand in a 15-ml Teflon-glass homogenizer to break up clumps. The homogenate was centrifuged 15 min at 5000 rpm in an SS-34 rotor (Sorvall, Newtown, CT) at 4°C. The supernatant was decanted, and nuclei were resuspended in 10 ml of buffer, centrifuged as above, and decanted. The pellet was resuspended in 0.3–0.5 ml of buffer, KCl was added to a final concentration of 400 mM, and the solution was gently mixed. Suspensions were incubated on ice for 1 h with periodic mixing. Debris was pelleted by centrifugation at 10 000 rpm for 10 min at 4°C in an SS-34 rotor, and the extract was divided into aliquots, frozen in liquid nitrogen, and stored at -80°C. Extracts of nuclei prepared in this manner retained binding activity for at least 6 mo. Protein concentrations were typically 5–10 µg/µl as determined by the Bio-Rad (Hercules, CA) protein assay.
Generation of Oligonucleotide Probes
Oligonucleotides containing the GC box were sense 5'-ccAGCAGGCAGTGGGCGGGGCTTGCGTG-3' and antisense 5'-ctAGCACGCAAGCCCCGCCCACTGCCTG-3'. Oligonucleotides containing the ATF/CREs were sense 5'-caTGCGTGCTGACGTTGACTTTGTGACGTTCCTTTTC-3' and antisense 5'-cCGGAAAAGGAACGTCACAAAGTCAACGTCAGCACG-3'. Lowercase letters indicate nonhomologous nucleotides added to facilitate labeling. Methylation was accomplished during synthesis (IDT, Coralville, IA).
Complementary oligonucleotides were annealed in STE (100 mM NaCl, 10 mM Tris·HCl, pH 8.0, 1 mM EDTA, pH 8.0) by heating to 95°C for 5 min and then cooling to room temperature over several hours. Oligonucleotide pairs were labeled by incubation with Klenow fragment for 15 min at 25°C in the presence of 30 µCi of [
-32P]dCTP and 33 µM each of unlabeled dATP, dGTP, and dTTP. Unlabeled dCTP was then added to a final concentration of 33 µM, and the reactions were incubated an additional 10 min at 25°C. Unincorporated nucleotides were removed by passage over a G-50 Sephadex spin column (Pharmacia, Peapack, NJ). Specific activity of each labeled probe was determined by scintillation counting.
Electrophoretic Mobility Shift Assays
Mouse testis or liver nuclear extract (7.5 µg) was incubated on ice for 15 min in the presence of binding buffer (13 mM Hepes, pH 7.9, 60 mM KCl, 0.13 mM EDTA, 2 mM DTT, 10% glycerol, and 0.2 mM PMSF), 1 µg poly dI·dC, and 1 µg boiled salmon sperm DNA. KCl present in the nuclear extracts was taken into account when calculating final KCl concentration. Probe (50 000 cpm) was added, and the reactions were incubated on ice an additional 30 min. Reactions were loaded onto 4% nondenaturing polyacrylamide gels that had been prerun at 150 V and 4°C for 45 min. Reactions were electrophoresed at 175 V and 4°C and then blotted onto paper support, dried, and exposed to film at -80°C for 5–24 h.
DNA Constructs
A 430-bp mldhc 5' promoter fragment (-425 to +10) was amplified by PCR using oligonucleotide Xhosens (5'-CCGCTCGAGGTCTACAGAGTTCCAGGACG-3') corresponding to -425 to -406 and incorporating an XhoI site and with MC +10 to -12 HindIII (5'-CCGAAGCTTATAACTGTTGGGTCCAGGAGCC-3') corresponding to +10 to -12 and incorporating a HindIII site. Template DNA was the pNAssß vector containing the mldhc promoter fragment (-425 to +10). The amplimer was digested with XhoI and HindIII and cloned into the corresponding restriction sites of the pßgal-Basic vector (Clontech). The resulting construct was termed pWT. Plasmid pWT was methylated in vitro by incubation with one unit of SssI methylase per microgram of plasmid DNA under conditions recommended by the manufacturer (New England Biolabs), resulting in pMethWT. Completeness of the methylation reaction was determined by resistance of the methylated plasmid to digestion with the methylation-sensitive restriction endonucleases AciI and Bsu15I.
Transfections and ß-Galactosidase Assay
Mouse L cells (gift from Prof. Dan Linzer, Northwestern University, Evanston, IL) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Cultured cells were grown to 50% confluency in six-well plates and then transfected with 2 µg of the reporter construct using Superfect (Qiagen, Valencia, CA). Cells were harvested 48 h after transfection and assayed for ß-galactosidase activity using the Tropix Galacto-Light Plus kit (Applera, Foster City, CA) according to the manufacturer's instructions. Plasmid pRL-TK (Promega), which expresses luciferase from a constitutive promoter, was cotransfected as an internal control to normalize transfection efficiencies. Luciferase assay was performed using the Luciferase Assay System (Promega) according to the manufacturer's instructions. Results reported are the average of three independent experiments.
RESULTS
GC Box and ATF/CREs Are Differentially Methylated in Expressing and Nonexpressing Tissues
A restriction endonuclease/PCR assay was used to determine the methylation status of the GC box and ATF/CREs located at -70 bp, -53 bp, and -39 bp, respectively, 5' to the mldhc transcription start site. Mouse testis and liver genomic DNA was digested with the methylation-sensitive restriction endonucleases AciI (for GC box, Fig. 1) or TaiI (for ATF/CREs, Fig. 2). PCR was then performed using primers specific for either the GC box (Fig. 1) or the ATF/CREs (Fig. 2). A methylated cytosine at a site results in inability of the restriction enzyme to cleave the DNA and, consequently, amplification of a PCR product of the correct size. Absence of methylation allows complete digestion of the DNA, and no PCR product is obtained. For both the GC box (Fig. 1, ‘promoter’ arrow) and ATF/CRE (Fig. 2, ‘promoter’ arrow) reactions, PCR amplified a band when liver but not testis genomic DNA was used as template, indicating methylation in liver but not in testis. Each reaction contained an internal positive control in the form of primers that amplified a product from intron I (Fig. 1, ‘Intron I’ arrow) or an exon (Fig. 2, ‘Coding’ arrow) of the mldhc cDNA regardless of tissue or whether the DNA was digested, demonstrating the competence of the reaction cocktails. A positive control reaction with a plasmid template containing the promoter fragment but not intron I or the mldhc cDNA produced a single band corresponding to the promoter, and a negative control reaction to which no template DNA had been added produced no amplimers (Figs. 1 and 2, ‘Plasmid (+)’ and ‘(-) Control’, respectively).
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Bisulfite Sequencing Precisely Maps Methylation Pattern in GC and ATF/CRE Region
The CpG methylation pattern in this region was mapped more precisely using the bisulfite sequencing technique (Fig. 3). Mouse testis and liver genomic DNA was isolated and treated with a mixture of sodium metabisulfite and hydroquinone and then exposed to NaOH. This procedure results in the conversion of cytosines to uracils. However, methylated cytosines are resistant to bisulfite treatment and remain unchanged. The sense strand of the region containing the GC box and the ATF/CREs was then amplified by PCR, and the amplimer was sequenced. All nonmethylated cytosines are read as thymidines, but methylated cytosines are not converted. All cytosines in the GC and ATF/CRE regions of testis genomic DNA were sequenced as thymidines, indicating their unmethylated status (Fig. 3, Testis). Sequencing of converted liver genomic DNA (Fig. 3, Liver) revealed that cytosines in CpG dinucleotides at -67 bp (GC box), -58 bp, and -36 bp (3' ATF/CRE) were methylated, whereas the CpG at -50 bp (5' ATF/CRE) was only partially methylated. Similar results to those reported for liver genomic DNA were also obtained using genomic DNA from kidney, heart, and skeletal muscle.
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Methylation Affects In Vitro Binding to the ATF/CREs but Not the GC Box
The effect of methylation on protein binding to the ATF/CREs and GC box was determined by comparing the binding patterns generated by incubation of mouse testis or liver nuclear extracts using methylated versus nonmethylated probes in an electrophoretic mobility shift assay (EMSA). Incubation of a radiolabeled oligonucleotide probe containing the GC box (Fig. 4B, 1) produced a single major shifted band using either liver or testis nuclear extract (Fig. 4A, set 1, lanes T and L). Although testis and liver nuclear extracts produced different patterns of minor shifted complexes, methylation of the CpG dinucleotide within the GC box (Fig. 4B, 2) did not change the binding pattern in either testis or liver nuclear extract compared with unmethylated probe (compare Fig. 4A, set 2, T and L, with set 1, T and L, respectively).
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We next investigated the effect of methylation on binding to the two ATF/CREs. Four oligonucleotide probes were synthesized with no methylation or methylation of the 5' ATF/CRE, the 3' ATF/CRE, or both ATF/CREs (Fig. 5B, 1–4, respectively). In EMSAs using testis nuclear extract, a single shifted complex was seen in all cases (Fig. 5A, sets 1–4, lane T). Incubation of liver nuclear extract with unmethylated probe (Fig. 5B, 1) resulted in the appearance of three major shifted complexes (Fig. 5A, set 1, lane L). Although methylation of the 5' ATF/CRE (Fig. 5B, 2) had no effect on binding (compare Fig. 5A, set 2, lane L, to set 1, lane L), methylation of the 3' ATF/CRE (Fig. 5B, 3) results in reduction in the intensity of the uppermost shifted complex (compare Fig. 5A, set 3, lane L, *, with set 1, lane L, *), and methylation of both ATF/CREs (Fig. 5B, 4) results in reduction in the intensity of the uppermost shifted complex and appearance of another complex with an even slower mobility (compare Fig. 5A, set 4, lane L, * and 
, respectively, with set 1, lane L, *).
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Methylation Silences the mldhc Promoter
The effect of methylation on transcriptional competence of the mldhc promoter was determined by transfecting mouse L cells with methylated or nonmethylated constructs containing 435 bp of mldhc promoter driving a lac Z reporter (Fig. 6). Methylation was accomplished by incubation with SssI methylase, which methylates all cytosine residues that are part of CpG dinucleotides. Treatment with SssI methylase results in a methylation pattern identical to that observed in wild-type liver (Fig. 3) except that the 5' ATF/CRE is completely rather than partially methylated. The level of ß-galactosidase expression was compared with that resulting from transfection of the pßgal-Basic reporter vector with no promoter. In vitro methylation of the construct (Fig. 6, methylated) reduces expression observed with the unmethylated construct (Fig. 6, wild type) to the level of the promoterless vector (Fig. 6, vector).
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DISCUSSION
The complex process of spermatogenesis requires the coordinated expression of many genes. The molecular mechanisms required to control this expression often involve DNA modifications of cis-elements located within the gene promoter. Methylation of cytosine residues in mammalian DNA is emerging as an important component of a multilevel control mechanism for testis-specific gene expression that may also include participation by testis-specific activator factors and somatic repressor factors.
To determine the role CpG methylation plays in regulation of the testis-specific expression of mldhc, we analyzed the methylation status of the mldhc promoter. CpG dinucleotides in the GC box and the 3' ATF/CRE of the mldhc promoter were hypermethylated in every somatic tissue examined and were hypomethylated in testis. Although the 5' ATF/CRE was hypomethylated in testis, sequencing of bisulfite-treated genomic DNA from this region showed that this region was much less heavily methylated in somatic tissues than were the other elements. The difference in methylation level between the 5' and 3' ATF/CREs in somatic tissues corresponds to the effect of methylation on in vitro protein binding to each element. Methylation of either the 3' ATF/CRE or both ATF/CREs changed the pattern of protein binding with somatic but not testis nuclear extract. Methylation of the 5' ATF/CRE alone, however, did not change the protein binding pattern in either somatic or testis nuclear extract, suggesting this element may not be involved in methylation-mediated modulation of mldhc transcription. Methylation of the GC box did not affect in vitro binding in either mouse testis or liver nuclear extract, suggesting that, like the 5' ATF/CRE, the GC box is not likely to be involved in methylation-dependent regulation of the mldhc gene. Incubation of unmethylated ATF/CRE probe with liver and testis extract resulted in entirely different banding patterns in the EMSAs, suggesting different factors may bind this sequence in each extract. The possibility exists, therefore, that this differential binding and not methylation is responsible for differences in transcription between testis and somatic tissues. Another possibility is that methylation of the ATF/CREs in somatic tissues allows binding of transcription factors that recognize the methylated CpG dinucleotides. Once occupied, these sites would no longer be available to activator factors, resulting in transcriptional silencing.
Complete CpG methylation of an mldhc promoter fragment extending to -435 bp 5' to the transcription start site was sufficient to reduce expression of a reporter gene driven by this promoter in mouse L cells to background. Although LDH-C4 is not normally expressed in L cells, a transfected nonmethylated mldhc-lac Z reporter construct demonstrated promoter activity, which was eliminated by methylation, clearly demonstrating the importance of this nucleotide modification in silencing this gene. All CpG dinucleotides in the reporter vector were methylated by SssI methylase. As indicated by bisulfite sequencing of the GC box and ATF/CREs, not all CpG dinucleotides were methylated to the same extent in vivo. Nevertheless, methylation by SssI methylase is the closest approximation to the native methylation pattern. The human ldhc promoter is also differentially methylated in testis and somatic tissues [2]. Although the mouse and human ldhc promoters share little sequence homology, methylation appears to be a common silencing mechanism.
Our conclusions are bolstered by recent findings of Iannello et al. [5]. These investigators determined that methylation perturbs in vitro binding to an ATF/CRE element in the promoter of the mouse Pdha-2 gene, a testis-specific gene for which, like mldhc, transcription begins in meiotic prophase I. Similar to our results, in vitro methylation of a promoter fragment of the Pdha-2 gene reduced transcription when transfected into tissue culture cells. Furthermore, methylation of the ATF/CRE was responsible for methylation-induced silencing of this gene, and the ATF/CRE was essential for transcription [4]. Like the mldhc promoter, the Pdha-2 promoter contains a GC box located just 5' to the ATF/CRE. In both promoters, the GC box is hypermethylated in somatic tissues and hypomethylated in testis. Methylation does not affect in vitro binding in either case, however, suggesting that this element may not be involved in silencing of either promoter.
In previous studies of the mldhc promoter [9, 18], a 31-bp palindromic sequence containing a TATA box and the transcription start site was identified and is thought to be involved in both positive and negative regulation of the mldhc gene through differential transcription factor binding. Those results and those obtained in the present study suggest that testis-specific transcription of the mldhc gene is regulated through multiple mechanisms.
The data presented here and those obtained by others [1–8] make a strong argument for somatic methylation of promoter sequences as a general mechanism for silencing testis-specific genes in somatic tissues.
ACKNOWLEDGMENTS
We thank Drs. Jorg Bungert and Scott Ness for helpful advice regarding the EMSAs.
FOOTNOTES
1 This work was supported by NIH grants HD05863 (to E.G.) and 5T32HD07068 (to D.J.M.) and by Fogarty International Center Sub 521215/D43-TW00654 (to P.J.). 
2 Correspondence: Erwin Goldberg, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Rd., Evanston, IL 60208. FAX: 847 467 1380; erv{at}northwestern.edu
3 Current address: University of California, San Francisco, CA 94143-0512.
4 Current address: Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660.
5 Current address: Emory University, Atlanta, GA 30322.
Accepted: June 20, 2001.
Received: March 6, 2001.
REFERENCES
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