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a Department of Pathology,/ L9 G, N7 h: `
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( B- v# j; ]$ V: l' p& mc Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA;. V, Z, b U! S+ h3 ?! U- u' p
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d Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA
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Key Words. Embryonic stem cell ? DNA methylation ? Differentiation ? Germ cell ? Adenine nucleotide translocase ? Gene repression
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Correspondence: Naohiro Terada, M.D., Ph.D., Department of Pathology, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, Florida 32610, USA. Telephone: 352-392-2696; Fax: 352-392-6249; e-mail: terada@pathology.ufl.edu
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: z1 e) ~) E* @ABSTRACT* o; D& l7 [' F. {
1 i+ p, x7 ^, h$ v% M/ bDNA methylation, or the addition of a methyl group to the 5'-position of cytosine within the CpG dinucleotide, is a heritable modification that contributes to gene silencing. Most CpG sites in mammalian cells are methylated in a nonrandom fashion. For instance, repetitive and parasitic elements tend to be hypermethylated, whereas CpG island–associated promoters are usually hypomethylated . The complex process of DNA methylation has been proven essential for normal development , X-chromosome inactivation , imprinting , and the suppression of parasitic DNA sequences . Although DNA methylation is a modification that promotes genomic integrity and ensures proper temporal and spatial gene expression during development, it also associates with malignancy when aberrantly controlled. Both hypermethylation and hypomethylation have been attributed to cancer development . DNA methylation is proposed to prevent the binding of transcription factors and recruit repressor complexes that induce the formation of inactive chromatin complexes . For example, it was recently shown that methylation of a CpG dinucleotide within the glial fibrillary acidic protein promoter prevents STAT3 binding . In another instance, DNA methylation –mediated gene silencing is dependent on the presence of methyl-CpG binding protein, MeCP2, which forms a complex with histone deacetylases and a repressor protein, mSin3A, to repress transcription in a methylation-dependent manner . It is still unclear in most cases, however, whether DNA methylation is a causal event in gene silencing or, rather, a consequence of gene silencing.
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$ h' F/ G9 [3 {3 l- UIt is becoming increasingly clear that epigenetic modifications play a critical role in the regulation of gene expression in many cellular processes . Studying changes in epigenetic modifications during stem cell differentiation will help us understand how cells maintain or lose differentiation potential. In the present study, we attempted to identify differentially methylated loci that are hypomethylated in undifferentiated embryonic stem cells (ESCs) and become hypermethylated after differentiation. Murine ESCs are originally derived from the inner cell mass of a developing blastocyst and have the ability to differentiate into all cell types of an adult animal . Pluripotency of ESCs can be maintained in vitro when the cells are cultured in a serum-containing medium supplemented with leukemia inhibitory factor (LIF) . When LIF is removed from the medium, the ESCs begin to differentiate in vitro into all three embryonic germ layers. This in vitro ESC differentiation system serves as an excellent model to study the regulation of gene expression required for stem cell self-renewal and pluripotency . Recent studies on molecules involved in epigenetic modifications have revealed a unique expression pattern of DNA methyltransferases , histone deacetylases , and methyl-binding proteins in ESCs. ESCs also have a differential genome-wide DNA methylation pattern compared with their descendant differentiated cells . However, exact genomic loci of such differentially methylated regions remain unknown.
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& K7 i4 ~9 p$ rUsing methylation-sensitive restriction fingerprinting (MSRF), we identified a novel gene encoding an adenine nucleotide (ADP/ATP) translocase homologue that is specifically expressed in undifferentiated ESCs and germ cells. Furthermore, we show that DNA methylation, but not the availability of transcription factors, is the dominant factor restricting the gene’s expression.
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4 L. ?4 [* p4 D7 A3 TMATERIALS AND METHODS% h" ?+ }* a' O# c; O) B2 u& ?
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Identification of a CpG-Rich DNA Sequence That Undergoes De Novo DNA Methylation During ESC Differentiation
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$ V, ^7 Q+ J! B4 M( G! A' J1 d0 rWe investigated changes in DNA methylation during the transition of ESCs into differentiated cell types using an MSRF method. Mouse ESCs were differentiated in a culture medium without LIF using a hanging drop method. DNA was extracted from undifferentiated ESCs (day 0) and differentiated EBs (days 5 and 10). The DNA was then digested by a combination of methylation-sensitive (BstUI) and insensitive (MseI) restriction enzymes and amplified by PCR using CpG-rich 10-mer primers (Fig. 1A). Using a combination of four different CpG-rich primers, we identified a total of eight bands that exhibited differential methylation patterns during ESC differentiation (data not shown). The bands were excised from the gels, and their DNA sequences were determined. Database searches revealed that one of these methylated fragments (methylated fragment 1, MF1 in Figs. 1B, 1C) mapped to the exon 1/intron 1 boundary of a previously uncharacterized gene (RIKEN cDNA 1700034J06). The other seven DNA fragments were localized outside of any CpG islands and were derived from various genomic regions. Because the MF1 DNA fragment mapped to a genomic region that partially overlapped with a CpG island, we decided to further analyze the associated gene.; w5 i, N2 A9 i5 B( s
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Figure 1. Identification of differentially methylated CpG-rich fragment in ES cells and EBs. (A): MSRF. Genomic DNA was prepared from undifferentiated ES cells and differentiating EBs (days 5 and 10). DNA was digested either by MseI alone or MseI and BstUI and subjected to PCR amplification using CpG-rich 10-mer primers in the presence of radiolabeled dCTP. Amplified DNA fragments were separated in 4.5% polyacrylamide gels. In MseI/BstUI double-digested samples, the intensity of a band indicated by arrowhead (MF1) increased after ES cell differentiation. (B): Nucleotide sequence of methylated fragment 1 (MF1). The DNA band was cloned into a PCR cloning vector and sequenced. Bold letters indicate primers used, whereas BstUI sites are underlined. MF1 corresponds to the exon 1/intron 1 boundary region of a previously uncharacterized gene on mouse chromosome 13; black solid boxes represent exons predicted by Expressed Sequence Tag and Ensemble mouse genome databases. The translation initiation site is marked as 1. The arrow represents the major transcription initiation site (–60 bp). Abbreviations: EB, embryoid body; ESC, embryonic stem cell; MseI, methylation-insensitive restriction enzyme; MSRF, methylation-sensitive restriction fingerprinting; PCR, polymerase chain reaction.6 U# a# R0 G! f! k0 W6 n
$ J/ R4 ]7 d& D% K1 dThe associated gene on mouse chromosome 13 did not contain a classic TATA box sequence at its 5'-flanking region. Because TATA-less genes are often associated with multiple transcriptional initiation sites, we performed a 5'RACE (rapid amplification of cDNA ends) reaction using cDNA isolated from undifferentiated ESCs. 5'RACE identified five different transcriptional initiation sites, four of which were previously reported in Expressed Sequence Tag (EST) databases (data not shown). Because there are multiple transcriptional initiation sites, nucleotide positions are marked relative to the translation initiation site ( 1) throughout the manuscript. The major transcript initiated at –60 bp, whereas the longest transcript initiated 304 bp upstream of the translation initiation site.' P" o/ m5 x2 m' ^
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The MF1-associated gene was predicted to encode a 320-aminoacid protein and shared high amino acid sequence homology with the other mouse adenine nucleotide translocase (Ant) proteins previously identified (70.1% and 69.1% overall amino acid identity to Ant1 and Ant2 isoform, respectively) (Fig. 2A). The MF1-associated gene also contained three tandem repeats of a domain of approximately 100 residues, each domain containing two transmembrane regions, a characteristic shared by all members of the Ant family . Because another isoform of Ant, ANT3, has been reported in human , we tentatively named the present gene adenine nucleotide translocase 4 (Ant4). All known mammalian Ant isoforms (unlike plant adenine nucleotide translocases) lack an N-terminal mitochondrial localization sequence, yet they localize to mitochondria . In agreement with this observation, Ant4 also does not contain a classic mitochondrial localization sequence. However, it did localize to mitochondria when N-terminal FLAG-tagged Ant4 was expressed in NIH3T3 fibroblast cells (data not shown).2 C9 N2 d0 r5 B- D; R+ F
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Figure 2. Ant4 encodes a novel isoform of adenine nucleotide translocase. (A): Deduced amino acid sequence of the mouse Ant4 gene is aligned with previously identified mouse Ant proteins (Ant1 and Ant2). (B): Deduced amino acid sequence of the mouse Ant4 gene is aligned with ANT4 human orthologue.
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The human orthologue of ANT4 is located on chromosome 4q28.1, and its deduced amino acid sequence shares 85.9% overall amino acid identity with mouse Ant4 (Fig. 2B). In addition, the genomic architecture with six exons is conserved between mouse and human (data not shown). It is notable that the human genome contains at least seven ANT pseudogenes on the X chromosome; however, in contrast to ANT4, such pseudogenes do not reveal conserved exon/intron architecture or translatable coding regions .0 ~" ]( q1 b, W
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Ant4 Promoter Locus Undergoes De Novo DNA Methylation After ESC Differentiation
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To determine DNA methylation patterns across the Ant4 promoter locus during ESC differentiation, genomic DNAs from ESCs or day-10 EBs were treated with bisulfite and subjected to sequence analysis. We analyzed the Ant4 promoter region that encompasses a total of 47 CpG dinucleotides, from 516 bp upstream of the translation initiation site to the 3'-end of exon 1. Sequencing of individual bisulfite-converted genomic DNAs revealed that the Ant4 promoter and associated CpG island region were mostly unmethylated in undifferentiated ESCs (Fig. 3). In contrast, EBs at day 10 showed significant hypermethylation around the Ant4 promoter region, indicating that after ESC differentiation, the Ant4 promoter undergoes de novo DNA methylation. The further upstream region (>1 kb) of the Ant4 promoter outside of the CpG island revealed hypermethylation regardless of the differentiation status of ESCs (Fig. 3). Ant4 promoter methylation was confirmed by the quantitative method of COBRA . The frequency of methylation at a specific CpG site overlapping an HhaI restriction site (–169 bp) was evaluated by the COBRA assay. In agreement with the bisulfite sequencing data, we detected low levels of DNA methylation in ESCs and high methylation levels (~75%) in EBs at day 10.
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2 r; i- {' X$ B3 f) m3 dFigure 3. Ant4 promoter region undergoes DNA methylation during ES cell differentiation. Summary of Ant4 methylation levels in ES cells and EBs (day 10) is illustrated. For each primer pair, up to 14 clones were sequenced after bisulfite treatment of either ES cells or EBs (day 10) to determine the rate of DNA methylation. Closed circles represent methylated CpG, whereas open circles represent unmethylated CpG. The y-axis of the graph represents percent methylation for each CpG residue. Diagram at the bottom represents relative position of CpG residues within the 5'-end regulatory region of Ant4 gene. Nucleotide positions are marked relative to the translation initiation site ( 1). Arrow represents a major transcription start site (–60 bp), black rectangle represents the first exon, and vertical lines mark the location of individual CpG residues. Abbreviations: EB, embryoid body; ESC, embryonic stem cell. H3 U- E9 X9 G( u" ~
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Ant4 mRNA Expression Is Downregulated After ESC Differentiation1 T" V9 A5 Z$ D, @- e8 H% j9 M
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We then examined Ant4 mRNA expression levels in ESCs and EBs using semiquantitative RT-PCR. Ant4 mRNA was easily detectable in undifferentiated ESCs, whereas the relative abundance of Ant4 mRNA decreased after ESC differentiation (Fig. 4A). ?-Actin expression was relatively unchanged during ESC differentiation. We then used the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) to study the effect of demethylation on transcription of the Ant4 gene. 5-aza-dC induces DNA demethylation in cells by depleting DNA methyltransferases through its covalent and irreversible binding to the enzymes. Notably, the Ant4 gene was readily derepressed by the addition of 5-aza-dC to differentiated EBs (day 10) (Fig. 4B). In contrast, expression of Oct-4 and Nanog, transcription factors selectively expressed in pluripotent ESCs and primordial germ cells, was not affected. Ant4 (but not Oct-4 or Nanog) was similarly derepressed by addition of 5-aza-dC in NIH3T3 cells and an immortalized endoderm precursor cell line (OBAT, unpublished data).3 o) w/ _: C! H& x9 ? l+ s+ Y
4 e- y! r! ?! l. e6 L5 |. uFigure 4. Ant4 is repressed during ES cell differentiation. (A): Total RNA was extracted from undifferentiated ES cells and differentiating EBs at days 5 and 10. RNA expression levels of Ant4 and ?-actin were evaluated by semiquantitative RT-PCR analysis using various cycles of DNA amplification (20 to 30 cycles). (B): Ant4 derepression by 5-aza-dC. EBs were treated with various concentrations (0–10 μM) of 5-aza-dC for 48 hours from day 8 and harvested at day 10. RNA expression levels of Ant4, Oct-4, Nanog, and ?-actin genes were examined by RT-PCR. Abbreviations: 5-aza-dC, 5-aza-2'-deoxycytidine; EB, embryoid body; ESC, embryonic stem cell; RT-PCR, reverse transcription–polymerase chain reaction.
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8 h* u, b1 r8 b" R! f* c2 NDNA Methylation of Ant4 Gene During ESC Differentiation Requires Dnmt3! h M' Q; V: W' H, ]
4 l R1 w- l* YDNA methyltransferase 3a and 3b (Dnmt3a and Dnmt3b) has been proposed to play a primary role in de novo methylation during murine embryonic development . To investigate the role of the Dnmt3 in the establishment of DNA methylation of the Ant4 promoter during ESC differentiation, we used ESCs homozygously deleted for both the Dnmt3a and Dnmt3b genes. Dnmt3a-Dnmt3b double-null ESCs (earlier passage cell lines) were subjected to the same differentiation protocol as parental (wild-type) J1 ESCs. Of interest, Ant4 expression was not suppressed but rather increased in differentiated double-null cells (Fig. 5A). In contrast, the pluripotent ESC marker Oct-4 was downregulated, whereas the primitive ectoderm marker fibroblast growth factor, Fgf-5, and endoderm marker transthyretin, Ttr, were upregulated after ESC differentiation. These results indicate that Dnmt3a-Dnmt3b double-null ESCs proceed with a normal differentiation pattern. COBRA assays confirmed that there was an absence of DNA methylation at the Ant4 promoter region (–169 bp) in the double knockout ESCs (Fig. 5B). These results indicate that functional Dnmt3 is required for repression of the Ant4 gene during ESC differentiation.
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7 Y1 g) c3 s7 I- y! FFigure 5. Dnmt3 is required for Ant4 repression during ESC differentiation. (A): Expression of the Ant4 gene in WT ES cells and Dnmt3a-Dnmt3b double-null ESCs. WT J1 ES cells and Dnmt3a-Dnmt3b double-null ESCs (Dnmt3a–/–, Dnmt3b–/–) were differentiated as described above. RNA expression was evaluated using RT-PCR as described above. Oct-4, Ttr, and Fgf-5 are markers for undifferentiated ESCs, early visceral endoderm, and early ectoderm, respectively. (B): Ant4 promoter DNA methylation determined by COBRA assay. DNA was extracted from WT ESCs and Dnmt3a-Dnmt3b-null ESCs (Dnmt3a–/–, Dnmt3b–/–) and treated with bisulfite. The Ant4 promoter region was amplified and subjected to overnight digestion with HhaI restriction enzyme, which cuts GCGC sites at –169 bp. DNA methylation of the CpG protects the site from bisulfite conversion; thus, the polymerase chain reaction fragments are digested by HhaI only when the template genomic DNA is methylated at the site. The digested DNA samples were separated in 4.5% polyacrylamide gels and visualized using a SyBr-green dye. (C): Expression of the Ant4 gene and Ant4 promoter methylation in Dnmt3a-null ESCs and Dnmt3b-null ESCs. Dnmt3a-null ESCs (Dnmt3a–/–) and Dnmt3b-null ESCs (Dnmt3b–/–) were differentiated, and Ant4 expression and DNA methylation of the Ant4 promoter were examined as described above. Abbreviations: COBRA, combined bisulfite restriction analysis; EB, embryoid body; ESC, embryonic stem cell; RT-PCR, reverse transcription–polymerase chain reaction; WT, wild-type.' X4 \& L" k. X. G
% W8 m) L- D' B8 \6 q' i* a: HIn addition, we examined which member of the Dnmt3 gene family, Dnmt3a or Dnmt3b, played a predominant role in Ant4 methylation using ESCs deleting either gene. As shown in Figure 5C, downregulation of Ant4 expression was incomplete when Dnmt3a-null ESCs or Dnmt3b-null ESCs were differentiated. Furthermore, both Dnmt3a-null ESCs and Dnmt3b-null ESCs demonstrated virtually no DNA methylation on the CpG site assayed by COBRA. These data indicated that both Dnmt3a and Dnmt3b are required for DNA methylation and repression of Ant4.9 f8 ?! e3 V' u+ k7 m
3 z: D/ J4 M( k, ]( R/ ~Hypomethylation of the Ant4 Promoter and Ant4 Expression Are Restricted to Testicular Germ Cells/ p3 H/ p6 E8 Z; a$ H; d* `5 d" U6 I6 [
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To determine if Ant4 is specifically expressed in ESCs, we initially used a Basic Local Alignment Search Tool (BLAST) search against EST databases. We used full-length Ant4 cDNA as a query sequence and found a total of eight cDNA clones (with scores of >200 bits). All clones identified were from testis. We then investigated expression patterns of Ant4 in adult mouse organs using Northern blot analysis. Ant4 mRNA was found specifically in testes, at the predicted approximately 1.6-kb transcript size, but was undetectable in any other organs examined in Figure 6A. There was no detectable Ant4 mRNA expression in stomach, small intestine, skeletal muscle, ovary, thymus, uterus, or placenta (data not shown). In contrast, the previously identified Ant isoforms, Ant1 and Ant2, were expressed in many non-germ cell organs at various levels but were low or undetectable in testes.
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Figure 6. Ant4 expression and promoter DNA methylation in adult organs. (A): Northern blot analysis of Ant4 mRNA expression in various organs from adult mice (8 weeks old). The blot was hybridized to specific cDNA probes for Ant4, Ant1, Ant2, and ?-actin. (B): Immunohistochemical analysis of Ant4 expression in testis, ovary, and liver. Mouse ovary, testis, and liver were harvested from 3-month-old mice, and frozen sections were stained with affinity-purified rabbit polyclonal anti-Ant4 antibodies and visualized using horse radish peroxidase (brown). Slides were counterstained using Light Green SF Yellowish. (C): Bisulfite analysis of the Ant4 promoter in various organs from adult mice (8 weeks old). DNA samples were extracted from the indicated mice organs and subjected to bisulfite conversion. Seven to eight individual clones were sequenced for each sample. Nucleotide positions are marked relative to the translation initiation site. Arrow represents a major transcription start site (–60 bp). (D): Ant4 mRNA is expressed in purified primordial germ cells. Primordial germ cells were obtained from E11.5 and 12.5-dpc genital ridges and purified using anti-SSEA1 magnetic beads. The immunodepleted fraction (dep) refers to cells not retained on the magnetic column. Individual samples were subjected to reverse transcription–polymerase chain reaction analysis.
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8 r3 f4 R: T+ U, ]( J0 I/ S8 p( \We then examined which cells within testis express the Ant4 protein using specific antibodies raised against mouse Ant4 (Fig. 6B). Results obtained from immunohistochemical analyses confirmed the presence of Ant4 protein within testicular tissue. The strong cytoplasmic staining of spermatagonia, spermatocytes, and spermatids within the seminiferous tubules, coupled with the lack of signal in interstitial, capillary, and capsular cells, suggests testicular germ cell–specific expression of Ant4. Of interest, it seems that mature sperm is absent for Ant4 expression. Although Northern blot analyses using whole ovaries did not detect any Ant4 gene expression (described above), immunohistochemical analyses revealed that Ant4 protein is selectively expressed in the cytoplasm of oocytes (Fig. 6B, middle panels). In contrast, staining performed on liver sections was negative. These data indicate that Ant4 is specifically expressed in developing gametes in testis and ovary. Similar data were obtained using in situ RNA hybridization with riboprobes against Ant4 transcript (data not shown).
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( _7 a' s G* ^# \Further, we determined DNA methylation levels across the Ant4 promoter region in adult mice organs. Only testis showed hypomethylation of the Ant4 promoter, whereas other tissues examined were hypermethylated (Fig. 6C). Additionally, Ant4 was expressed in purified primordial germ cells, obtained from E11.5 and 12.5-dpc genital ridges when they were purified using anti-SSEA1 magnetic beads (Fig. 6D). The data indicate that Ant4 is expressed in premeiotic fetal germ cells as well.2 j+ |7 U" S& y% w2 n/ U* u
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The authors thank Drs. Thomas Yang, Paul Oh, Jorg Bungert, Keith Robertson, and Michael Rutenberg for helpful discussions and critical reading of the manuscript. This work was supported in part by National Institutes of Health grants DK59699 and RR17001 (to N.T.).
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9 _% m2 _2 m# h& pN.T. owns stock in RegenMed, Inc.3 Z7 x; u7 m h! U8 Y" z5 j
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