- Email: [email protected]
Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells
Biochemical and Biophysical Research Communications 359 (2007) 536–542 www.elsevier.com/locate/ybbrc
Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells Seungeun Yeo
a,b
, Sangkyun Jeong a, Janghwan Kim a, Jee-Soo Han a, Yong-Mahn Han Yong-Kook Kang a,b,*
c,* ,
a Center for Regenerative Medicine, KRIBB, Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea Department of Functional Genomics, University of Science and Technology, Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea Department of Biological Sciences, Korean Advanced Institute of Science and Technology, Guseong-Dong, Yuseong-Gu, Daejeon 305-701, Republic of Korea b
c
Received 9 May 2007 Available online 25 May 2007
Abstract Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Human embryonic stem cell; DNA methylation; Oct4; Nanog
The pluripotent nature of human embryonic stem cells (ESCs) that can differentiate into multi-lineage cell types supports their use as potential therapeutic resources in cell transplantation and tissue repair [1]. ESCs exist in a transcriptionally permissive state, allowing self-renewal without imposing ‘restrictions’ until differentiation into diverse cell types [2]. One of the typical and mitotically heritable ‘restrictions’ of the ESC genome accompanying differentiation is DNA methylation. Differentiation-associated accumulation of DNA methylation gradually restrains pluripotency and the self-renewing ability of ESCs.
*
Corresponding authors. Address: Center for Regenerative Medicine, KRIBB, Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea. Fax: +82 42 860 4608 (Y.-K. Kang). E-mail addresses: [email protected] (Y.-M. Han), ykkang@kribb. re.kr (Y.-K. Kang). 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.120
The expression of several regulatory proteins, such as OCT4, SOX2, FOXD3, NANOG, and REX1, is necessary for maintaining the pluripotent properties of stem cells [3–6]. Differentiation of ESCs inevitably involves repression of these stem cell marker (SCM) genes. Here, we investigate the mechanistic relationship between the two characteristic events accompanying ESC differentiation, specifically, repression of SCM genes and changes in DNA methylation. If DNA methylation imposes ‘restrictions’ on regulatory regions of SCM genes during differentiation, it may alter the surrounding chromatin structures and transform genomic loci into a transcriptionally incompetent state. A number of earlier studies show that some, not all, SCM genes exhibit DNA methylation [7–9]. In this case, the DNA methylation states of specific SCM genes may serve as an effective indicator of the degree of ESC differentiation. To this purpose, we examined the DNA methylation of the regulatory regions of SCM genes within the
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
ESC genome. As references, several representative genomic repeats (LINE-1, alpha satellite and RTVL-I-like LTR sequences) were analyzed to monitor genome-wide alterations of the DNA methylation state during differentiation of human ESCs. Our data show that the putative regulatory sequences of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, become methylated as ESCs differentiate into embryoid bodies (EBs) or retinoic acid-driven differentiation. Based on these results, we propose that the DNA methylation states of OCT4 and NANOG sequences may be applied as a barometer to determine the extent of hESC differentiation under specific experimental or culture conditions. Materials and methods Cell lines, cultures and harvest. HeLa cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM) (Gibco-BRL, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal calf serum, 100 U/ml gentamycin, and 1% non-essential amino acids (Gibco-BRL, Carlsbad, CA, USA) at 37 °C, 5% CO2, in air. Human embryonic stem cell culture and embryonic body production. Miz-hES1 (passage 70), SNU-hES3 (130 passages), and Cha4-hES (passage 70) cell lines were analyzed. Cells were maintained in an undifferentiated state, as described previously [10]. ESCs were cultured in DMEM/ F12 medium (Invitrogen, Carlsbad, CA, USA) containing 20% knockout serum replacement (Invitrogen, Carlsbad, CA, USA), 1% non-essential amino acids (Invitrogen, Carlsbad, CA, USA), 0.1 mM mercaptoethanol (Sigma, MO, USA), 100 U/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA), and 4 ng/ml FGF (Invitrogen, Carlsbad, CA, USA) on mitomycin C-treated mouse embryonic fibroblast (MEF) feeders at 37 °C, 5% CO2 in air. ESCs were detached mechanically using the glass micropipette. A single ES colony was mechanically divided to 4–5 parts, and clumps were separately plated on a fresh MEF feeder layer. Human embryoid bodies (EBs) were generated according to a previous report [11]. Briefly, to form EBs, clumps of ESCs were cultured under differentiation conditions on bacterial dishes without feeder cells for 3 days, and further incubated at 37 °C and 5% CO2 in air for 3 weeks. Alkaline phosphatase staining. Alkaline phosphatase (AP) staining was performed in ESCs using a kit from Sigma (Sigma, MO, USA), following the manufacturer’s instructions. mRNA extraction and RT-PCR. ESCs and EBs were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Poly(A+) RNA was prepared from total RNA using the Oligotex mRNA kit (Qiagen, Valencia, CA, USA). Poly(A+) RNA samples (100 ng) were used to generate first-strand cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo(dT) primers. First-strand cDNA (0.5 ll) was applied in a 20 ll PCR mix. The PCR consisted of an initial step at 95 °C for 3 min, 30 cycles of 30 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C, and a final elongation step at 72 °C for 10 min. Glyceraldehyde-3phosphate dehydrogenase (GAPDH), was used as the positive control. The primer are presented in Supplementary Table 1. Real-time PCR. Real-time PCR was performed with the 7500 Realtime PCR System (Applied biosystems, CA, USA), according to the manufacturer’s instructions. The following conditions were employed for amplification using a SYBR Green PCR master mix (Applied Biosystems, CA, USA): initial denaturation for 10 min, followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The relative quantification of gene expression between multiple samples was achieved by normalization against endogenous GAPDH using the DCT method of quantification. Fold changes were calculated as 2 (SDCT CDCT). The same primers were employed for both real-time RT-PCR and RT-PCR. Genomic DNA extraction, bisulfite sequencing, and HpaII-sensitive PCR. ESCs, EBs, and HeLa cells were lysed in lysis buffer containing 0.5% SDS, 0.1 M EDTA, 10 mM Tris–Cl, pH 8.0, and 100 ng/ml proteinase K,
537
and incubated at 55 °C for 16 h. Genomic DNA was purified by phenol/ chloroform extraction, and digested with BamH1 for 2 days at 37 °C prior to denaturation with 0.3 N NaOH. Two hundred thirty-four microliters of 5 M sodium bisulfite (pH 5) and 13.5 ll of 10 mM hydroquinone were added to digested DNA to convert unmethylated cytosines to uracils and mixture was incubated at 55 °C for 16 h. Converted genomic DNA was recovered with a PCR purification kit (Qiagen, Valencia, CA, USA). To remove sulfate, 3 N NaOH was added to the eluted DNA to a final concentration of 0.3 N. Following ethanol precipitation, genomic DNA was resuspended in 35 ll of distilled water (DW). Amplification was performed in 20 ll reaction mixture containing 2 ll of bisulfite-treated genomic DNA for 40 cycles. The primers are presented in Supplementary Table 1. Amplified products were cloned into a pGEM-T easy vector (Promega, Madison, WI). Individual clones were sequenced using an automatic sequencer (ABI PRISM 377). For HS-PCR, 2 lg of genomic DNA from ESCs, EB or HeLa cells was digested with MspI and HpaII restriction enzymes (New England Biolabs, Ipswich, MA, USA) for 2 days. Amplification was performed in 20 ll of reaction mixture comprising 14 ng of digested genomic DNA for 40 cycles.
Results DNA methylation of repetitive sequences is stably sustained during the differentiation of embryonic stem cells into embryonic bodies To determine DNA methylation changes during early differentiation of ESCs, we analyzed the methylation states of various genomic regions, both in undifferentiated ESCs and their EB derivatives. EBs were prepared by growing detached ES colonies in ESC culture medium without fibroblast growth factor (FGF) supplement on bacterial dishes for three weeks [12]. As shown in Supplementary Fig. 1, undifferentiated ESC colonies stained positive for alkaline phosphatase (AP), and displayed well-defined edges. Expression of SCM maker genes was abundant in ESCs, but substantially decreased in EBs, signifying differentiation and loss of stemness (Supplementary Fig. 1B). The methylation states of three repetitive sequences (retroviral long terminal repeat (LTR) sequence of minisatellite MS32, alpha satellites, and long-interspersed element1 (LINE-1)) were determined by sequencing the individual PCR amplicons from bisulfite-treated genomic DNA. Bisulfite deaminates unmethylated, but not methylated cytosine, converting it to uridine, which makes it possible to discern between unmethylated and methylated cytosine residues in amplification and sequencing analyses. We included the methylation profiles of HeLa cells as a representative control of differentiated cells. Bisulfite sequencing analysis of the MS32 LTR repeat encompassing six CpG dinucleotides revealed 71.7%, 84.6% and 63.4% methylation frequencies in ESCs, EBs and HeLa cells, respectively (Fig. 1). Alpha satellite fragments (210 bp) containing four CpG dinucleotides displayed similar levels of methylation to MS32 LTR in both ESCs and EBs. However HeLa cells exhibited higher methylation of alpha satellites than MS32 LTR (85.0% vs. 63.4%). In contrast, a 215 bp fragment of LINE-1 containing 15 CpG dinucleotides exhibited relatively low methylation levels in all three samples, ranging from 32.5% to
538
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
RTVL-1 like LTR of minisatellite MS32
Alpha satellite x2 x1 x1 x1 x1 x1 x3 x1 x1 x2 x3 x1 x1 x8
ESCs x2 x10 x6
71% (76/106)
x1 x1 x1 x1 x1 x1 x1 x2 x1 x6
HeLa
63.4% (59/93)
x1 x1 x1 x1 x1 x1 x1 x1 x1 x1
x1 x1 x1 x2 x3 x6 x2 x4 x16
34.6% (46/133)
81.9% (118/144)
84.6% (99/117)
x1 x1 x1 x1 x1 x1 x1 x1 x1 x1
33.3% (45/135)
73.1% (76/104)
x1 x1 x1 x3 x2 x1 x11
EBs
LINE-1
x1 x1 x1 x1 x1 x1 x1 x1 x1 x1 x1 x1 x1
x1 x1 x3 x3 x2 x1 x1 x17
85.0% (108/127)
32.5% (62/191)
Fig. 1. DNA methylation of each repetitive sequence is maintained at a certain level during ESCs differentiation. The DNA methylation states of RTVL1-like LTR of minisatellite MS32, Alpha satellite and LINE-1 were determined by bisulfite sequencing. Filled circles denote methylated cytosines and open circles denote unmethylated cytosines in CpG dinucleotides. Representative patterns are shown with the copy numbers presented on the right. The methylation level of each repeat sequence is presented as the percentage of filled circles over total circles for a given repeat in a genomic DNA preparation.
34.6%. It appears that ESCs and EBs possess similar DNA methylation signatures for genomic repeats. Our data suggest that overall, the methylation states of repetitive sequences are stably maintained during differentiation of ESCs into EBs. OCT4 and NANOG genes acquire a certain level of DNA methylation on their putative regulatory loci during differentiation into embryoid bodies Next, we examined the DNA methylation states around the promoter regions of several SCM genes (Oct4, NANOG, SOX2, FOXD3 and REX1). In addition to bisulfite sequencing analysis, we performed HpaII restrictionsensitive PCR (HS-PCR) to determine methylation states. HS-PCR exploits the differential sensitivity of the substrate
to HpaII, depending on methylation modifications. The enzyme digests unmethylated CCGG, but not the methylated version (CCmGG). Thus, a DNA fragment containing methylated HpaII site(s) should be amplified by PCR, even following HpaII digestion. Sequence analysis of the promoter regions of SCM genes led to the identification of canonical CpG islands, the elements characterized by high CpG dinucleotide frequency within a GC-rich context, except in OCT4 and NANOG. OCT4 expression is driven by the associated activities of an enhancer centered at 2500 bp and the promoter region (Fig. 2A) [13]. Bisulfite sequencing analysis revealed that these cis elements were almost free of cytosine methylation in ES cells, but became slightly methylated upon differentiation into EBs with frequencies of 23.2% and 14.3% in the enhancer and promoter regions, respec-
c Fig. 2. Methylation states on the promoter regions of SCM genes were analyzed using bisulfite sequencing and HS-PCR. (A) Promoter regions of SCM genes are depicted on a schematic map to highlight the transcription initiation site (arrow) and relative positions of CpG–dinucleotides (vertical lines). Regions analyzed for methylation states are specified on the map with horizontal bars (open form for HS-PCR or filled for bisulfite sequencing). CCGG on HS-PCR fragments are depicted with stars (*). (B) Genomic DNA was initially digested to completion with either MspI or HpaII restriction nucleases, and subjected to PCR to amplify interested region. Amplification of HpaII digested genomic DNA provides evidence of cytosine methylation at the recognition sequence. No amplified product was expected from MspI-digested genomic DNA. S, ESCs; B, EBs; H, HeLa cells; C, undigested genomic DNA control. (C) The DNA methylation states of SCM genes were determined by bisulfite sequencing. The methylation level is presented as the percentage of filled circles denoting methylated cytosines over total circles showing cytosine in CpG for a given locus in the genomic DNA preparation.
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
OCT4
NANOG NAN-D1
Oct-P1
Oct-D1 x8 x1 x1 x1
ESCs 6.3%( 3/48)
0% (0/72)
23.2% (13/56)
HeLa
76.9%(40/52)
7.5% (6/80) x6 x3 x3 x1 x1
9.6% (5/52)
SOX2
5.6% (3/54)
58.9% (33/56)
2.8% (1/36)
Fox-P1
Fox-P2
EBs
x5 x1
HeLa 92.7% (102.110)
3.0% (13/435)
x9
0% (0/153) x1 x1 x1 x1 x3 x1 x4 x1 x1 x1
x10
0% (0/150)
x12
0% (0/204) x5 x1 x3 x3 x1 x2
x10
x1 x1 x3
31.7% (19/60)
Rex-P1
3.4% (16/464)
0% (0/150)
0.8% (1/132)
x3 x1 x2 x1 x2 x1 x1 x1
x3 x1 x2 x2 x5 x1 x2
x7
0% ( 0/105)
0.8% (1/132)
6.7% (6/90)
REX1
ESCs x5 x1
x13 x1 x1 x1 x1 x1
x12
0% (0/36)
FOXD3
Sox-E1
x13 x1
1.4% (1/70)
x11 x1
x11 x1 x2
x8
83.3%(40/48)
NAN-P1 x15 x1 x2
x8 x3 x2
14.3% (12/84)
x1 x2 x1 x1 x2 x1 x5
NAN-M1 x14 x6
x12
x2 x2 x3 x6 x1
EBs
539
62.8% (273/435)
x9
0% (0/153)
540
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
Change of mRNA expression
Control
RA
1.2 1 0.8
x3 x1 x1 x1 x2 x1 x3 x1
0.6
OCT4
0.4 0.2 x12
0
Control
NANOG
1 Day
3 Day
5 Day
0% (0/72 )
1.4 1.2 1 0.8 0.6 0.4 0.2 0
16.9% (13/77) x2 x2 x2 x1 x2 x4 x1 x1 x1 x1 x2 x3
x16 x1 x1
Control
1 Day
3 Day
5 Day
2.2% (2/90)
43.6% (48/110)
Fig. 3. Expression of SCM genes and methylation patterns on the promoter regions were analyzed in differentiating ESCs by treatment with retinoic acid. Relative expression of SCM genes in cells on specific days, compared to that in control cells, was determined with the DCT method using real-time PCR. Genomic DNA prepared from RA-induced differentiated cells on day 5 was subjected to bisulfite sequencing to determine the methylation states of the SCM gene promoter regions. The methylation level of each region is presented as the percentage of filled circles over total circles for a given locus in the genomic DNA.
tively. In contrast, these elements were heavily methylated in HeLa cells (Fig. 2C). Similar results were obtained with HS-PCR (Fig. 2B). In view of the finding that OCT4 expression is downregulated upon differentiation of ESCs into EBs, and absent in HeLa cells, we propose that cytosine methylation of these cis elements is associated with transcriptional activity of the OCT4 gene. NANOG is also devoid of CpG islands near the transcription initiation site (TIS). Three regions (centered at 0.4, 1.0 and 1.7 kb from the TIS) were arbitrarily selected for methylation analysis (Fig. 2A). As shown in Fig. 3C, most CpGs in the sequences examined were not methylated in ESCs (ranging from 1.4% to 7.5% of the methylation rate), and maintained at this level with no significant changes during differentiation into EBs (2.8–9.6% methylation frequency). Distinct results were obtained with HS-PCR (Fig. 2B). Two selected regions of NANOG were differentially methylated between ESCs and EBs. Unfortunately, the scarcity of HpaII sites in the NANOG promoter regions prevented a detailed investigation of differentiation-induced alterations in DNA methylation in EBs. Other SCM genes (SOX2, FOXD3 and REX1) are associated with CpG islands around their TISs. Bisulfite sequencing analyses revealed that the CpG-rich first exon sequence of SOX2 was almost completely unmethylated in both ESCs (0.8%, 1/132) and EBs (0.8%, 1/132) (Figs. 2A and C). In contrast, genomic DNA in HeLa calls was heavily methylated in the locus. Two other loci, one in the proximal promoter and the other further upstream, were almost devoid of methylation in both ESCs and EBs, as determined by HS-PCR (Fig. 3B). Similar low or no methylation was observed in the promoter regions of FOXD3 encompassing a
2 kb region upstream of TIS (Fig. 2A) in ESCs and EBs. Finally, a CpG-rich region proximal to the promoter of REX1 exhibited an unmethylated state in both ESCs and EBs. However, an upstream locus with low CpG density was heavily methylated in all cell lines examined using HS-PCR (Fig. 2B). These results indicate that the promoters of SOX2, FOXD3 and REX1 were maintained in a methylation-free state during differentiation of ESCs into EBs. The promoter regions of OCT4 and NANOG genes undergo methylation during retinoic acid-induced differentiation Since the promoters of OCT4 and NANOG genes undergo cytosine methylation during differentiation of ESCs into EBs, we ascertained whether these methylation changes are replicated in chemically induced differentiation. The methylation states of SCM genes were analyzed in ESCs subjected to retinoic acid (RA) differentiation. Data presented in Fig. 3 were obtained from ESCs treated with 1 lM RA for 5 days. One micromolar RA stimulated ESCs resistance to AP staining and to change morphology from compact association between individual cells to released association (data not shown). Quantitative real-time PCR results revealed a general down-regulation of SCM genes expression to a level of almost complete cessation. In addition, imposition of methylation on the promoters of OCT4 and NANOG genes became evident as ESCs underwent RA-induced differentiation, with frequencies of 18.6% and 43.6%, respectively. Consistent with EB differentiation data, the promoters of other SCM genes (SOX2 and FOXD3) did not undergo methylation modifications during RA-induced differentiation (data not shown).
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
Discussion Differentiation involves various cellular changes, including physiology, structural architecture and functions. In addition, derivation of specific somatic cells from pluripotent stem cells occurs in a well organized and programmed manner. Every event in the course of differentiation should be accompanied by coordinated expression and/or repression of certain subsets of genes. DNA methylation is one of the epigenetic means playing key roles in stable and heritable setting up chromatin structures, but reversible in case of requirement for differentiation-associated gene expression/repression. Therefore, elucidating the mechanistic relationship between DNA methylation and gene regulation during ESC differentiation should improve our understanding of the differentiation-associated cellular changes, which may facilitate the manipulation of stem cell differentiation into a desired cell type. We initially analyzed the methylation profiles of several repetitive sequences in both undifferentiated and differentiated ESCs. These sequences do not appear to undergo any notable changes in the methylation level during differentiation of ESCs into EBs. In addition, the methylation patterns of HeLa are similar to those of ESCs and EBs. The LINE-1 repeat sequence constitutes about 17% of the human genome [14]. Alpha satellites are positioned to centromeric regions of human chromosomes, and occupy 3–5% of the chromosome content [15]. Thus, changes in the methylation levels of highly repetitive sequences should significantly affect the overall integrity of a genome. In this regard, differentiation doesn’t seem to trigger alteration of the global DNA methylation level on the genome. Approximately half of the human genes are associated with CpG islands at their promoter regions [16]. Earlier studies indicate that CpG islands are virtually free of cytosine methylation. However, recent reports show that a subset of the islands undergoes methylation in a tissue-, disease- or differentiation-specific way [17–19], suggesting that cytosine methylation may play key roles in differentiation by modulating gene expression. Thus, the issue of whether the differentiation-associated repression of SCM genes is subjected to epigenetic regulation is of particular interest. Of the SCM genes analyzed, OCT4 and NANOG do not have typical CpG islands around their transcription initiation sites. Nonetheless, the cytosine residue of CpG dinucleotides around the promoter region undergoes limited methylation modifications during differentiation. These epigenetic changes coincide with the down-regulation of OCT4 and NANOG expression. In keeping with this finding, Lee et. al. reported that DNA and histone H3-lys9 methylation of the OCT4 promoter region accompany gene repression in differentiating mouse ES cells upon LIF removal [20]. An association between epigenetic modification and gene
541
expression changes in OCT4 and NANOG promoters was additionally observed in embryonal carcinoma cells (NT2) differentiating into neuronal cells [7]. In our study, OCT4 and NANOG levels were almost completely repressed at 4 and 6 days of RA treatment, respectively, whereas gain of methylation on the promoters appeared weak, relative to the degree of gene repression, even after longer RA treatment. In view of the substantial reduction in gene expression versus low rate of methylation modification, we conclude that the imposition of methylation on promoter regions is not the primary cause of expression repression. Rather, the methylation pattern may arise as a consequence of accumulation of occasional hits by DNA methyltransferase activities on transcriptionally inactive loci. Consistent with this theory, HeLa cells display high methyl cytosine content in these loci, possibly due to the prolonged accumulation of such hits. All the other SCM genes examined were associated with a CpG island in their promoter regions. CpG islands were essentially devoid of methylation in ESCs and EBs, confirming that methylation is not the cause of gene repression in differentiating ESCs. One report addressed the reactivation of SCM genes, including SOX2 in EBs, by the demethylating agent, 5-azacytidine (5-AzaC) [9]. Since our EBs displayed virtually no methylation of SOX2, 5-AzaC-mediated gene reactivation seems to be exerted indirectly. However, heavy methylation of SOX2 and FOXD3 islands in HeLa cells indicates a requirement for epigenetic modification on those loci for stable gene repression. In summary, our results support the repression of stem cell marker genes in differentiating ESCs by mechanisms that do not involve DNA methylation as a primary operator. However, two representative genes, OCT4 and NANOG, which do not contain canonical CpG islands within the promoter region, display some degree of methylation upon differentiation. In particular, NANOG shows varying degrees of methylation in response to discrete differentiation protocols, supporting the theory that the quality or degree of differentiation can be determined by methylation analysis. Acknowledgments This research was supported by grants (SC2090) from the Stem Cell Research Center of the 21st Centry Frontier Research Program, KRIBB Research Initiative Program, and KOSEF Stem Cell Research Program (M1064102 000206N410200210). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2007.05.120.
542
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
References [1] J.A. Thomson, J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, J.M. Jones, Embryonic stem cell lines derived from human blastocysts, Science 282 (1998) 1145–1147. [2] M.A. Surani, Reprogramming of genome function through epigenetic inheritance, Nature 414 (2001) 122–128. [3] M. Boiani, S. Eckardt, H.R. Scholer, K.J. McLaughlin, Oct4 distribution and level in mouse clones: consequences for pluripotency, Genes Dev. 16 (2002) 1209–1219. [4] L.A. Hanna, R.K. Foreman, I.A. Tarasenko, D.S. Kessler, P.A. Labosky, Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo, Genes Dev. 16 (2002) 2650–2661. [5] K. Mitsui, Y. Tokuzawa, H. Itoh, K. Segawa, M. Murakami, K. Takahashi, M. Maruyama, M. Maeda, S. Yamanaka, The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells, Cell 113 (2003) 631–642. [6] H. Niwa, Molecular mechanism to maintain stem cell renewal of ES cells, Cell Struct. Funct. 26 (2001) 137–148. [7] P. Deb-Rinker, D. Ly, A. Jezierski, M. Sikorska, P.R. Walker, Sequential DNA Methylation of the Nanog and Oct-4 Upstream Regions in Human NT2 Cells during Neuronal Differentiation, J. Biol. Chem. 280 (2005) 6257–6260. [8] N. Hattori, K. Nishino, Y.G. Ko, J. Ohgane, S. Tanaka, K. Shiota, Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells, J. Biol. Chem. 279 (2004) 17063– 17069. [9] K. Tsuji-Takayama, T. Inoue, Y. Ijiri, T. Otani, R. Motoda, S. Nakamura, K. Orita, Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells, Biochem. Biophys. Res. Commun. 323 (2004) 86–90. [10] J.H. Park, S.J. Kim, E.J. Oh, S.Y. Moon, S.I. Roh, C.G. Kim, H.S. Yoon, Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line, Biol. Reprod. 69 (2003) 2007–2014.
[11] N. Sato, I.M. Sanjuan, M. Heke, M. Uchida, F. Naef, A.H. Brivanlou, Molecular signature of human embryonic stem cells and its comparison with the mouse, Dev. Biol. 260 (2003) 404– 413. [12] J.Y. Rho, K. Yu, J.S. Han, J.I. Chae, D.B. Koo, H.S. Yoon, S.Y. Moon, K.K. Lee, Y.M. Han, Transcriptional profiling of the developmentally important signalling pathways in human embryonic stem cells, Hum. Reprod. 21 (2006) 405–412. [13] Y.I. Yeom, G. Fuhrmann, C.E. Ovitt, A. Brehm, K. Ohbo, M. Gross, K. Hubner, H.R. Scholer, Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells, Development 122 (1996) 881–894. [14] A.F. Smit, The origin of interspersed repeats in the human genome, Curr. Opin. Genet. Dev. 6 (1996) 743–748. [15] K.H. Choo, B. Vissel, A. Nagy, E. Earle, P. Kalitsis, A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence, Nucleic Acids Res. 19 (1991) 1179–1182. [16] I.P. Ioshikhes, M.Q. Zhang, Large-scale human promoter mapping using CpG islands, Nat. Genet. 26 (2000) 61–63. [17] M. Bibikova, E. Chudin, B. Wu, L. Zhou, E.W. Garcia, Y. Liu, S. Shin, T.W. Plaia, J.M. Auerbach, D.E. Arking, R. Gonzalez, J. Crook, B. Davidson, T.C. Schulz, A. Robins, A. Khanna, P. Sartipy, J. Hyllner, P. Vanguri, S. Savant-Bhonsale, A.K. Smith, A. Chakravarti, A. Maitra, M. Rao, D.L. Barker, J.F. Loring, J.B. Fan, Human embryonic stem cells have a unique epigenetic signature, Genome Res. 16 (2006) 1075–1083. [18] K.D. Robertson, DNA methylation and human disease, Nat. Rev. Genet. 6 (2005) 597–610. [19] K. Shiota, DNA methylation profiles of CpG islands for cellular differentiation and development in mammals, Cytogenet. Genome. Res. 105 (2004) 325–334. [20] J.H. Lee, S.R. Hart, D.G. Skalnik, Histone deacetylase activity is required for embryonic stem cell differentiation, Genesis 38 (2004) 32– 38.
Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells Seungeun Yeo
a,b
, Sangkyun Jeong a, Janghwan Kim a, Jee-Soo Han a, Yong-Mahn Han Yong-Kook Kang a,b,*
c,* ,
a Center for Regenerative Medicine, KRIBB, Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea Department of Functional Genomics, University of Science and Technology, Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea Department of Biological Sciences, Korean Advanced Institute of Science and Technology, Guseong-Dong, Yuseong-Gu, Daejeon 305-701, Republic of Korea b
c
Received 9 May 2007 Available online 25 May 2007
Abstract Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Human embryonic stem cell; DNA methylation; Oct4; Nanog
The pluripotent nature of human embryonic stem cells (ESCs) that can differentiate into multi-lineage cell types supports their use as potential therapeutic resources in cell transplantation and tissue repair [1]. ESCs exist in a transcriptionally permissive state, allowing self-renewal without imposing ‘restrictions’ until differentiation into diverse cell types [2]. One of the typical and mitotically heritable ‘restrictions’ of the ESC genome accompanying differentiation is DNA methylation. Differentiation-associated accumulation of DNA methylation gradually restrains pluripotency and the self-renewing ability of ESCs.
*
Corresponding authors. Address: Center for Regenerative Medicine, KRIBB, Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea. Fax: +82 42 860 4608 (Y.-K. Kang). E-mail addresses: [email protected] (Y.-M. Han), ykkang@kribb. re.kr (Y.-K. Kang). 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.120
The expression of several regulatory proteins, such as OCT4, SOX2, FOXD3, NANOG, and REX1, is necessary for maintaining the pluripotent properties of stem cells [3–6]. Differentiation of ESCs inevitably involves repression of these stem cell marker (SCM) genes. Here, we investigate the mechanistic relationship between the two characteristic events accompanying ESC differentiation, specifically, repression of SCM genes and changes in DNA methylation. If DNA methylation imposes ‘restrictions’ on regulatory regions of SCM genes during differentiation, it may alter the surrounding chromatin structures and transform genomic loci into a transcriptionally incompetent state. A number of earlier studies show that some, not all, SCM genes exhibit DNA methylation [7–9]. In this case, the DNA methylation states of specific SCM genes may serve as an effective indicator of the degree of ESC differentiation. To this purpose, we examined the DNA methylation of the regulatory regions of SCM genes within the
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
ESC genome. As references, several representative genomic repeats (LINE-1, alpha satellite and RTVL-I-like LTR sequences) were analyzed to monitor genome-wide alterations of the DNA methylation state during differentiation of human ESCs. Our data show that the putative regulatory sequences of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, become methylated as ESCs differentiate into embryoid bodies (EBs) or retinoic acid-driven differentiation. Based on these results, we propose that the DNA methylation states of OCT4 and NANOG sequences may be applied as a barometer to determine the extent of hESC differentiation under specific experimental or culture conditions. Materials and methods Cell lines, cultures and harvest. HeLa cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM) (Gibco-BRL, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal calf serum, 100 U/ml gentamycin, and 1% non-essential amino acids (Gibco-BRL, Carlsbad, CA, USA) at 37 °C, 5% CO2, in air. Human embryonic stem cell culture and embryonic body production. Miz-hES1 (passage 70), SNU-hES3 (130 passages), and Cha4-hES (passage 70) cell lines were analyzed. Cells were maintained in an undifferentiated state, as described previously [10]. ESCs were cultured in DMEM/ F12 medium (Invitrogen, Carlsbad, CA, USA) containing 20% knockout serum replacement (Invitrogen, Carlsbad, CA, USA), 1% non-essential amino acids (Invitrogen, Carlsbad, CA, USA), 0.1 mM mercaptoethanol (Sigma, MO, USA), 100 U/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA), and 4 ng/ml FGF (Invitrogen, Carlsbad, CA, USA) on mitomycin C-treated mouse embryonic fibroblast (MEF) feeders at 37 °C, 5% CO2 in air. ESCs were detached mechanically using the glass micropipette. A single ES colony was mechanically divided to 4–5 parts, and clumps were separately plated on a fresh MEF feeder layer. Human embryoid bodies (EBs) were generated according to a previous report [11]. Briefly, to form EBs, clumps of ESCs were cultured under differentiation conditions on bacterial dishes without feeder cells for 3 days, and further incubated at 37 °C and 5% CO2 in air for 3 weeks. Alkaline phosphatase staining. Alkaline phosphatase (AP) staining was performed in ESCs using a kit from Sigma (Sigma, MO, USA), following the manufacturer’s instructions. mRNA extraction and RT-PCR. ESCs and EBs were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Poly(A+) RNA was prepared from total RNA using the Oligotex mRNA kit (Qiagen, Valencia, CA, USA). Poly(A+) RNA samples (100 ng) were used to generate first-strand cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo(dT) primers. First-strand cDNA (0.5 ll) was applied in a 20 ll PCR mix. The PCR consisted of an initial step at 95 °C for 3 min, 30 cycles of 30 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C, and a final elongation step at 72 °C for 10 min. Glyceraldehyde-3phosphate dehydrogenase (GAPDH), was used as the positive control. The primer are presented in Supplementary Table 1. Real-time PCR. Real-time PCR was performed with the 7500 Realtime PCR System (Applied biosystems, CA, USA), according to the manufacturer’s instructions. The following conditions were employed for amplification using a SYBR Green PCR master mix (Applied Biosystems, CA, USA): initial denaturation for 10 min, followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The relative quantification of gene expression between multiple samples was achieved by normalization against endogenous GAPDH using the DCT method of quantification. Fold changes were calculated as 2 (SDCT CDCT). The same primers were employed for both real-time RT-PCR and RT-PCR. Genomic DNA extraction, bisulfite sequencing, and HpaII-sensitive PCR. ESCs, EBs, and HeLa cells were lysed in lysis buffer containing 0.5% SDS, 0.1 M EDTA, 10 mM Tris–Cl, pH 8.0, and 100 ng/ml proteinase K,
537
and incubated at 55 °C for 16 h. Genomic DNA was purified by phenol/ chloroform extraction, and digested with BamH1 for 2 days at 37 °C prior to denaturation with 0.3 N NaOH. Two hundred thirty-four microliters of 5 M sodium bisulfite (pH 5) and 13.5 ll of 10 mM hydroquinone were added to digested DNA to convert unmethylated cytosines to uracils and mixture was incubated at 55 °C for 16 h. Converted genomic DNA was recovered with a PCR purification kit (Qiagen, Valencia, CA, USA). To remove sulfate, 3 N NaOH was added to the eluted DNA to a final concentration of 0.3 N. Following ethanol precipitation, genomic DNA was resuspended in 35 ll of distilled water (DW). Amplification was performed in 20 ll reaction mixture containing 2 ll of bisulfite-treated genomic DNA for 40 cycles. The primers are presented in Supplementary Table 1. Amplified products were cloned into a pGEM-T easy vector (Promega, Madison, WI). Individual clones were sequenced using an automatic sequencer (ABI PRISM 377). For HS-PCR, 2 lg of genomic DNA from ESCs, EB or HeLa cells was digested with MspI and HpaII restriction enzymes (New England Biolabs, Ipswich, MA, USA) for 2 days. Amplification was performed in 20 ll of reaction mixture comprising 14 ng of digested genomic DNA for 40 cycles.
Results DNA methylation of repetitive sequences is stably sustained during the differentiation of embryonic stem cells into embryonic bodies To determine DNA methylation changes during early differentiation of ESCs, we analyzed the methylation states of various genomic regions, both in undifferentiated ESCs and their EB derivatives. EBs were prepared by growing detached ES colonies in ESC culture medium without fibroblast growth factor (FGF) supplement on bacterial dishes for three weeks [12]. As shown in Supplementary Fig. 1, undifferentiated ESC colonies stained positive for alkaline phosphatase (AP), and displayed well-defined edges. Expression of SCM maker genes was abundant in ESCs, but substantially decreased in EBs, signifying differentiation and loss of stemness (Supplementary Fig. 1B). The methylation states of three repetitive sequences (retroviral long terminal repeat (LTR) sequence of minisatellite MS32, alpha satellites, and long-interspersed element1 (LINE-1)) were determined by sequencing the individual PCR amplicons from bisulfite-treated genomic DNA. Bisulfite deaminates unmethylated, but not methylated cytosine, converting it to uridine, which makes it possible to discern between unmethylated and methylated cytosine residues in amplification and sequencing analyses. We included the methylation profiles of HeLa cells as a representative control of differentiated cells. Bisulfite sequencing analysis of the MS32 LTR repeat encompassing six CpG dinucleotides revealed 71.7%, 84.6% and 63.4% methylation frequencies in ESCs, EBs and HeLa cells, respectively (Fig. 1). Alpha satellite fragments (210 bp) containing four CpG dinucleotides displayed similar levels of methylation to MS32 LTR in both ESCs and EBs. However HeLa cells exhibited higher methylation of alpha satellites than MS32 LTR (85.0% vs. 63.4%). In contrast, a 215 bp fragment of LINE-1 containing 15 CpG dinucleotides exhibited relatively low methylation levels in all three samples, ranging from 32.5% to
538
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
RTVL-1 like LTR of minisatellite MS32
Alpha satellite x2 x1 x1 x1 x1 x1 x3 x1 x1 x2 x3 x1 x1 x8
ESCs x2 x10 x6
71% (76/106)
x1 x1 x1 x1 x1 x1 x1 x2 x1 x6
HeLa
63.4% (59/93)
x1 x1 x1 x1 x1 x1 x1 x1 x1 x1
x1 x1 x1 x2 x3 x6 x2 x4 x16
34.6% (46/133)
81.9% (118/144)
84.6% (99/117)
x1 x1 x1 x1 x1 x1 x1 x1 x1 x1
33.3% (45/135)
73.1% (76/104)
x1 x1 x1 x3 x2 x1 x11
EBs
LINE-1
x1 x1 x1 x1 x1 x1 x1 x1 x1 x1 x1 x1 x1
x1 x1 x3 x3 x2 x1 x1 x17
85.0% (108/127)
32.5% (62/191)
Fig. 1. DNA methylation of each repetitive sequence is maintained at a certain level during ESCs differentiation. The DNA methylation states of RTVL1-like LTR of minisatellite MS32, Alpha satellite and LINE-1 were determined by bisulfite sequencing. Filled circles denote methylated cytosines and open circles denote unmethylated cytosines in CpG dinucleotides. Representative patterns are shown with the copy numbers presented on the right. The methylation level of each repeat sequence is presented as the percentage of filled circles over total circles for a given repeat in a genomic DNA preparation.
34.6%. It appears that ESCs and EBs possess similar DNA methylation signatures for genomic repeats. Our data suggest that overall, the methylation states of repetitive sequences are stably maintained during differentiation of ESCs into EBs. OCT4 and NANOG genes acquire a certain level of DNA methylation on their putative regulatory loci during differentiation into embryoid bodies Next, we examined the DNA methylation states around the promoter regions of several SCM genes (Oct4, NANOG, SOX2, FOXD3 and REX1). In addition to bisulfite sequencing analysis, we performed HpaII restrictionsensitive PCR (HS-PCR) to determine methylation states. HS-PCR exploits the differential sensitivity of the substrate
to HpaII, depending on methylation modifications. The enzyme digests unmethylated CCGG, but not the methylated version (CCmGG). Thus, a DNA fragment containing methylated HpaII site(s) should be amplified by PCR, even following HpaII digestion. Sequence analysis of the promoter regions of SCM genes led to the identification of canonical CpG islands, the elements characterized by high CpG dinucleotide frequency within a GC-rich context, except in OCT4 and NANOG. OCT4 expression is driven by the associated activities of an enhancer centered at 2500 bp and the promoter region (Fig. 2A) [13]. Bisulfite sequencing analysis revealed that these cis elements were almost free of cytosine methylation in ES cells, but became slightly methylated upon differentiation into EBs with frequencies of 23.2% and 14.3% in the enhancer and promoter regions, respec-
c Fig. 2. Methylation states on the promoter regions of SCM genes were analyzed using bisulfite sequencing and HS-PCR. (A) Promoter regions of SCM genes are depicted on a schematic map to highlight the transcription initiation site (arrow) and relative positions of CpG–dinucleotides (vertical lines). Regions analyzed for methylation states are specified on the map with horizontal bars (open form for HS-PCR or filled for bisulfite sequencing). CCGG on HS-PCR fragments are depicted with stars (*). (B) Genomic DNA was initially digested to completion with either MspI or HpaII restriction nucleases, and subjected to PCR to amplify interested region. Amplification of HpaII digested genomic DNA provides evidence of cytosine methylation at the recognition sequence. No amplified product was expected from MspI-digested genomic DNA. S, ESCs; B, EBs; H, HeLa cells; C, undigested genomic DNA control. (C) The DNA methylation states of SCM genes were determined by bisulfite sequencing. The methylation level is presented as the percentage of filled circles denoting methylated cytosines over total circles showing cytosine in CpG for a given locus in the genomic DNA preparation.
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
OCT4
NANOG NAN-D1
Oct-P1
Oct-D1 x8 x1 x1 x1
ESCs 6.3%( 3/48)
0% (0/72)
23.2% (13/56)
HeLa
76.9%(40/52)
7.5% (6/80) x6 x3 x3 x1 x1
9.6% (5/52)
SOX2
5.6% (3/54)
58.9% (33/56)
2.8% (1/36)
Fox-P1
Fox-P2
EBs
x5 x1
HeLa 92.7% (102.110)
3.0% (13/435)
x9
0% (0/153) x1 x1 x1 x1 x3 x1 x4 x1 x1 x1
x10
0% (0/150)
x12
0% (0/204) x5 x1 x3 x3 x1 x2
x10
x1 x1 x3
31.7% (19/60)
Rex-P1
3.4% (16/464)
0% (0/150)
0.8% (1/132)
x3 x1 x2 x1 x2 x1 x1 x1
x3 x1 x2 x2 x5 x1 x2
x7
0% ( 0/105)
0.8% (1/132)
6.7% (6/90)
REX1
ESCs x5 x1
x13 x1 x1 x1 x1 x1
x12
0% (0/36)
FOXD3
Sox-E1
x13 x1
1.4% (1/70)
x11 x1
x11 x1 x2
x8
83.3%(40/48)
NAN-P1 x15 x1 x2
x8 x3 x2
14.3% (12/84)
x1 x2 x1 x1 x2 x1 x5
NAN-M1 x14 x6
x12
x2 x2 x3 x6 x1
EBs
539
62.8% (273/435)
x9
0% (0/153)
540
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
Change of mRNA expression
Control
RA
1.2 1 0.8
x3 x1 x1 x1 x2 x1 x3 x1
0.6
OCT4
0.4 0.2 x12
0
Control
NANOG
1 Day
3 Day
5 Day
0% (0/72 )
1.4 1.2 1 0.8 0.6 0.4 0.2 0
16.9% (13/77) x2 x2 x2 x1 x2 x4 x1 x1 x1 x1 x2 x3
x16 x1 x1
Control
1 Day
3 Day
5 Day
2.2% (2/90)
43.6% (48/110)
Fig. 3. Expression of SCM genes and methylation patterns on the promoter regions were analyzed in differentiating ESCs by treatment with retinoic acid. Relative expression of SCM genes in cells on specific days, compared to that in control cells, was determined with the DCT method using real-time PCR. Genomic DNA prepared from RA-induced differentiated cells on day 5 was subjected to bisulfite sequencing to determine the methylation states of the SCM gene promoter regions. The methylation level of each region is presented as the percentage of filled circles over total circles for a given locus in the genomic DNA.
tively. In contrast, these elements were heavily methylated in HeLa cells (Fig. 2C). Similar results were obtained with HS-PCR (Fig. 2B). In view of the finding that OCT4 expression is downregulated upon differentiation of ESCs into EBs, and absent in HeLa cells, we propose that cytosine methylation of these cis elements is associated with transcriptional activity of the OCT4 gene. NANOG is also devoid of CpG islands near the transcription initiation site (TIS). Three regions (centered at 0.4, 1.0 and 1.7 kb from the TIS) were arbitrarily selected for methylation analysis (Fig. 2A). As shown in Fig. 3C, most CpGs in the sequences examined were not methylated in ESCs (ranging from 1.4% to 7.5% of the methylation rate), and maintained at this level with no significant changes during differentiation into EBs (2.8–9.6% methylation frequency). Distinct results were obtained with HS-PCR (Fig. 2B). Two selected regions of NANOG were differentially methylated between ESCs and EBs. Unfortunately, the scarcity of HpaII sites in the NANOG promoter regions prevented a detailed investigation of differentiation-induced alterations in DNA methylation in EBs. Other SCM genes (SOX2, FOXD3 and REX1) are associated with CpG islands around their TISs. Bisulfite sequencing analyses revealed that the CpG-rich first exon sequence of SOX2 was almost completely unmethylated in both ESCs (0.8%, 1/132) and EBs (0.8%, 1/132) (Figs. 2A and C). In contrast, genomic DNA in HeLa calls was heavily methylated in the locus. Two other loci, one in the proximal promoter and the other further upstream, were almost devoid of methylation in both ESCs and EBs, as determined by HS-PCR (Fig. 3B). Similar low or no methylation was observed in the promoter regions of FOXD3 encompassing a
2 kb region upstream of TIS (Fig. 2A) in ESCs and EBs. Finally, a CpG-rich region proximal to the promoter of REX1 exhibited an unmethylated state in both ESCs and EBs. However, an upstream locus with low CpG density was heavily methylated in all cell lines examined using HS-PCR (Fig. 2B). These results indicate that the promoters of SOX2, FOXD3 and REX1 were maintained in a methylation-free state during differentiation of ESCs into EBs. The promoter regions of OCT4 and NANOG genes undergo methylation during retinoic acid-induced differentiation Since the promoters of OCT4 and NANOG genes undergo cytosine methylation during differentiation of ESCs into EBs, we ascertained whether these methylation changes are replicated in chemically induced differentiation. The methylation states of SCM genes were analyzed in ESCs subjected to retinoic acid (RA) differentiation. Data presented in Fig. 3 were obtained from ESCs treated with 1 lM RA for 5 days. One micromolar RA stimulated ESCs resistance to AP staining and to change morphology from compact association between individual cells to released association (data not shown). Quantitative real-time PCR results revealed a general down-regulation of SCM genes expression to a level of almost complete cessation. In addition, imposition of methylation on the promoters of OCT4 and NANOG genes became evident as ESCs underwent RA-induced differentiation, with frequencies of 18.6% and 43.6%, respectively. Consistent with EB differentiation data, the promoters of other SCM genes (SOX2 and FOXD3) did not undergo methylation modifications during RA-induced differentiation (data not shown).
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
Discussion Differentiation involves various cellular changes, including physiology, structural architecture and functions. In addition, derivation of specific somatic cells from pluripotent stem cells occurs in a well organized and programmed manner. Every event in the course of differentiation should be accompanied by coordinated expression and/or repression of certain subsets of genes. DNA methylation is one of the epigenetic means playing key roles in stable and heritable setting up chromatin structures, but reversible in case of requirement for differentiation-associated gene expression/repression. Therefore, elucidating the mechanistic relationship between DNA methylation and gene regulation during ESC differentiation should improve our understanding of the differentiation-associated cellular changes, which may facilitate the manipulation of stem cell differentiation into a desired cell type. We initially analyzed the methylation profiles of several repetitive sequences in both undifferentiated and differentiated ESCs. These sequences do not appear to undergo any notable changes in the methylation level during differentiation of ESCs into EBs. In addition, the methylation patterns of HeLa are similar to those of ESCs and EBs. The LINE-1 repeat sequence constitutes about 17% of the human genome [14]. Alpha satellites are positioned to centromeric regions of human chromosomes, and occupy 3–5% of the chromosome content [15]. Thus, changes in the methylation levels of highly repetitive sequences should significantly affect the overall integrity of a genome. In this regard, differentiation doesn’t seem to trigger alteration of the global DNA methylation level on the genome. Approximately half of the human genes are associated with CpG islands at their promoter regions [16]. Earlier studies indicate that CpG islands are virtually free of cytosine methylation. However, recent reports show that a subset of the islands undergoes methylation in a tissue-, disease- or differentiation-specific way [17–19], suggesting that cytosine methylation may play key roles in differentiation by modulating gene expression. Thus, the issue of whether the differentiation-associated repression of SCM genes is subjected to epigenetic regulation is of particular interest. Of the SCM genes analyzed, OCT4 and NANOG do not have typical CpG islands around their transcription initiation sites. Nonetheless, the cytosine residue of CpG dinucleotides around the promoter region undergoes limited methylation modifications during differentiation. These epigenetic changes coincide with the down-regulation of OCT4 and NANOG expression. In keeping with this finding, Lee et. al. reported that DNA and histone H3-lys9 methylation of the OCT4 promoter region accompany gene repression in differentiating mouse ES cells upon LIF removal [20]. An association between epigenetic modification and gene
541
expression changes in OCT4 and NANOG promoters was additionally observed in embryonal carcinoma cells (NT2) differentiating into neuronal cells [7]. In our study, OCT4 and NANOG levels were almost completely repressed at 4 and 6 days of RA treatment, respectively, whereas gain of methylation on the promoters appeared weak, relative to the degree of gene repression, even after longer RA treatment. In view of the substantial reduction in gene expression versus low rate of methylation modification, we conclude that the imposition of methylation on promoter regions is not the primary cause of expression repression. Rather, the methylation pattern may arise as a consequence of accumulation of occasional hits by DNA methyltransferase activities on transcriptionally inactive loci. Consistent with this theory, HeLa cells display high methyl cytosine content in these loci, possibly due to the prolonged accumulation of such hits. All the other SCM genes examined were associated with a CpG island in their promoter regions. CpG islands were essentially devoid of methylation in ESCs and EBs, confirming that methylation is not the cause of gene repression in differentiating ESCs. One report addressed the reactivation of SCM genes, including SOX2 in EBs, by the demethylating agent, 5-azacytidine (5-AzaC) [9]. Since our EBs displayed virtually no methylation of SOX2, 5-AzaC-mediated gene reactivation seems to be exerted indirectly. However, heavy methylation of SOX2 and FOXD3 islands in HeLa cells indicates a requirement for epigenetic modification on those loci for stable gene repression. In summary, our results support the repression of stem cell marker genes in differentiating ESCs by mechanisms that do not involve DNA methylation as a primary operator. However, two representative genes, OCT4 and NANOG, which do not contain canonical CpG islands within the promoter region, display some degree of methylation upon differentiation. In particular, NANOG shows varying degrees of methylation in response to discrete differentiation protocols, supporting the theory that the quality or degree of differentiation can be determined by methylation analysis. Acknowledgments This research was supported by grants (SC2090) from the Stem Cell Research Center of the 21st Centry Frontier Research Program, KRIBB Research Initiative Program, and KOSEF Stem Cell Research Program (M1064102 000206N410200210). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2007.05.120.
542
S. Yeo et al. / Biochemical and Biophysical Research Communications 359 (2007) 536–542
References [1] J.A. Thomson, J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, J.M. Jones, Embryonic stem cell lines derived from human blastocysts, Science 282 (1998) 1145–1147. [2] M.A. Surani, Reprogramming of genome function through epigenetic inheritance, Nature 414 (2001) 122–128. [3] M. Boiani, S. Eckardt, H.R. Scholer, K.J. McLaughlin, Oct4 distribution and level in mouse clones: consequences for pluripotency, Genes Dev. 16 (2002) 1209–1219. [4] L.A. Hanna, R.K. Foreman, I.A. Tarasenko, D.S. Kessler, P.A. Labosky, Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo, Genes Dev. 16 (2002) 2650–2661. [5] K. Mitsui, Y. Tokuzawa, H. Itoh, K. Segawa, M. Murakami, K. Takahashi, M. Maruyama, M. Maeda, S. Yamanaka, The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells, Cell 113 (2003) 631–642. [6] H. Niwa, Molecular mechanism to maintain stem cell renewal of ES cells, Cell Struct. Funct. 26 (2001) 137–148. [7] P. Deb-Rinker, D. Ly, A. Jezierski, M. Sikorska, P.R. Walker, Sequential DNA Methylation of the Nanog and Oct-4 Upstream Regions in Human NT2 Cells during Neuronal Differentiation, J. Biol. Chem. 280 (2005) 6257–6260. [8] N. Hattori, K. Nishino, Y.G. Ko, J. Ohgane, S. Tanaka, K. Shiota, Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells, J. Biol. Chem. 279 (2004) 17063– 17069. [9] K. Tsuji-Takayama, T. Inoue, Y. Ijiri, T. Otani, R. Motoda, S. Nakamura, K. Orita, Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells, Biochem. Biophys. Res. Commun. 323 (2004) 86–90. [10] J.H. Park, S.J. Kim, E.J. Oh, S.Y. Moon, S.I. Roh, C.G. Kim, H.S. Yoon, Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line, Biol. Reprod. 69 (2003) 2007–2014.
[11] N. Sato, I.M. Sanjuan, M. Heke, M. Uchida, F. Naef, A.H. Brivanlou, Molecular signature of human embryonic stem cells and its comparison with the mouse, Dev. Biol. 260 (2003) 404– 413. [12] J.Y. Rho, K. Yu, J.S. Han, J.I. Chae, D.B. Koo, H.S. Yoon, S.Y. Moon, K.K. Lee, Y.M. Han, Transcriptional profiling of the developmentally important signalling pathways in human embryonic stem cells, Hum. Reprod. 21 (2006) 405–412. [13] Y.I. Yeom, G. Fuhrmann, C.E. Ovitt, A. Brehm, K. Ohbo, M. Gross, K. Hubner, H.R. Scholer, Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells, Development 122 (1996) 881–894. [14] A.F. Smit, The origin of interspersed repeats in the human genome, Curr. Opin. Genet. Dev. 6 (1996) 743–748. [15] K.H. Choo, B. Vissel, A. Nagy, E. Earle, P. Kalitsis, A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence, Nucleic Acids Res. 19 (1991) 1179–1182. [16] I.P. Ioshikhes, M.Q. Zhang, Large-scale human promoter mapping using CpG islands, Nat. Genet. 26 (2000) 61–63. [17] M. Bibikova, E. Chudin, B. Wu, L. Zhou, E.W. Garcia, Y. Liu, S. Shin, T.W. Plaia, J.M. Auerbach, D.E. Arking, R. Gonzalez, J. Crook, B. Davidson, T.C. Schulz, A. Robins, A. Khanna, P. Sartipy, J. Hyllner, P. Vanguri, S. Savant-Bhonsale, A.K. Smith, A. Chakravarti, A. Maitra, M. Rao, D.L. Barker, J.F. Loring, J.B. Fan, Human embryonic stem cells have a unique epigenetic signature, Genome Res. 16 (2006) 1075–1083. [18] K.D. Robertson, DNA methylation and human disease, Nat. Rev. Genet. 6 (2005) 597–610. [19] K. Shiota, DNA methylation profiles of CpG islands for cellular differentiation and development in mammals, Cytogenet. Genome. Res. 105 (2004) 325–334. [20] J.H. Lee, S.R. Hart, D.G. Skalnik, Histone deacetylase activity is required for embryonic stem cell differentiation, Genesis 38 (2004) 32– 38.