PRDM14 suppresses expression of differentiation marker genes in human embryonic stem cells

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Biochemical and Biophysical Research Communications 367 (2008) 899–905 www.elsevier.com/locate/ybbrc

PRDM14 suppresses expression of differentiation marker genes in human embryonic stem cells Norihiro Tsuneyoshi a,b, Tomoyuki Sumi a, Hiroaki Onda c, Hiroshi Nojima c,d, Norio Nakatsuji b,e, Hirofumi Suemori a,* a

Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan b Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan c Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan d DNA-chip Development Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan e Institute for Integrated Cell-Material Sciences, Kyoto University, 69 Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan Received 3 December 2007 Available online 14 January 2008

Abstract PRDM14 was identified by microarray analysis and was expressed in specifically undifferentiated human ES cells. PRDM14 protein is thought to regulate gene transcription in human ES cells, as it contains a PR domain, a subtype of the SET domain which catalyzes histone methylation. To analyze the function of PRDM14, we performed knock-down and forced expression of PRDM14 in human ES cells. Knock-down of PRDM14 by siRNA induced expression of early differentiation marker genes. Forced expression of PRDM14 suppressed expression of differentiation marker genes in the embryoid body. These results suggest that PRDM14 is involved in the maintenance of the self-renewal of human ES cells by suppression of gene expression. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Human ES cells; Self-renewal; Pluripotency; PRDM14; PFM11; PR domain; SET domain

Embryonic stem (ES) cell lines were first established from the inner cell mass (ICM) of mouse blastocysts [1,2]. ES cells can be indefinitely propagated while retaining their potency to differentiate into cells and tissues of almost all types, and they are used in research into developmental biology and molecular genetics. Since human ES cell lines were established [3], they have been expected to be a valuable source of functional cells such as neurons, cardiac muscle, and insulin producing cells for cell-transplantation therapy and pharmaceutical research. Indefinite proliferation of ES cells is considered a fundamental feature for

*

Corresponding author. Fax: +81 75 751 3890. E-mail address: [email protected] (H. Suemori).

0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.189

such purposes, and therefore the molecular mechanism sustaining the self-renewal of ES cells has been studied as an important issue in ES cell biology. Mouse ES cells have long been used to analyze the mechanism for the selfrenewal of ES cells, and numerous factors are reported to be involved in maintenance of ES cell self-renewal. For example, NANOG, OCT-4 and SOX2 have been reported as core-transcriptional factors of mouse ES cell selfrenewal [4–7], and they are also expressed in human ES cells and play essential roles in human ES cell self-renewal [8–10]. Although some factors, including these transcription factors, share similar functions in the self-renewal of mouse and human ES cells, others do not appear to. Leukemia inhibitory factor (LIF) supports mouse ES cell selfrenewal [11,12] but not human ES cell self-renewal [13]. It

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has been also shown that STAT-3 and c-myc, which are considered downstream factors of LIF signaling [14,15], do not sustain human ES cell self-renewal [13,16]. In contrast, bFGF and Activin A are capable of maintaining human ES cell self-renewal [17,18], whereas Activin A is involved in proliferation of mouse ES cells but does not participate in the pluripotency [19]. Thus, the molecular mechanisms of human ES cell self-renewal are largely unclear and need to be explored. To elucidate the molecular mechanism of human ES cell self-renewal, we identified genes expressed specifically in undifferentiated human ES cells but not in somatic cells by microarray analysis. Among these genes, we focused on PRDM14 (PR domain [PRDI-BF1 and RIZ homologous region] containing 14), a member of the PRDM family proteins considered to be involved in histone methylation [20], because there has been increasing evidence that epigenetic modifications, such as histone modification and DNA methylation, regulate self-renewal and differentiation lineage choice of stem cells [21,22]. Our data suggest that PRDM14 is involved in the self-renewal of human ES cells. Materials and Methods Cell culture. The human ES cell lines, KhES-1, KhES-2, and KhES-3, were maintained in human ES cell medium on a feeder layer of mouse embryonic fibroblasts (MEF) inactivated with mitomycin C (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as previously described [16,23]. The MEF-conditioned medium (CM) was prepared as previously described [16]. For feeder-free culture, human ES cells were dissociated into small clumps and plated on dishes coated with Matrigel (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The cells were cultured in CM or 105 M retinoic acid (RA) (Sigma–Aldrich Co., St. Louis, MO, USA) for 5 days. For embryoid body (EB) differentiation, human ES cells were dissociated and transferred into plastic petri dishes in human ES cell medium to form EBs. The TIG-3 cell line, a human fetal lung normal diploid fibroblast line [24], was purchased from The Health Science Research Resources Bank (HSRRB) (Osaka, Japan, Cell No. JCRB0506). TIG-3 cells were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS) (Sigma). Immunocytochemical and immunofluorescence analysis. For staining of alkaline phosphatase (ALP) activity, human ES cells were fixed in 3.7% formaldehyde and detected with Vector Blue substrate (Vector Laboratories, Inc., Burlingame, CA, USA). For OCT-4 staining, human ES cells were fixed, permeabilized with 0.2% Triton X-100/PBS for 5 min and incubated with anti-OCT-4 antibody (C-10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Dako A/S, Glostrup, Denmark), antigens were visualized with 3,30 -diaminobenzidine (DAB) (Sigma). For immunofluorescence, human ES cells were fixed, permeabilized and incubated with the primary antibodies anti-PRDM14 (AP1214a, Abgent, San Diego, CA, USA) and anti-OCT-4 using Can Get Signal immunostain (TOYOBO Co., Ltd., Osaka, Japan). After incubation with Alexa Fluor 488 or 546-conjugated secondary antibodies (Invitrogen Corporation, Carlsbad, CA, USA), nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and examined under the fluorescent microscope Axio Imager Z1 (Carl Zeiss AG, Oberkochen, Germany).

Immunoblot analysis. Lysates of human ES cells were separated by SDS–PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and probed with the following primary antibodies: anti-PRDM14 using Can Get Signal (TOYOBO) and anti-a-Tubulin (DM1A, Sigma). After incubation with HRP-conjugated secondary antibody, proteins were detected using Western Blotting Luminol Reagent (Santa Cruz). RNA interference. siRNAs for human PRDM14 (Stealth Select RNAi, oligo ID HSS127282 and HSS127283) and negative control siRNA (Stealth RNAi Negative Control LO GC) were purchased from Invitrogen. Transfection for human ES cells was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. siRNA was transfected at a final concentration of 40 nM. The second transfection was carried out 24 h after the first transfection. Quantitative real-time PCR and semi-quantitative PCR analysis. Total RNA was extracted using the RNeasy Micro kit (QIAGEN GmbH, Hilden, Germany) and then 0.5–2 lg total RNA was used for reverse transcription with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocols. For quantitative real-time PCR analysis, PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems). Relative quantitation was performed using the comparative method (2DDCT method) [25] and normalized against GAPDH or 18S rRNA. For semi-quantitative PCR analysis, PCR was performed with TaKaRa Ex Taq (TaKaRa Bio Inc., Shiga, Japan). PCR was optimized to allow semiquantitative comparisons within the log phase of amplification. The primer sequences for quantitative and semi-quantitative PCR are shown in Table 1 and S1. Expression vector construction and establishing stable cell clones. Human PRDM14 cDNA was cloned by PCR using PrimeSTAR HS DNA Polymerase (TaKaRa) and the following primers: forward, 50 CCCGGGATGGCTCTACC-30 ; and reverse, 50 -AGGGCTAGTAGT CTTCATGAAAC-30 . The PCR product was ligated into a pCR-Blunt vector (Invitrogen). An EcoRI fragment containing hPRDM14 was ligated into an EcoRI-digested pCAG-IRES-Puro expression vector [16,26]. The sequences were confirmed by nucleotide sequence analysis. To establish stable ES cell clones, human ES cells were transfected with hPRDM14 expression vector (pCAG-hPRDM14-IRES-Puro) or empty vector (pCAG-IRES-Puro) and selected using 1 lg/ml puromycin with conditioned medium on Matrigel-coated dishes. After 2 weeks of selection, resistant colonies were isolated and plated on MEF feeder layers.

Results Identification of genes specifically expressed in undifferentiated human ES cells Genes that are specifically expressed in undifferentiated human ES cells have the potential to play important roles in human ES cell self-renewal. To find genes that can elucidate the mechanism of human ES cell self-renewal, we conducted microarray analysis (data not shown) using the total RNA from KhES-1 cells (a human ES cell line) and TIG-3 cells (a fetal lung fibroblast cell line, as differentiated cells), and identified 89 genes whose mRNA levels are upregulated in KhES-1 cells compared to TIG-3 cells by RT-PCR analysis (Fig. S1 and Table S1). To further confirm genes with expression closely related to the undifferentiated state of human ES cells, we examined their expression level by RT-PCR analysis using undifferentiated human ES cells and cells differentiated from human ES cells with retinoic acid (RA) treatment for 5 days.

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Table 1 PCR Primers Forward primer (50 -30 )

Reverse primer (50 -30 )

Size (bps)

Ref.

For quantitative real-time PCR PRDM14 TGAGCCTTCAGGTCACAGAG NANOG CTGCTGAGATGCCTCACACG POU5F1 TCTCGCCCCCTCCAGGT SOX2 GTATCAGGAGTTGTCAAGGCAGAG GATA6 TGTGCAATGCTTGTGGACTC GATA4 CCTGGCCTGTCATCTCACTAC SOX7 GGCCAAGGACGAGAGGAAAC CXCR4 AGGGCCTGAGTGCTCCAGTAG T TGCTTCCCTGAGACCCAGTT GSC GAGGAGAAAGTGGAGGTCTGGTT MIXL1 CCGAGTCCAGGATCCAGGTA FOXF1 ACAGCGGCGCCTCTTATATC PAX6 GCTTCACCATGGCAAATAACC CDX2 CCGAACAGGGACTTGTTTAGAG CGA GTTTCTGCATGTTCTCCATTC CSH1 CAGCTCACCTAGTGGCAATG GAPDH GAAGGTGAAGGTCGGAGTC 18S rRNA CGGCTACCACATCCAAGGAA

ATTTCCTATCGCCCTTGTCC TGCCTTTGGGACTGGTGGA GCCCCACTCCAACCTGG TCCTAGTCTTAAAGAGGCAGCAAAC AGTTGGAGTCATGGGAATGG AGAGGACAGGGTGGATGGA TCCACGTACGGCCTCTTCTG ATAGTCCCCTGAGCCCATTTC GATCACTTCTTTCCTTTGCATCAAG CTCTGATGAGGACCGCTTCTG CTCTGACGCCGAGACTTGG CTCCTTTCGGTCACACATGC GGCAGCATGCAGGAGTATGA CTCTGGCTTGGATGTTACACAG GTGGACTCTGAGGTGACGT CTTGGAGCATAGCGTGGTCA GAAGATGGTGATGGGATTTC GCTGGAATTACCGCGGCT

162 99 219 78 161 100 123 113 121 72 58 194 76 198 195 144 226 187

— [23] [8] [10] — — — — [35] [35] [35] — — [8] [8] — [23] [36]

For semi-quantitative PCR PRDM14 CCTGCACCATGCGATTTCAGG (exo) PRDM14 TCTGACTGACCGCGTTACTCCCACA GAPDH GGATTTGGCCGTATTGG

TGCATGAGCAGCCATCATCCTC CAGGGCAGATCGTAGAGAGG TCATGGATGACCTTGGC

992 370 454

— — [16]

Gene symbol

Annealing Temperature at 58 °C (except for Semi-quantitative GAPDH primers; 56 °C)

Expression of NANOG, POU5F1, SOX2, LEFTY1, and TDGF1, which have important roles in undifferentiated human ES cells [8–10,27], was markedly reduced by RA treatment (Fig. 1). In addition, PRDM14, BEX1, C14orf115, PCSK9, OGDHL, PIM2, LECT1, ABHD12B, HEYL, GAL3ST1, DLEU7, ATCAY, SFRP2, TNNT1, HAS3, PIF1, POU5F1P4 (putative pseudogene), and TDGF3 (putative pseudogene), of which functions in

human ES cells are yet unknown, were also markedly down-regulated (Fig. 1). PRDM14 was further examined, because it contains a PR domain, a subtype of the SET domain that is involved in histone methylation [20]. Histone methylation is thought to play important roles in control of gene expression, and it has been shown that such modifications are important for ES cell self-renewal [21,22]. Immunocytochemical analysis

KhES-2

KhES-2

KhES-2

KhES-2

KhES-2

KhES-2

CM RA

CM RA

CM RA

CM RA

CM RA

CM RA

PODXL

AK091547

HRASLS3

C9orf58

SPINT2

DNMT3B

LEFTY1

RARRES2

OGDHL

MBD2

SFRP2

SOX2

AL157455

PRDM14

KIAA1553

HEYL

FAM83D

CDH1

DPPA4

KRTCAP3

KRTCAP3

FLJ35773

TNNT1

TDGF3

L1TD1

SPP1

ZIC3

PCDHB2

GAL3ST1

FAM137A

RAB11FIP4

MYCN

F11R

PIM2

NEFL

HAS3

LINGO1

CKMT1B

EGLN3

FAAH2

PHF17

PIF1

POU5F1P3

BMP7

IQGAP2

LECT1

CXCL14

C10orf35

TDGF1

MARVELD3

SALL4

CTSL2

CXADR

MCM3

POU5F1P4

KRT18

LEFTY2

RAB17

SPINT1

TPD52 EDNRB

BI088569

POU5F1

SEMA6A

ABHD12B

DLEU7

BEX1

KIRREL2

SLC16A10

FZD5

RASL11B

LCP1

CD24

FGF13

BC064610

NAT8L

ATCAY

ARID3B

PDPN

SCG3

BEX2

TMEM125

CHST6

C12orf35

NANOG

C14orf115

PCSK9

FA2H

LRRN1

GAPDH

Fig. 1. Comparison of gene expressions between undifferentiated KhES-2 cells and cells differentiated from KhES-2 cells. Genes were identified by microarray analysis. KhES-2 cells were cultured in conditioned medium (CM) or 105 M retinoic acid (RA) for 5 days. GAPDH was used as the internal control. The annealing temperature and amplification cycles used in the RT-PCRs are denoted in Table S1.

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revealed that expression of PRDM14 was expressed in undifferentiated human ES cells and that this expression was reduced during differentiation. Furthermore, PRDM14 was localized in the nucleus, suggesting that it functions in the nucleus (Fig. 2).

A

PRDM14 (at 48 hr) 1.0

C

siNegative Control

siPRDM14

0.5 0 N si1 si2

Knock-down of PRDM14 induced expression of differentiation marker genes

B

N

si1 si2

PRDM14

To analyze the function of PRDM14 in human ES cells, we performed knock-down experiments using two different siRNAs for hPRDM14 in KhES-1 cells. Knock-down efficiency was analyzed at 48 h after transfection of siRNA, and at that time PRDM14 expression had decreased to 0.25- and 0.33-fold of the cells transfected with control siRNA (Fig. 3A). Western blot analysis revealed that the PRDM14 protein was detected in control cells, whereas PRDM14 was markedly decreased in PRDM14 knockdown cells (Fig. 3B). The control cells retained typical undifferentiated states regarding morphology, namely tightly packed colonies with a high nucleo/cytoplasmic ratio, whereas PRDM14 knock-down human ES cells displayed the flattened morphology typical of differentiating cells (Fig. 3C). We then examined changes in the expression levels of several marker genes. Expression of NANOG, POU5F1, and SOX2, markers for undifferentiated human ES cells, was down-regulated to about 70–80% of the control cell level (Fig. 3D). Expression of early differentiation marker genes such as GATA6 for the endoderm, T, GSC, CXCR4 for mesoderm and CSH1 for the trophectoderm was upregulated (Fig. 3D). There was no significant effect RA

Nuclei

PRDM14 / OCT-4

OCT-4

PRDM14

CM

Fig. 2. PRDM14 was expressed in undifferentiated human ES cells. Immunofluorescence analysis of PRDM14 expression. KhES-2 cells were cultured in conditioned medium (CM) or 105M retinoic acid (RA) for 5 days. OCT-4 (POU5F1) is a marker for undifferentiated ES cells. The nuclei were counterstained with DAPI. Scale bar = 100 lm.

α-Tubulin

D

NANOG

POU5F1

SOX2

1.0

1.0

1.0

0.5

0.5

0.5

0

0 N si1 si2 GATA6

T

5.0

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3.0

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CXCR4 3.0

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1.0

1.0 0 N si1 si2

N si1 si2

CSH1

N si1 si2 2.0

PAX6

2.0 1.0

2.0 1.0

1.0 0

0 N si1 si2

0 N si1 si2

N si1 si2

Fig. 3. Knock-down of PRDM14 induced expression of differentiation marker genes. (A) Quantitative real-time PCR analysis of PRDM14 expression at 48 h after transfection. (B) Western blot analysis of PRDM14 at 72 h after transfection. (C) Morphology of KhES-1 cells at 72 h after transfection. (D) Quantitative real-time PCR analysis of gene expression levels at 72 h. The relative quantitation was normalized against GAPDH, and the range was calibrated to the value of siNegative Control (=1.0). N, siNegative Control; si1, siPRDM14 (HSS127282); si2, siPRDM14 (HSS128273). Scale bars = 100 lm.

on expression of PAX6, a marker for ectoderm (Fig. 3D). Similar results were obtained from KhES-2 cells, another human ES cell line (data not shown). These results indicate that PRDM14 is essential for the maintenance of human ES cell self-renewal. PRDM14 inhibits differentiation in the embryoid body To further examine the functions of PRDM14, human ES cell lines stably expressing PRDM14 were established. Exogenous PRDM14 expression was confirmed by RTPCR, and transgenic cell lines with 1.5- to 2.5-fold PRDM14 expression were obtained (Fig. 4A). PRDM14 protein levels were increased in transgenic cell lines (Fig. 4B). PRDM14 transgenic clones were indistinguishable from their parental cells with respect to morphology, expression of markers (Fig. 4C). To examine the effect of

N. Tsuneyoshi et al. / Biochemical and Biophysical Research Communications 367 (2008) 899–905

A

B

KhES-2

KhES-2

wt vector PRDM14

(exogenous) PRDM14 (total) PRDM14

wt vector PRDM14 PRDM14 α-Tubulin

GAPDH

Phase

ALP

OCT-4

vector

PRDM14

PRDM14

C

D

E

PRDM14

3.0 2.0 1.0 0

0.5

0 ES

0 ES

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GATA6 90 60 30 0

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20 0 ES

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EB MIXL1

0 EB

150 100 50 0 ES

CGA

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CDX2 80

12

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EB PAX6

5000

0

EB FOXF1

18 12 6 0

500 250

EB SOX7

40

T

ES

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GATA4 75 50 25 0

ES

POU5F1 1.0

0.5

ES

EB

parental or empty vector transfected cells (Fig. 4D). In the EBs of parental (wt) and empty vector transfected cell lines (vector), the marker genes of all germ layers were induced, namely GATA6, GATA4, SOX7 for endoderm, T, MIXL1, FOXF1 for mesoderm, PAX6 for ectoderm and CDX2, CGA for trophectoderm, while undifferentiated ES cell markers such as NANOG and POU5F1 were downregulated and PRDM14 was also decreased (Fig. 4E). However, in PRDM14 transgenic EBs, we did not observe upregulation of any of these differentiation marker genes, although NANOG and POU5F1 were down-regulated (Fig. 4E). Similar results were obtained using transgenic cell lines produced from KhES-3 cells, another human ES cell line (data not shown). Discussion

NANOG 1.0

903

ES

EB

wt vector PRDM14

Fig. 4. PRDM14 suppressed expression of differentiation marker genes in the embryoid body. (A) RT-PCR analysis of PRDM14 expression in PRDM14 transgenic clones. (B) Western blot analysis of PRDM14 transgenic clones. (C) Morphology and ALP and OCT-4 staining of PRDM14 transgenic clones cultured in CM for 5 days. (D) Morphology of EBs produced from clones transfected with PRDM14 or empty vector by culturing in suspension for 10 days. (E) Quantitative real-time PCR analysis for expression of marker genes. The relative quantitation was normalized against 18S rRNA, and the range was calibrated to the value of wt ES (=1.0). ES, human ES cells cultured in CM for 4 days; EB, 10day-old EBs; wt, parental cell line; vector, empty vector transfected clone; PRDM14, PRDM14 transgenic clones. Scale bars = 100 lm.

constitutive PRDM14 expression on differentiation, transgenic clones were induced to differentiate by embryoid body formation for 10 days. The morphology of the PRDM14 transgenic EBs appeared similar to that of the

In the present study, we performed a microarray analysis to find genes that might be involved in maintenance of human ES cell self-renewal, and identified 89 candidate genes whose expressions are upregulated in human ES cells compared to TIG-3 cells (Fig. S1 and Table S1). Since many of the genes that play important roles in maintenance of ES cell self-renewal, such as NANOG, POU5F1, SOX2, TDGF1, LEFTY1, LEFTY2, ZIC3, and SALL4 [4–10,27– 29], are specifically expressed in undifferentiated ES cells, we expect that at least some of the genes identified here might also play important roles in maintenance of human ES cell self-renewal, and future research will address this possibility. PRDM14, one of the genes identified here, contains a PR/SET domain and is thought to be involved in epigenetic control of gene expression in undifferentiated ES cells. To analyze the function of PRDM14 in human ES cells, we performed knock-down and stable expression of PRDM14 in human ES cells. Knock-down of PRDM14 caused a significant change to the cell morphology observed during differentiation and induced expression of early differentiation marker genes, whereas expression of NANOG, POU5F1, and SOX2 was slightly decreased. Transgenic ES cell lines stably expressing PRDM14 showed remarkable suppression of expression of differentiation marker genes when induced to differentiate by embryoid body formation, although expression of NANOG and POU5F1 in transgenic EBs was decreased to the similar level as in EBs from empty vector transfected and parental cells. Therefore, our study suggests that PRDM14 plays crucial roles in the human ES cell self-renewal and differentiation through inhibiting the induction of differentiation genes. A recent report showed that there are binding sites for NANOG, OCT-4, and SOX2 in a putative promoter region of the human PRDM14 gene [9], suggesting that hPRDM14 functions under control of NANOG, OCT-4, and SOX2. Given that PRDM14 could regulate differentiation marker genes but not undifferentiated marker genes, constitutive expression of PRDM14 alone might not be sufficient for maintaining undifferentiated states of human ES cells. Indeed,

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PRDM14 transgenic clones did not maintain their undifferentiated state in the condition of feeder layer free culture (data not shown), which could not support ES cells in an undifferentiated state [18]. Altogether, it was concluded that PRDM14 is necessary but not sufficient alone for maintenance of human ES cell self-renewal, although it plays some role in maintenance of the undifferentiated state of human ES cells. Histone modification plays an important role in selfrenewal and lineage determination of stem cells, such as ES cells, hematopoietic stem cells, and neural stem cells [21,22,30,31]. PRDM14 belongs to the PRDM family, which contains BLIMP1 (PRDI-BF1, PRDM1), RIZ1 (PRDM2), and Meisetz (PRDM9) [20]. RIZ1 has been shown to specifically methylate lysine 9 of histone H3 (H3K9) to repress gene transcription [32,33]. In contrast, Meisetz methylates at H3K4 and activates transcription [34]. Thus, PRDM14 might have the histone methyltransferase activity, and suppress the differentiation marker genes via histone modifications. Future studies are necessary to clarify the functions of PRDM14 and to provide valuable information on the self-renewal of human ES cells. Acknowledgments We thank Takahiro Tougan of Osaka University and the members of the laboratories of N. Nakatsuji and H. Suemori for valuable discussions and technical assistance. This work was supported in part by the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, the Japan Society for the Promotion of Science, and by New Energy and Industrial Technology Development Organization (NEDO) of Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc. 2007.12.189. References [1] M.J. Evans, M.H. Kaufman, Establishment in culture of pluripotential cells from mouse embryos, Nature 292 (1981) 154–156. [2] G.R. Martin, Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells, Proc. Natl. Acad. Sci. USA 78 (1981) 7634–7638. [3] 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. [4] H. Niwa, J. Miyazaki, A.G. Smith, Quantitative expression of Oct3/4 defines differentiation, dedifferentiation or self-renewal of ES cells, Nat. Genet. 24 (2000) 372–376. [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.

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