E. DEPARTMENT OF INTEGRATED GENETICS
E-d. Division of Applied Genetics - Kunio Shiota Group

RESEARCH ACTIVITIES

(1) The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice

Jun Ohagane¹, Teruhiko Wakayama^{2, 3}, Sho Senda¹, Yukiko Yamazaki³, Kimiko Inoue², Atsuo Ogura², Joel Marh³, Satoshi Tanaka¹, Ryuzo Yanagimashi³ and Kunio Shiota¹ (¹Univ. Tokyo; ²RIKEN; ³Hawaii Univ.)

--Cloned offspring develop a variety of abnormal phenotypes such as increased body weight (large fetus syndrome), pulmonary hypertension, placental overgrowth, respiratory problems and early death. We had identified several aberrantly methylated loci in the tissues of full-term cloned fetuses. Interestingly, each cloned animal has a different DNA methylation pattern and the extent of hyper- or hypo-methylation varies among the individuals (Ohgane et al., Genesis 30, 45-50, 2001). In contrast, overgrowth of the placenta is one of the commonly observed symptoms in all cloned mice regardless of the sex and strain of animal and the type of donor cell. Thus, there may be genomic loci associated with the abnormal placental development in cloned mice and the genomic loci frequently associated with the epigenetic error have been explored in the cloned animals. We identified the Spalt-like gene 3 (Sall3) locus as a hypermethylated region in the placental genome of cloned mice. The Sall3 locus has a CpG island containing a T-DMR. The T-DMR sequence is conserved in the human genome at the SALL3 locus of chromosome 18q23, which has been suggested to be involved in the 18q deletion syndrome. Intriguingly, larger placentas were more heavily methylated at the Sall3 locus in cloned mice. This epigenetic error was found in all cloned mice examined regardless of sex, mouse strain and the type of donor cells. In contrast, the placentas of in vitro fertilized (IVF) and intracytoplasmic sperm injected (ICSI) mice did not show such hypermethylation, suggesting that aberrant hypermethylation at the Sall3 locus is associated with abnormal placental development caused by nuclear transfer of somatic cells. Thus the Sall3 locus is the area with frequent epigenetic errors in cloned mice. These data suggest that there exists at least genetic locus that is highly susceptible to epigenetic error caused by nuclear transfer.

(2) Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells

Naoko Hattori¹, Koichiro Nishino^{1, 2}, Yeoung-Gyu Ko¹, Naka Hattori^{1, 2}, Jun Ohgane¹, Satoshi Tanaka¹ and Kunio Shiota¹ (¹Univ. Tokyo; ²Bio-oriented Technology Research Advancement Institution)

--The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2'-deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct-4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1(n/n) mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression.

(3) DNA methylation-mediated control of Sry gene expression in mouse gonadal development

Koichiro Nishino^{1, 2}, Naoko Hattori¹, Satoshi Tanaka¹ and Kunio Shiota¹ (¹Univ. Tokyo; ²Bio-oriented Technology Research Advancement Institution)

--DNA methylation at CpG sequences is involved in tissue-specific and developmentally regulated gene expression. The Sry (sex determining region on the Y chromosome) gene encodes a master protein for initiating testis differentiation in mammals, and its expression is restricted to gonadal somatic cells at 10.5-12.5 days post coitum (dpc) in the mouse. We found that in vitro methylation of the 5'-flanking region of the Sry gene caused suppression of reporter activity, implying that Sry gene expression could be regulated by DNA methylation-mediated gene silencing. Bisulfite restriction mapping and sodium bisulfite sequencing revealed that the 5'-flanking region of the Sry gene was hypermethylated in the 8.5 dpc embryos in which the Sry gene was not expressed. Importantly, this region was specifically hypomethylated in the gonad at 11.5 dpc, while the hypermethylated status was maintained in tissues that do not express the Sry gene. We concluded that expression of the Sry gene is under the control of an epigenetic mechanism mediated by DNA methylation.

(4) Skewed X-inactivation in cloned mice

Sho Senda¹, Teruhiko Wakayama², Yukiko Yamazaki², Jun Ohgane¹, Naka Hattori¹, Satoshi Tanaka¹, Ryuzo Yanagimachi² and Kunio Shiota¹ (¹Univ. Tokyo; ²Hawaii Univ.)

--In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P<0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders.

(5) Non-coding RNA directed DNA demethylation of Sphk1 CpG island

Takuya Imamura¹, Soshi Yamamoto¹, Jun Ohgane¹, Naka Hattori¹, Satoshi Tanaka¹ and Kunio Shiota¹ (¹Univ. Tokyo)

--The formation of DNA methylation patterns is one of the epigenetic events that underlie mammalian development. The Sphk1 CpG island is a target for tissue-dependent DNA methylation as well as a template for generating multiple subtypes. The number of mammalian non-coding RNA genes is rapidly expanding. In this study, we found endogenous antisense transcripts, Khps1 subtypes with different sizes (600-20,000nt). A subtype, Khps1a, was a 1290-bp, non-coding, 5'-capped and 3'-polyadenylated RNA that originated from the CpG island and overlapped with a tissue-dependent differentially methylated region (T-DMR) of Sphk1. Intriguingly, overexpression of two fragments of Khps1 caused demethylation of CG sites in the T-DMR. Furthermore, this RNA-directed demethylation was associated with DNA methylation at three CC(A/T)GG sites in the T-DMR. The link between the RNA-directed CG demethylation and non-CG methylation provides a novel mechanism of epigenetic regulation and potential tool for epigenetic manipulation of mammalian cells.

(6) Preference of DNA methyltransferases for CpG islands in normal cells

Naka Hattori¹, Tetsuya Abe¹, Masako Suzuki¹, Tomooki Matsuyama², Shigeo Yoshida², En Li³ and Kunio Shiota¹ (¹Univ. Tokyo; ²RIKEN; ³Massachusets General Hospital)

--In vitro studies indicated that DNA methyltransferase1 (Dnmt1) prefers hemimethylated DNA compared to unmethylated DNA, while Dnmt3a/3b methylate CpG dinucleotides without preference for hemimethylated or unmethylated DNA. Collectively, the fact that Dnmt1 is localized to DNA replication foci and associated with the methyl-CpG binding protein, MeCP2, which directs DNA methyltransferase activity to hemimethylated DNA²⁾, implies that Dnmt1 is involved in maintenance methylation in vivo to preserve methylation patterns in genomic DNA, and that Dnmt3a/3b function as de novo methyltransferases. However, a direct association between DNMTs was reported, and it was demonstrated that DNMT1 and DNMT3B function cooperatively for maintenance methylation in the human cancer cell line. Therefore, categorizing Dnmts into maintenance and de novo DNA methylation may not be appropriate when attempting to characterize in vivo mechanisms involved in determining or establishing DNA methylation profiles in the genome. To address the question how the T-DMR of CpG islands are regulated by Dnmts in vivo, we analyzed the genome-wide DNA methylation pattern focussing on CpG islands and found that each Dnmt has target preferences depending the genomic component (in preparation).

(7) Stage-by-stage change in DNA methylation status of DNA methyltransferase 1 (Dnmt1) locus during mouse early development

Yeoung-Gyu Ko¹, Koichiro Nishino¹, Naoko Hattori¹, Yoshikazu Arai¹, Satoshi Tanaka¹ and Kunio Shiota¹ (¹Univ. Tokyo)

--Methylation of DNA is involved in tissue-specific gene control, and establishment of DNA methylation pattern in the genome is thought to be essential for embryonic development. Three isoforms of DNA methyltransferase 1 (Dnmt1) transcripts, Dnmt1s, Dnmt1o and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1s is expressed in somatic cells. Dnmt1p is found only in pachytene spermatocytes, whereas Dnmt1o is specific to oocytes and preimplantation embryos. Here we determined that there is a tissue-dependent differentially methylated region (T-DMR) in the 5' region of Dnmt1o but not in that of the Dnmt1s/1p. The methylation status of the Dnmt1o T-DMR was distinctively different in the oocyte from that in the sperm and adult somatic tissues, and changed at each stage from fertilization to blastocyst stage, suggesting that active methylation and demethylation occur during preimplantation development. The T-DMR was highly methylated in somatic cells and ES cells. Analysis using Dnmt-deficient ES cell lines revealed that each of Dnmt1, Dnmt3a and Dnmt3b is partially responsible for maintenance of methylation of Dnmt1o T-DMR. In particular, there are compensatory and cooperative roles between Dnmt3a and Dnmt3b. Thus, the regulatory region of Dnmt1o, but not of Dnmt1s/1p, appeared to be a target of DNA methylation. The present study also suggested that the DNA methylation status of the gene region dynamically changes during embryogenesis independently of the change in the bulk DNA methylation status.

PUBLICATIONS

Papers
1. Ohgane, J., Wakayama, T., Senda, S., Yamazaki, Y., Inoue, K., Ogura, A., Marh, J., Tanaka, S., Yanagimachi, R. and Shiota, K. (2004). The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice. Genes to Cells, 9, 253-260.
2. Hattori, N., Nishino, K., Ko, Y.G., Ohgane, J., Tanaka, S. and Shiota, K. (2004). Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J. Biol. Chem., 279, 17063-17069.
3. Nishino, K., Hattori, N., Tanaka, S. and Shiota, K. (2004). DNA methylation-mediated control of Sry gene expression in mouse gonadal development. J. Biol. Chem., 279, 22306-22313.
4. Senda, S., Wakayama, T., Yamazaki, Y., Ohgane, J., Hattori, N., Tanaka, S., Yanagimachi, R. and Shiota, K. (2004). Skewed X-inactivation in cloned mice. Biochem. Biophys. Res. Commun., 321, 38-44.
5. Imamura, T., Yamamoto, S., Ohgane, J., Hattori, N., Tanaka, S. and Shiota, K. (2004). Non-coding RNA directed DNA demethylation of Sphk1 CpG island. Biochem. Biophys. Res. Commun., 322, 593-600.
6. Hattori, N., Abe, T., Suzuki, M., Matsuyama, T., Yoshida, S., Li, E. and Shiota, K. (2004). Preference of DNA methyltransferases for CpG islands in mouse embryonic stem cells. Genome Res., 14, 1733-1740.
7. Ko, Y. G., Nishino, K., Hattori, N., Arai, Y., Tanaka, S. and Shiota, K. (2005). Stage-by-stage change in DNA methylation status of DNA methyltransferase 1 (Dnmt1) locus during mouse early development. J. Biol. Chem., in press.

Reviews
8. Shiota, K. (2004). DNA methyaltion profiles of CpG islands for Cellular differentiation and development in mammals. Cytogenetic and Genome Res., 105, 325-334.
9. Ohgane, J., Hattori, N. and Shiota, K. (2004). Analysis of tissue-specific DNA methylation during development. Methods Mol. Biol., 289, 371-382.
10. 坂本英樹,塩田邦郎（2004）発生プログラムと組織・細胞特異的DNAメチル化プロファイルの形成,わかる実験医学シリーズ「注目のエピジェネティクスがわかる」（羊土社）,90-95.
11. 鈴木雅子,塩田邦郎（2004）発生と治療用クローニングのエピジェネティックス.Medical Science Digest（ニュー・サイエンス社）,30, 26-30.

EDUCATION

1. Dr. K. Shiota was invited to give a seminar on “Cell- and tissue-specific DNA methylation profiles in mammalian genome: Epigenetics of Embryo Development", at Canadian Workshop on “Human Reproduction & Reproductive Biology", Ottawa, Canada, May 3-5, 2004.
2. Dr. K. Shiota was invited to give a lecture on “DNA methylation profiles of CpG islands during cellular differentiation and development in mammals." At McGill University, Reproductive and Developmental Biology Seminar, Montreal, Canada, May 6, 2004.
3. Dr. K. Shiota was invited to give a lecture on “DNA methylation profiles of CpG islands for cellular-differentiation and development in mammals", at 13th Conference of the International Society of Differentiation, Honolulu, USA, September 5-9, 2004.
4. Dr. K. Shiota was invited to give a seminar on “Epigenetic marks by DNA methylation specific to cell types", at IX International Congress of Reproductive Immunology, Hakone, Japan, October 11-15, 2004.
5. Dr. K. Shiota was invited to give a seminar on “DNA methylation profiles for evaluation of epigenetic risk", at International Symposium on Environmental Endocrine Disrupters 2004, Nagoya, Japan, December 15-17, 2004.

SOCIAL CONTRIBUTIONS AND OTHERS

1. 東京大学大学院農学生命科学研究科 教授
2. 東京大学大学院農学生命科学研究科応用動物科学専攻
3. 日本繁殖生物学会 理事
4. 生殖免疫学会 評議員
5. 日本獣医学会生理・生化学分科会 副会長
6. 日本再生医療学会 評議員