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Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis
Mechanisms of Development 118 (2002) 187–190 www.elsevier.com/locate/modo
Gene expression pattern
Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis Daisuke Watanabe a,1, Isao Suetake b,1, Takashi Tada c, Shoji Tajima b,* a
Institute of Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Japan Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan c Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan b
Received 8 April 2002; received in revised form 16 May 2002; accepted 4 July 2002
Abstract DNA methylation is essential for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, contribute to the creation of DNA methylation patterns in embryos. We demonstrated that the Dnmt3a and Dnmt3b proteins are expressed at different stages of embryogenesis. Dnmt3b is specifically expressed in totipotent embryonic cells, such as inner cell mass, epiblast and embryonic ectoderm cells, whilst Dnmt3a is significantly and ubiquitously expressed after E10.5. The difference in the expression stages of the Dnmt3a and Dnmt3b proteins may contribute to their distinct functions during the embryogenesis. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mouse embryogenesis; DNA methylation pattern; Dnmt3a; Dnmt3b; De novo-type DNA methyltransferase
1. Results and discussion In vertebrates, the methylation of cytosine in genomic DNA plays a crucial role in gene silencing, and is essential for development (Li et al., 1992; Okano et al., 1999). Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of methylation patterns at an early stage of embryogenesis (Okano et al., 1999). When Dnmt3a or Dnmt3b is targeted, residual Dnmt3b or Dnmt3a, respectively, partly compensates for the lack of the gene (Okano et al., 1999), suggesting that Dnmt3a and Dnmt3b possess similar functional specificities. On the other hand, targeting of Dnmt3b leads to a severer phenotype than that of Dnmt3a, and a mutation in DNMT3b, a human homologue of Dnmt3b, leads to hypomethylation of the satellite 2 and 3 regions of specific chromosomes (Miniou et al., 1997), which is the cause of ICF syndrome (Okano et al., 1999; Xu et al., 1999; Hansen et al., 1999). These reports suggest that Dnmt3a and Dnmt3b possess distinct functions. We deduced that the difference in the timing of cell-type specific expression of the Dnmt3a and Dnmt3b proteins was one of the reasons for the different functions. To address this issue, we determined Dnmt3a and Dnmt3b proteins in mouse embryos using specific antibodies recognizing the amino-terminal sequence of each * Corresponding author. Tel.: 181-6-6879-8627; fax: 181-6-6879-8629. E-mail address: [email protected] (S. Tajima). 1 These authors contribute equally to this work.
protein, where no homology exists between the proteins (Aoki et al., 2001). Surprisingly, Dnmt3a was not significantly detected in E4.5–8.5 embryos (Fig. 1A). On the other hand, Dnmt3b existed at E4.5–7.0, in the inner cell mass (ICM) at E4.5, the epiblast at E5.5, and the embryonic ectoderm at E7.0. In E8.5 embryos, Dnmt3b was weakly detected in the head folds, but at very low level in E9.5 embryos (Fig. 1A). The localization of Dnmt3b in early embryos at E4.5–E7.0 resembled that of Oct3/4, a transcription factor specifically expressed in totipotent cells (Fig. 1B) (Pesce et al., 1998). Dnmt3b was the major de novo DNA methyltransferase in E4.5–7.5 embryos, in which genomewide DNA methylation occurs (Monk, 1990). The Dnmt3a protein was below the detection level until E9.5, but was detected after E10.5. Typical immunostaining of the hind limb and tail regions at E12.5, and the forelimb region at E14.5 is shown in Fig. 2. Dnmt3a was detected ubiquitously in mesenchymal cells. In contrast, the Dnmt3b protein was not detected in E10.5–14.5 embryos. The amounts of Dnmt3a and Dnmt3b in E7.0–E16.5 embryos were determined using recombinant Dnmt3a and Dnmt3b as standards (Fig. 3). Dnmt3a was below the detection level in E7.0–9.5 embryos (,0.07 ng/10 mg protein), significantly detected in E10.5 ones (0.45 ng/10 mg protein), and was detected thereafter. Inversely, Dnmt3b was highly expressed in E7.0 embryos (7.2 ng/10 mg protein), decreased thereafter, and was below the detection level after E10.5 (,0.07 ng/10 mg protein). Judging from its
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00242-3
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D. Watanabe et al. / Mechanisms of Development 118 (2002) 187–190
Fig. 1. Expression of Dnmt3a and Dnmt3b in mouse embryos. (A) ICR mouse embryos at E4.5, 5.5, 7.0, 8.5, and 9.5 were stained with anti-Dnmt3a (3a), antiDnmt3b (3b), or anti-Oct 3/4 (Oct) antibodies, and/or DAPI (DAPI, blue). The antibodies were detected with anti-rabbit antibodies coupled with ALEXA568. The ICM and trophectoderm (T) at E4.5 are indicated by arrows and arrowheads, respectively. The epiblast (E) at E5.5, and the embryonic ectoderm (EE) and extraembryonic region (EX) at E7.0 are indicated by square brackets. The head folds at E8.5 and E9.5 are indicated by arrows. (B) High magnification images of an E5.5 embryo stained with anti-Dnmt3b, Oct3/4, and DAPI are shown. Note that Dnmt3b was not detected in primitive endoderm cells surrounding the epiblast, which is similar to the expression pattern of Oct3/4. Arrowheads and arrows indicate the nuclei of primitive endoderm and epiblast cells, respectively.
mobility, the major Dnmt3b in embryos was Dnmt3b1, a translation product of one of the splicing isoforms. De novotype DNA methyltransferase switches from Dnmt3b to Dnmt3a at around E9.5–10.5. Embryonic stem (ES) cells express both Dnmt3a and Dnmt3b mRNAs at high levels compared to those in established somatic cells with no totipotency (Okano et al., 1998). As ES cells reflect the state of totipotent cells in embryos, we determined the levels of the Dnmt3a and Dnmt3b proteins in ES cells, R1. Dnmt3b was detected in the nuclei of all R1 cells, but Dnmt3a was not highly expressed (Fig. 4). Interestingly, only a portion, i.e. not all, of the Dnmt3b was localized to the heterochromatin. The localization of the endogenous Dnmt3b was not identical to that of ectopically expressed Dnmt3b, which has been reported to be co-localized with heterochromatin (Bachman et al., 2001). The amounts of Dnmt3a and Dnmt3b in R1 cells were determined as in Fig. 3. R1 cells significantly expressed Dnmt3b (5 ng/
10 mg protein), but not much Dnmt3a (0.3 ng/10 mg protein). The amount of Dnmt3a was similar to that in cultured fibroblasts, C3H10T1/2 (1.0 ng/10 mg protein). Another ES cell line, TMA-S5, showed similar expression profiles for the Dnmt3a and Dnmt3b proteins as those in R1. Not the Dnmt3a but the Dnmt3b protein was highly expressed in ES cells. The results of the present study suggest that the different expression profiles of the Dnmt3a and Dnmt3b proteins are one of the causes of their distinct functions in an early stage of embryogenesis.
2. Methods ICR mouse embryos at E4.5–E9.5 were fixed in cold acetone. Embryos at E12.5 and E14.5 were mounted in TissueTek OCT compound (Miles Scientific, IL, USA), cryosec-
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Fig. 2. The expression of Dnmt3b decreased and that of Dnmt3a increased as embryogenesis proceeded. Serial sections (8 mm-thick) of E12.5 or E14.5 embryos mounted in Tissue-Tek OCT compound were immunostained with anti-Dnmt3a (3a), or anti-Dnmt3b (3b) antibodies, or without first antibodies (-). The hind limb (HL) and tail (T) regions of E12.5 embryos, and the forelimb region of E14.5 embryos were observed. The strong signals for the epidermis in E14.5 embryos (indicated by asterisks) were non-specific. No significant expression of Dnmt3b was detected, whilst Dnmt3a was significantly expressed in all the mesenchymal cells.
Fig. 3. Western blotting of Dnmt3a and Dnmt3b, and semi-quantitative determination of their amounts. (A) Whole embryos were solubilized by sonication or homogenization in the presence of 0.1% SDS. In dissected embryos (10 mg protein) at E7.0–16.5, the Dnmt3a (3a) and Dnmt3b isoforms (3b1, 3b2, and 3b3), of which the cDNAs were transiently expressed in 293T cells, were determined with specific antibodies. (B) The amount of each band material was determined using the recombinant Dnmt3a and Dnmt3b as standards. The density of each of the immunodetected bands was determined and plotted. Insets show Western blotting of Dnmt3a and Dnmt3b. The Dnmt3a expressed in E7.0, 8.5, and 9.5 embryos amounted to less than 0.07 ng, in E10.5 embryos 0.45 ng, in E12.5 embryos 0.70 ng, in E14.5 embryos 0.60 ng, and in E16.5 embryos 0.48 ng per 10 mg embryonic tissue protein. The Dnmt3b expressed in E7.0 embryos amounted to 7.2 ng, in E8.5 embryos 2.5 ng, in E9.5 embryos 0.2 ng, and in E10.5, E12.5, E14.5, and E16.5 embryos less than 0.07 ng per 10 mg tissue protein.
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Fig. 4. Immunostaining of Dnmt3a and Dnmt3b in ES cells. (A) ES cells of R1 were immunostained with anti-Dnmt3a (red) or anti-Dnmt3b (red) antibodies, and then with Hoechst (blue). (B) In the cells immunostained with anti-Dnmt3b antibodies (red), Hoechst-stained regions (blue) are indicated by arrowheads and arrows (Merge). The arrowheads indicate the regions that were co-stained with anti-Dnmt3b antibodies, and the arrows indicate the region stained not with anti-Dnmt3b but with Hoechst. Bars indicate 10 mm (A) and 5 mm (B).
tioned, and fixed with paraformaldehyde. Samples were reacted with anti-Dnmt3a, anti-Dnmt3b (Aoki et al., 2001), or anti-Oct3/4 antibodies (Saijoh et al., 1996), and then stained with anti-rabbit IgG conjugated with ALEXA568 (Molecular Probe, OR, USA). ES cells (Tada et al., 2001) were immunostained as described previously (Inano et al., 2000). All the samples except those for whole-mount immunostaining were observed under a laser scanning confocal microscope (Zeiss LSM 510). The whole-mount samples were observed under a Nikon TE 300 stereoscope. Embryos or cells were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Dnmt3a and Dnmt3b were immunodetected as described elsewhere (Aoki et al., 2001). The amounts of Dnmt3a and Dnmt3b were determined using standard curves for recombinant Dnmt3a and Dnmt3b. Acknowledgements We wish to thank Drs Hiroshi Hamada and Yukio Saijoh (Osaka University) for providing us with the anti-Oct3/4 antibodies and the discussion on the expression profile of Oct3/4. This work was supported by the Promotion of Basic Research Activities for Innovative Biosciences of Japan. References Aoki, A., Suetake, I., Miyagawa, J., Fujio, T., Chijiwa, T., Sasaki, S., Tajima, S., 2001. Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 29, 3506–3512. Bachman, K.E., Rountree, M.R., Baylin, S.B., 2001. Dnmt3a and Dnmt3b
are transcriptional repressors that exhibit unique localization properties to heterochromatin. J. Biol. Chem. 276, 32282–32287. Hansen, R.S., Wijmenga, C., Luo, P., Stanek, A.M., Canfield, T.K., Weemaes, C.M.R., Gartler, S.M., 1999. The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc. Natl Acad. Sci. USA 96, 14412–14417. Inano, K., Suetake, I., Ueda, T., Miyake, Y., Nakamura, M., Okada, M., Tajima, S., 2000. Maintenance-type DNA methyltransferase is highly expressed in post-mitotic neurons and localized in the cytoplasmic compartment. J. Biochem. 128, 315–321. Li, E., Bestor, T.H., Jaenisch, R., 1992. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926. Miniou, P., Jeanpierre, M., Bourc’his, D., Barbosa, A.C.C., Blanquet, V., Viegas-Pequignot, E., 1997. a-Satellite DNA methylation in normal individuals and in ICF patients: heterogeneous methylation of constitutive heterochromatin in adult and fetal tissues. Hum. Genet. 99, 738–745. Monk, M., 1990. Changes in DNA methylation during mouse embryonic development in relation to X-chromosome activity and imprinting. Phil. Trans. R. Soc. Lond. 326, 299–312. Okano, M., Xie, S., Li, E., 1998. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19, 219–220. Okano, M., Bell, D.W., Haber, D.A., Li, E., 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257. Pesce, M., Gross, M.K., Scho¨ ler, H.R., 1998. In line with our ancestors: Oct-4 and the mammalian germ. BioEssays 20, 722–732. Saijoh, Y., Fujii, H., Meno, C., Sato, M., Hirota, Y., Nagamatsu, S., Ikeda, M., Hamada, H., 1996. Identification of putative downstream genes of Oct3, a pluripotent cell-specific transcription factor. Genes Cells 1, 239–252. Tada, M., Takahama, Y., Abe, K., Nakatsuji, N., Tada, T., 2001. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr. Biol. 11, 1553–1558. Xu, G.L., Bestor, T.H., Bourc’his, D., Hsieh, C.L., Tommerup, N., Bugge, M., Hulten, M., Qu, X., Russo, J.J., Viegas-Pequignot, E., 1999. Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402, 187–191.
Gene expression pattern
Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis Daisuke Watanabe a,1, Isao Suetake b,1, Takashi Tada c, Shoji Tajima b,* a
Institute of Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Japan Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan c Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan b
Received 8 April 2002; received in revised form 16 May 2002; accepted 4 July 2002
Abstract DNA methylation is essential for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, contribute to the creation of DNA methylation patterns in embryos. We demonstrated that the Dnmt3a and Dnmt3b proteins are expressed at different stages of embryogenesis. Dnmt3b is specifically expressed in totipotent embryonic cells, such as inner cell mass, epiblast and embryonic ectoderm cells, whilst Dnmt3a is significantly and ubiquitously expressed after E10.5. The difference in the expression stages of the Dnmt3a and Dnmt3b proteins may contribute to their distinct functions during the embryogenesis. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mouse embryogenesis; DNA methylation pattern; Dnmt3a; Dnmt3b; De novo-type DNA methyltransferase
1. Results and discussion In vertebrates, the methylation of cytosine in genomic DNA plays a crucial role in gene silencing, and is essential for development (Li et al., 1992; Okano et al., 1999). Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of methylation patterns at an early stage of embryogenesis (Okano et al., 1999). When Dnmt3a or Dnmt3b is targeted, residual Dnmt3b or Dnmt3a, respectively, partly compensates for the lack of the gene (Okano et al., 1999), suggesting that Dnmt3a and Dnmt3b possess similar functional specificities. On the other hand, targeting of Dnmt3b leads to a severer phenotype than that of Dnmt3a, and a mutation in DNMT3b, a human homologue of Dnmt3b, leads to hypomethylation of the satellite 2 and 3 regions of specific chromosomes (Miniou et al., 1997), which is the cause of ICF syndrome (Okano et al., 1999; Xu et al., 1999; Hansen et al., 1999). These reports suggest that Dnmt3a and Dnmt3b possess distinct functions. We deduced that the difference in the timing of cell-type specific expression of the Dnmt3a and Dnmt3b proteins was one of the reasons for the different functions. To address this issue, we determined Dnmt3a and Dnmt3b proteins in mouse embryos using specific antibodies recognizing the amino-terminal sequence of each * Corresponding author. Tel.: 181-6-6879-8627; fax: 181-6-6879-8629. E-mail address: [email protected] (S. Tajima). 1 These authors contribute equally to this work.
protein, where no homology exists between the proteins (Aoki et al., 2001). Surprisingly, Dnmt3a was not significantly detected in E4.5–8.5 embryos (Fig. 1A). On the other hand, Dnmt3b existed at E4.5–7.0, in the inner cell mass (ICM) at E4.5, the epiblast at E5.5, and the embryonic ectoderm at E7.0. In E8.5 embryos, Dnmt3b was weakly detected in the head folds, but at very low level in E9.5 embryos (Fig. 1A). The localization of Dnmt3b in early embryos at E4.5–E7.0 resembled that of Oct3/4, a transcription factor specifically expressed in totipotent cells (Fig. 1B) (Pesce et al., 1998). Dnmt3b was the major de novo DNA methyltransferase in E4.5–7.5 embryos, in which genomewide DNA methylation occurs (Monk, 1990). The Dnmt3a protein was below the detection level until E9.5, but was detected after E10.5. Typical immunostaining of the hind limb and tail regions at E12.5, and the forelimb region at E14.5 is shown in Fig. 2. Dnmt3a was detected ubiquitously in mesenchymal cells. In contrast, the Dnmt3b protein was not detected in E10.5–14.5 embryos. The amounts of Dnmt3a and Dnmt3b in E7.0–E16.5 embryos were determined using recombinant Dnmt3a and Dnmt3b as standards (Fig. 3). Dnmt3a was below the detection level in E7.0–9.5 embryos (,0.07 ng/10 mg protein), significantly detected in E10.5 ones (0.45 ng/10 mg protein), and was detected thereafter. Inversely, Dnmt3b was highly expressed in E7.0 embryos (7.2 ng/10 mg protein), decreased thereafter, and was below the detection level after E10.5 (,0.07 ng/10 mg protein). Judging from its
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00242-3
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D. Watanabe et al. / Mechanisms of Development 118 (2002) 187–190
Fig. 1. Expression of Dnmt3a and Dnmt3b in mouse embryos. (A) ICR mouse embryos at E4.5, 5.5, 7.0, 8.5, and 9.5 were stained with anti-Dnmt3a (3a), antiDnmt3b (3b), or anti-Oct 3/4 (Oct) antibodies, and/or DAPI (DAPI, blue). The antibodies were detected with anti-rabbit antibodies coupled with ALEXA568. The ICM and trophectoderm (T) at E4.5 are indicated by arrows and arrowheads, respectively. The epiblast (E) at E5.5, and the embryonic ectoderm (EE) and extraembryonic region (EX) at E7.0 are indicated by square brackets. The head folds at E8.5 and E9.5 are indicated by arrows. (B) High magnification images of an E5.5 embryo stained with anti-Dnmt3b, Oct3/4, and DAPI are shown. Note that Dnmt3b was not detected in primitive endoderm cells surrounding the epiblast, which is similar to the expression pattern of Oct3/4. Arrowheads and arrows indicate the nuclei of primitive endoderm and epiblast cells, respectively.
mobility, the major Dnmt3b in embryos was Dnmt3b1, a translation product of one of the splicing isoforms. De novotype DNA methyltransferase switches from Dnmt3b to Dnmt3a at around E9.5–10.5. Embryonic stem (ES) cells express both Dnmt3a and Dnmt3b mRNAs at high levels compared to those in established somatic cells with no totipotency (Okano et al., 1998). As ES cells reflect the state of totipotent cells in embryos, we determined the levels of the Dnmt3a and Dnmt3b proteins in ES cells, R1. Dnmt3b was detected in the nuclei of all R1 cells, but Dnmt3a was not highly expressed (Fig. 4). Interestingly, only a portion, i.e. not all, of the Dnmt3b was localized to the heterochromatin. The localization of the endogenous Dnmt3b was not identical to that of ectopically expressed Dnmt3b, which has been reported to be co-localized with heterochromatin (Bachman et al., 2001). The amounts of Dnmt3a and Dnmt3b in R1 cells were determined as in Fig. 3. R1 cells significantly expressed Dnmt3b (5 ng/
10 mg protein), but not much Dnmt3a (0.3 ng/10 mg protein). The amount of Dnmt3a was similar to that in cultured fibroblasts, C3H10T1/2 (1.0 ng/10 mg protein). Another ES cell line, TMA-S5, showed similar expression profiles for the Dnmt3a and Dnmt3b proteins as those in R1. Not the Dnmt3a but the Dnmt3b protein was highly expressed in ES cells. The results of the present study suggest that the different expression profiles of the Dnmt3a and Dnmt3b proteins are one of the causes of their distinct functions in an early stage of embryogenesis.
2. Methods ICR mouse embryos at E4.5–E9.5 were fixed in cold acetone. Embryos at E12.5 and E14.5 were mounted in TissueTek OCT compound (Miles Scientific, IL, USA), cryosec-
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Fig. 2. The expression of Dnmt3b decreased and that of Dnmt3a increased as embryogenesis proceeded. Serial sections (8 mm-thick) of E12.5 or E14.5 embryos mounted in Tissue-Tek OCT compound were immunostained with anti-Dnmt3a (3a), or anti-Dnmt3b (3b) antibodies, or without first antibodies (-). The hind limb (HL) and tail (T) regions of E12.5 embryos, and the forelimb region of E14.5 embryos were observed. The strong signals for the epidermis in E14.5 embryos (indicated by asterisks) were non-specific. No significant expression of Dnmt3b was detected, whilst Dnmt3a was significantly expressed in all the mesenchymal cells.
Fig. 3. Western blotting of Dnmt3a and Dnmt3b, and semi-quantitative determination of their amounts. (A) Whole embryos were solubilized by sonication or homogenization in the presence of 0.1% SDS. In dissected embryos (10 mg protein) at E7.0–16.5, the Dnmt3a (3a) and Dnmt3b isoforms (3b1, 3b2, and 3b3), of which the cDNAs were transiently expressed in 293T cells, were determined with specific antibodies. (B) The amount of each band material was determined using the recombinant Dnmt3a and Dnmt3b as standards. The density of each of the immunodetected bands was determined and plotted. Insets show Western blotting of Dnmt3a and Dnmt3b. The Dnmt3a expressed in E7.0, 8.5, and 9.5 embryos amounted to less than 0.07 ng, in E10.5 embryos 0.45 ng, in E12.5 embryos 0.70 ng, in E14.5 embryos 0.60 ng, and in E16.5 embryos 0.48 ng per 10 mg embryonic tissue protein. The Dnmt3b expressed in E7.0 embryos amounted to 7.2 ng, in E8.5 embryos 2.5 ng, in E9.5 embryos 0.2 ng, and in E10.5, E12.5, E14.5, and E16.5 embryos less than 0.07 ng per 10 mg tissue protein.
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Fig. 4. Immunostaining of Dnmt3a and Dnmt3b in ES cells. (A) ES cells of R1 were immunostained with anti-Dnmt3a (red) or anti-Dnmt3b (red) antibodies, and then with Hoechst (blue). (B) In the cells immunostained with anti-Dnmt3b antibodies (red), Hoechst-stained regions (blue) are indicated by arrowheads and arrows (Merge). The arrowheads indicate the regions that were co-stained with anti-Dnmt3b antibodies, and the arrows indicate the region stained not with anti-Dnmt3b but with Hoechst. Bars indicate 10 mm (A) and 5 mm (B).
tioned, and fixed with paraformaldehyde. Samples were reacted with anti-Dnmt3a, anti-Dnmt3b (Aoki et al., 2001), or anti-Oct3/4 antibodies (Saijoh et al., 1996), and then stained with anti-rabbit IgG conjugated with ALEXA568 (Molecular Probe, OR, USA). ES cells (Tada et al., 2001) were immunostained as described previously (Inano et al., 2000). All the samples except those for whole-mount immunostaining were observed under a laser scanning confocal microscope (Zeiss LSM 510). The whole-mount samples were observed under a Nikon TE 300 stereoscope. Embryos or cells were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Dnmt3a and Dnmt3b were immunodetected as described elsewhere (Aoki et al., 2001). The amounts of Dnmt3a and Dnmt3b were determined using standard curves for recombinant Dnmt3a and Dnmt3b. Acknowledgements We wish to thank Drs Hiroshi Hamada and Yukio Saijoh (Osaka University) for providing us with the anti-Oct3/4 antibodies and the discussion on the expression profile of Oct3/4. This work was supported by the Promotion of Basic Research Activities for Innovative Biosciences of Japan. References Aoki, A., Suetake, I., Miyagawa, J., Fujio, T., Chijiwa, T., Sasaki, S., Tajima, S., 2001. Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 29, 3506–3512. Bachman, K.E., Rountree, M.R., Baylin, S.B., 2001. Dnmt3a and Dnmt3b
are transcriptional repressors that exhibit unique localization properties to heterochromatin. J. Biol. Chem. 276, 32282–32287. Hansen, R.S., Wijmenga, C., Luo, P., Stanek, A.M., Canfield, T.K., Weemaes, C.M.R., Gartler, S.M., 1999. The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc. Natl Acad. Sci. USA 96, 14412–14417. Inano, K., Suetake, I., Ueda, T., Miyake, Y., Nakamura, M., Okada, M., Tajima, S., 2000. Maintenance-type DNA methyltransferase is highly expressed in post-mitotic neurons and localized in the cytoplasmic compartment. J. Biochem. 128, 315–321. Li, E., Bestor, T.H., Jaenisch, R., 1992. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926. Miniou, P., Jeanpierre, M., Bourc’his, D., Barbosa, A.C.C., Blanquet, V., Viegas-Pequignot, E., 1997. a-Satellite DNA methylation in normal individuals and in ICF patients: heterogeneous methylation of constitutive heterochromatin in adult and fetal tissues. Hum. Genet. 99, 738–745. Monk, M., 1990. Changes in DNA methylation during mouse embryonic development in relation to X-chromosome activity and imprinting. Phil. Trans. R. Soc. Lond. 326, 299–312. Okano, M., Xie, S., Li, E., 1998. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19, 219–220. Okano, M., Bell, D.W., Haber, D.A., Li, E., 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257. Pesce, M., Gross, M.K., Scho¨ ler, H.R., 1998. In line with our ancestors: Oct-4 and the mammalian germ. BioEssays 20, 722–732. Saijoh, Y., Fujii, H., Meno, C., Sato, M., Hirota, Y., Nagamatsu, S., Ikeda, M., Hamada, H., 1996. Identification of putative downstream genes of Oct3, a pluripotent cell-specific transcription factor. Genes Cells 1, 239–252. Tada, M., Takahama, Y., Abe, K., Nakatsuji, N., Tada, T., 2001. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr. Biol. 11, 1553–1558. Xu, G.L., Bestor, T.H., Bourc’his, D., Hsieh, C.L., Tommerup, N., Bugge, M., Hulten, M., Qu, X., Russo, J.J., Viegas-Pequignot, E., 1999. Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402, 187–191.