한국발생생물학회지:발생과생식 (Development and Reproduction)

  • 제14권2호
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  • Pages.123-129
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  • 2010
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  • 2465-9525(pISSN)
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  • 2465-9541(eISSN)
한국발생생물학회 (The Korean Society of Developmental Biology)
권호 표지

OCT4와 SOX2에 의한 인간 Nanog 유전자의 전사 조절

Transcriptional Regulation of Human Nanog Gene by OCT4 and SOX2

  • 석현정 (충북대학교 자연과학대학 생화학과) ;
  • 김영은 (충북대학교 자연과학대학 생화학과) ;
  • 박정아 (충북대학교 자연과학대학 생화학과) ;
  • 이영희 (충북대학교 자연과학대학 생화학과)
  • Seok, Hyun-Jeong (Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University) ;
  • Kim, Young-Eun (Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University) ;
  • Park, Jeong-A (Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University) ;
  • Lee, Young-Hee (Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University)
  • 투고 : 2010.03.26
  • 심사 : 2010.05.09
  • 발행 : 2010.06.30
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초록

배아 줄기세포는 미분화상태에서 자가 재생을 유지할 수 있다. 자가 재생은 OCT4, SOX2와 NANOG와 같은 많은 인자들이 작용한다. 생쥐 배아 줄기세포에서 OCT4와 SOX2가 Nanog 프로모터에 결합하여 Nanog 유전자의 발현을 촉진한다는 사실은 생쥐 promoter에 관한 정밀분석으로 알려져 있다. 본 연구에서는 인간 Nanog promoter를 정밀 분석하기 위해 연속적인 결손 돌연변이를 가진 promoter-reporter construct를 제조하였다. Promoter의 최대 활성은 0.6 kb(-253/+365) promoter-reporter construct에서 발견되었으며, 이 construct에는 OCT4 및 SOX2의 결합부위가 포함된다. OCT4와 SOX2의 기여도를 확인하기 위하여 OCT4 및 SOX2의 결합부위에 자리 특이적 돌연변이를 유도하고 promoter 활성에 미치는 영향을 조사한 결과, OCT4나 SOX2 어느 한 군데라도 돌연변이가 존재하면 promoter 활성이 현저히 저해되었다. 본 연구 결과를 통해 인간 Nanog 유전자 발현에 있어 OCT4 및 SOX2가 필수적임을 직접적으로 확인할 수 있었다.

Embryonic stem (ES) cells can self-renew maintaining the undifferentiated state. Self-renewal requires many factors such as OCT4, SOX2, and NANOG. It is previously known that OCT4 and SOX2 can bind to NANOG promoter and support Nanog gene expression in mouse ES cells by the detailed studies using the mouse Nanog promoter. Here, we constructed serial deletion mutant promoter-reporter constructs to investigate the human Nanog gene promoter in detail. The highest promoter activity was obtained in the 0.6 kb (-253/+365) promoter-reporter construct which includes the binding sites of OCT4 and SOX2. To further confirm contribution of OCT4 and SOX2 in Nanog gene expression, we introduced site- directed mutation(s) in the OCT4 and/or SOX2 binding sites of the human Nanog promoter 0.6 kb (-253/+365) and checked the influence of the mutation on the promoter activity using human EC cell line NCCIT. Mutation either in OCT4 binding site or SOX2 binding site significantly reduced the activity of Nanog promoter which directly confirmed that OCT4 and SOX2 binding is essential in human Nanog gene expression.

키워드

참고문헌

  1. Babaie Y, Herwig R, Greber B, Brink TC, Wruck W, Groth D, Lehrach H, Burdon T, Adjaye J (2007) Analysis of OCT4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells 25:500-510. https://doi.org/10.1634/stemcells.2006-0426
  2. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643-655. https://doi.org/10.1016/S0092-8674(03)00392-1
  3. Chan KK, Zhang J, Chia NY, Chan YS, Sim HS, Tan KS, Oh SK, Ng HH, Choo AB (2009) KLF4 and PBX1 directly regulate NANOG expression in human embryonic stem cells. Stem Cells 27:2114-2125. https://doi.org/10.1002/stem.143
  4. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154-156. https://doi.org/10.1038/292154a0
  5. Han MK, Song EK, Guo Y, Ou X, Mantel C, Broxmeyer HE (2008) SIRT1 regulates apoptosis and Nanog expression in mouse embryonic stem cells by controlling p53 subcellular localization. Stem Cells 2: 241-251. https://doi.org/10.1016/j.stem.2008.01.002
  6. Jaenisch R, Young R (2008) Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132:567-582. https://doi.org/10.1016/j.cell.2008.01.015
  7. Kuroda T, Tada M, Kubota H, Kimura H, Hatano SY, Suemori H, Nakatsuji N, Tada T (2005) Octamer and Sox elements are required for transcriptional cis regu lation of Nanog gene expression. Mol Cell Biol 25: 2475-2485. https://doi.org/10.1128/MCB.25.6.2475-2485.2005
  8. Lin T, Chao C, Saito S, Mazur SJ, Murphy ME, Appella E, Xu Y (2005) p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nat Biotechnol 7:165-171.
  9. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631-642. https://doi.org/10.1016/S0092-8674(03)00393-3
  10. Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24:372-376. https://doi.org/10.1038/74199
  11. Odorico JS, Kaufman DS, Thomson JA (2001) Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19:193-204. https://doi.org/10.1634/stemcells.19-3-193
  12. Pan G, Thomson JA (2007) Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res 17:42-49. https://doi.org/10.1038/sj.cr.7310125
  13. Pan GJ, Pei DQ (2003) Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog. Cell Res 6:499-502.
  14. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399-405. https://doi.org/10.1038/74447
  15. Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P (2005) Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 280:24731-24737. https://doi.org/10.1074/jbc.M502573200
  16. Shi W, Wang H, Pan G, Geng Y, Guo Y, Pei D (2006) Regulation of the pluripotency marker rex-1 by nanog and sox2. J Biol Chem 281:23319-23325. https://doi.org/10.1074/jbc.M601811200
  17. Shultz WA, Hoffmann MJ (2007) Transcriptional networks in embryonic stem cells and testicular cancer and the definition of epigenetics. Epigenetics 2:37-42. https://doi.org/10.4161/epi.2.1.4067
  18. Silva J, Chambers I, Pollard S, Smith A (2006) Nanog promotes transfer of pluripotency after cell fusion. Nature 441:997-1001. https://doi.org/10.1038/nature04914
  19. Singh AM, Hamazaki T, Hankowski KE, Terada N (2007) A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem Cells 25:2534-2542. https://doi.org/10.1634/stemcells.2007-0126
  20. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145-1147. https://doi.org/10.1126/science.282.5391.1145
  21. Torres J, Watt FM (2008) Nanog maintains pluripotency of mouse embryonic stem cells by inhibiting NF-${\kappa}B$B and cooperating with Stat3. Nat Cell Biol 10:194-201. https://doi.org/10.1038/ncb1680
  22. Yamanaka S (2007) Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1:39-49. https://doi.org/10.1016/j.stem.2007.05.012