Tumor Biology

, Volume 37, Issue 1, pp 877–885 | Cite as

Genome-wide profiling of DNA methylation reveals preferred sequences of DNMTs in hepatocellular carcinoma cells

  • Hong Fan
  • Zhujiang Zhao
  • Yuchao Cheng
  • He Cui
  • Fengchang Qiao
  • Ling Wang
  • Jiaojiao Hu
  • Huzhang Wu
  • Wei Song
Research Article


Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in developmental process and diseases including cancer. To elucidate the functional preferred site of DNMTs, we analyzed the feature of distinct methylated sequences and established the defined relationship between DNMTs and preference genomic DNA sequences. Small interfering RNA (siRNA) construct of DNTM1, DNMT3A, and DNMT3B was transfected into the human hepatocellular carcinoma cell line SMMC-7721, respectively. Distinguishing methylated fragments pool was enriched by SHH method in cells which is knocked down DNMT1, DNMT3A, DNMT3B, separately. The defined binding transcription factors (TFs) containing of 5′CpG islands were obtained with bioinformatics software and website. In SMMC-7721 hepatocellular carcinoma (HCC) cell line, DNMT1, DNMT3A, and DNMT3B were specific suppressed by their corresponding siRNA construct, separately. A 46, 42, 67 distinctive methylated fragments from three different DNMTs were evaluated according to genomic DNA database. Those separated fragments were distributed among genomic DNA regions of all chromosome complements, including coding genes, repeat sequences, and genes with unknown function. The majority of coding genes contain CpG islands in their promoter region. Cluster analysis demonstrated all of preference sequences identified by three DNMTs shares their own conserved sequences. In depleting of different DNMTs cells, 80 % of 103 upregulation genes induced by DNMT1 knock-down contain CpG sites; 76 % of 25 upregulation genes induced by DNMT3A knock-down contain CpG sites; 63 % of 126 upregulation genes induced by DNMT3B knock-down contain CpG sites. Our findings suggested that distinctive DNMTs targeted DNA methylation site to their preference sequences, and this targeting might be associated with diverse roles of DNMTs in tumorigenesis. Meanwhile, the analysis of preference sequences provides an alternative way to find out the individual function of DNMTs.


DNA methyltranferases Preference sequences 5′CpG site 



Hepatocellular carcinoma


DNA methyltransferases


Small interfering RNA


Subtractive suppression hybridization



This work was supported by The National Natural Science Foundation of China, No. No.91229107. This work was also supported by National 973 Basic Research Program of China 2013CB911302, No.81171915, No. 81472548 and 30470950.

Conflicts of interest


Author’s contributions

FH was responsible for designing and execution, collation of study materials, and verification of the data and final manuscript writing; ZZJ designed and performed the siRNA of DNMTs in HCC cell lines and analyzed the microarray data; CH contributed to experiment performing and data collection. CYC designed and performed SSH. QFC, WL, HJJ, WHZ, and SW participated in the completion of the study. All the authors read and approved the final manuscript.

Supplementary material

13277_2015_3202_Fig8_ESM.jpg (903 kb)
Figure 1

Schematic representation of DNMTs mRNA structure and their siRNA positions (JPEG 903 kb)

13277_2015_3202_Fig9_ESM.jpg (859 kb)
Figure 2

Restoring expression of representative genes by depletion of different DNMTs in HCC SMMC-7721 cells. (A) Analysis of expression level of representative up-regulated gene CDH1 by Quantitative-PCR (left) and Western Blot (right) in DNMT1-depleted cell. (B) Analysis of expression level of representative up-regulated gene PTEN by Quantitative-PCR (left) and Western Blot (right) in DNMT3A-depleted cell. (C) Analysis of expression level of representative up-regulated gene PDCD4 by Quantitative-PCR (left) and Western Blot (right) in DNMT3B-depleted cell. (JPEG 858 kb)

13277_2015_3202_MOESM1_ESM.doc (74 kb)
Table 1 The information of sequences recognized by DNMT1 (DOC 74 kb)
13277_2015_3202_MOESM2_ESM.doc (71 kb)
Table 2 The information of sequences recognized by DNMT3A (DOC 71 kb)
13277_2015_3202_MOESM3_ESM.doc (101 kb)
Table 3 The information of sequences recognized by DNMT3B (DOC 101 kb)
13277_2015_3202_MOESM4_ESM.doc (110 kb)
Table 4 Genes altered ≥ 2-fold after DNMT1 knocked down treatment in hepatocellular carcinoma cell line SMMC-7721 (DOC 110 kb)
13277_2015_3202_MOESM5_ESM.doc (49 kb)
Table 5 Genes altered ≥ 2-fold after DNMT3A knocked-down treatment in hepatocellular carcinoma cell line SMMC-7721 (DOC 49 kb)
13277_2015_3202_MOESM6_ESM.doc (154 kb)
Table 6 Genes altered ≥ 2-fold after DNMT3B knocked-down treatment in hepatocellular carcinoma cell line SMMC-7721 (DOC 154 kb)


  1. 1.
    Bird A. DNA methylation de novo. Science. 1999;286:2287–8.CrossRefPubMedGoogle Scholar
  2. 2.
    Jones PA. DNA methylation errors and cancer. Cancer Res. 1996;56:2463.PubMedGoogle Scholar
  3. 3.
    Laird PW, Jackson-Grusby L, Fazeli A, Dickinson SL, Jung WE, Li E, et al. Suppression of intestinal neoplasia by DNA hypomethylation. Cell. 1995;81:197.CrossRefPubMedGoogle Scholar
  4. 4.
    Dodge JE, Okano M, Dick F, Tsujimoto N, Chen T, Wang S, et al. Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem. 2005;280:17986–91.CrossRefPubMedGoogle Scholar
  5. 5.
    Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, et al. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature. 2004;429:900–3.CrossRefPubMedGoogle Scholar
  6. 6.
    Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999;99:247–57.CrossRefPubMedGoogle Scholar
  7. 7.
    Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer. 2004;4:143–53.CrossRefPubMedGoogle Scholar
  8. 8.
    Jones PA. Epigenetics in carcinogenesis and cancer prevention. Ann N Y Acad Sci. 2003;983:213–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Lin C-H, Hsieh S-Y, Sheen I-S, Lee W-C, Chen T-C, Shyu W-C, et al. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res. 2001;61:4238–43.PubMedGoogle Scholar
  10. 10.
    Nishida N, Kudo M, Nagasaka T, Ikai I, Goel A. Characteristic patterns of altered DNA methylation predict emergence of human hepatocellular carcinoma. Hepatology. 2012;56(3):994–1003.CrossRefPubMedGoogle Scholar
  11. 11.
    Shen J, Wang S, Zhang Y-J, Wu H-C, Kibriya MG, Jasmine F, et al. Exploring genome-wide DNA methylation profiles altered in hepatocellular carcinoma using infinium humanmethylation 450 beadchips. Epigenetics. 2013;8:1.CrossRefGoogle Scholar
  12. 12.
    Fernandez AFHC, Fraga MF. De novo DNA methyltransferases: Oncogenes, tumor suppressors, or both? Trends Genet. 2012;28:474–9.CrossRefPubMedGoogle Scholar
  13. 13.
    Ji W, Wright MB, Cai L, Flament A, Lindpaintner K. Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics. 2002;3:12.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Gruenbaum Y, Stein R, Cedar H, Razin A. Methylation of CpG sequences in eukaryotic DNA. FEBS Lett. 1981;124:67.15.CrossRefGoogle Scholar
  15. 15.
    Razin A, Webb C, Szyf M, Yisraeli J, Rosenthal A, Naveh-Many T, et al. Variations in DNA methylation during mouse cell differentiation in vivo and in vitro. Proc Natl Acad Sci. 1984;81:2275–9.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Cross SH, Bird AP. CpG islands and genes. Curr Opin Genet Dev. 1995;5:309–14.CrossRefPubMedGoogle Scholar
  17. 17.
    Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683–92.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol. 2010;28:1057–68.CrossRefPubMedGoogle Scholar
  19. 19.
    Handa V, Jeltsch A. Profound flanking sequence preference of Dnmt3a and Dnmt3b mammalian DNA methyltransferases shape the human epigenome. J Mol Biol. 2005;348:1103–12.CrossRefPubMedGoogle Scholar
  20. 20.
    Waterston RH, Lander ES, Sulston JE. On the sequencing of the human genome. Proc Natl Acad Sci. 2002;99:3712–6.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921.CrossRefPubMedGoogle Scholar
  22. 22.
    Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et al. The sequence of the human genome. Science. 2001;291:1304–51.CrossRefPubMedGoogle Scholar
  23. 23.
    Song J, Teplova M, Ishibe-Murakami S, Patel DJ. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science. 2012;335:709–12.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001;293:1089–93.CrossRefPubMedGoogle Scholar
  25. 25.
    Branco MR, Oda M, Reik W. Safeguarding parental identity: Dnmt1 maintains imprints during epigenetic reprogramming in early embryogenesis. Genes Dev. 2008;22:1567–71.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Wienholz BL, Kareta MS, Moarefi AH, Gordon CA, Ginno PA, Chédin F. DNMT3L modulates significant and distinct flanking sequence preference for DNA methylation by DNMT3A and DNMT3B in vivo. PLoS Genet. 2010;6:e1001106.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Oka M, Rodić N, Graddy J, Chang L-J, Terada N. CpG sites preferentially methylated by Dnmt3a in vivo. J Biol Chem. 2006;281:9901–8.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Hong Fan
    • 1
    • 2
  • Zhujiang Zhao
    • 1
    • 2
  • Yuchao Cheng
    • 1
    • 2
  • He Cui
    • 1
    • 2
  • Fengchang Qiao
    • 1
    • 2
  • Ling Wang
    • 1
    • 2
  • Jiaojiao Hu
    • 1
    • 2
  • Huzhang Wu
    • 1
    • 2
  • Wei Song
    • 1
    • 2
  1. 1.Department of Medical Genetics and Developmental BiologyMedical School of Southeast UniversityNanjingChina
  2. 2.The Key Laboratory of Developmental Genes and Human Diseases, Ministry of EducationSoutheast UniversityNanjingChina

Personalised recommendations