Differential DNA Methylation Patterns in Endo-siRNAs Mediated Silencing of LINE-1 Retrotransposons

  • Long Chen
  • Jane E Dahlstrom
  • Danny RangasamyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1173)


Analyzing differences in DNA methylation is a powerful tool for assessing the effect of endo-siRNAs expression in the human genome. Here, we present a simple genome-wide DNA methylation assay that allows for a precise quantitative analysis of differences in the promoter of human long interspersed nuclear element 1 (LINE-1 or L1) retrotransposons in response to endogenous and exogenous expression of endo-siRNAs. Using the DNA bisulfite modification sequencing, we have optimized the method to detect small changes in heterogeneously methylated L1 repeats at multiple regions across the genome. We also provide guidance for analysis of primary bisulfite sequencing data and interpretation of the methylation status using the Web-based bisulfite sequencing DNA methylation (BISMA) analysis. This refined and reproducible assay can be performed even using a small amount of genomic DNA and is suitable for the analysis of clinical tissue samples.


Endo-siRNAs LINE-1 Breast cancer cells DNA methylation DNA sequencing PCR Bisulfite data analysis 



This work was supported by a grant from the Canberra Hospital Private Practice Fund and the ACT Health and Medical Research Support Program.


  1. 1.
    Ehrlich M (2002) DNA methylation in cancer: too much, but also too little. Oncogene 21:5400–5413PubMedCrossRefGoogle Scholar
  2. 2.
    Beck CR, Collier P, Macfarlane C, Malig M, Kidd JM, Eichler EE, Badge RM, Moran JV (2010) LINE-1 retrotransposition activity in human genomes. Cell 141:1159–1170PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Roman-Gomez J, Jimenez-Velasco A, Agirre X, Cervantes F, Sanchez J, Garate L, Barrios M, Castillejo JA, Navarro G, Colomer D, Prosper F, Heiniger A, Torres A (2005) Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia. Oncogene 24:7213–7223PubMedCrossRefGoogle Scholar
  4. 4.
    Chen L, Dahlstrom JE, Lee SH, Rangasamy D (2012) Naturally occurring endo-siRNA silences LINE-1 retrotransposons in human cells through DNA methylation. Epigenetics 7:758–771PubMedCrossRefGoogle Scholar
  5. 5.
    van Hoesel AQ, van de Velde CJ, Kuppen PJ, Liefers GJ, Putter H, Sato Y, Elashoff DA, Turner RR, Shamonki JM, de Kruijf EM, van Nes JG, Giuliano AE, Hoon DS (2012) Hypomethylation of LINE-1 in primary tumor has poor prognosis in young breast cancer patients: a retrospective cohort study. Breast Cancer Res Treat 134:1103–1114PubMedCrossRefGoogle Scholar
  6. 6.
    Chen L, Dahlstrom JE, Chandra A, Board P, Rangasamy D (2012) Prognostic value of LINE-1 retrotransposon expression and its subcellular localization in breast cancer. Breast Cancer Res Treat 136:129–142PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Estecio MR, Gharibyan V, Shen L, Ibrahim AE, Doshi K, He R, Jelinek J, Yang AS, Yan PS, Huang TH, Tajara EH, Issa JP (2007) LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability. PLoS One 2:e399PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP (2004) A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 32:e38PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Brouha B, Schustak J, Badge RM, Lutz-Prigge S, Farley AH, Moran JV, Kazazian HH Jr (2003) Hot L1s account for the bulk of retrotransposition in the human population. Proc Natl Acad Sci U S A 100:5280–5285PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Cordaux R, Batzer MA (2009) The impact of retrotransposons on human genome evolution. Nat Rev 10:691–703CrossRefGoogle Scholar
  11. 11.
    Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y, Chiba H, Kohara Y, Kono T, Nakano T, Surani MA, Sakaki Y, Sasaki H (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453:539–543PubMedCrossRefGoogle Scholar
  12. 12.
    Okamura K, Chung WJ, Ruby JG, Guo H, Bartel DP, Lai EC (2008) The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453:803–806PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Moazed D (2009) Small RNAs in transcriptional gene silencing and genome defence. Nature 457:413–420PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Rohde C, Zhang Y, Reinhardt R, Jeltsch A (2010) BISMA–fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences. BMC Bioinformatics 11:230PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Long Chen
    • 1
  • Jane E Dahlstrom
    • 1
    • 2
  • Danny Rangasamy
    • 1
    Email author
  1. 1.John Curtin School of Medical ResearchThe Australian National UniversityCanberraAustralia
  2. 2.Department of Anatomical PathologyThe Canberra HospitalGarranAustralia

Personalised recommendations