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Comparative Genomics-Based Identification and Analysis of Cis-Regulatory Elements

  • Hajime OginoEmail author
  • Haruki Ochi
  • Chihiro Uchiyama
  • Sarah Louie
  • Robert M. Grainger
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 917)

Abstract

Identification of cis-regulatory elements, such as enhancers and promoters, is very important not only for analysis of gene regulatory networks but also as a tool for targeted gene expression experiments. In this chapter, we introduce an easy but reliable approach to predict enhancers of a gene of interest by comparing mammalian and Xenopus genome sequences, and to examine their activity using a co-transgenesis technique in Xenopus embryos. Since the bioinformatics analysis utilizes publically available web tools, bench biologists can easily perform it without any need for special computing capability. The co-transgenesis assay, which directly uses polymerase chain reaction products, quickly screens for the activity of the candidate elements in a cloning-free manner.

Key words

Cis-regulatory elements Enhancer Comparative genomics Multiple alignment Phylogenetic footprinting Transcription factor-binding motif Xenopus transgenesis 

Notes

Acknowledgements

This work was supported by the Global COE Program in NAIST (Frontier Biosciences: strategies for survival and adaptation in a changing global environment), Grant-in-Aid for Scientific Research (C) (20579002 and 23570256) from JSPS and Grant-in-Aid for Scientific Research on Innovative Areas (21200064) from MEXT, Japan, to H. Ogino, by Grant-in-Aid for Young Scientists (B) (21770234) from JSPS and Research for Promoting Technological Seeds (A) (10-099) from JST, Japan, to H. Ochi, and by CREST (JST). This work was also supported by NIH grants EY00675, EY017400, EY018000, and RR013221 to R. Grainger.

References

  1. 1.
    Wasserman WW, Sandelin A (2004) Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet 5:276–287PubMedCrossRefGoogle Scholar
  2. 2.
    Pennacchio LA et al (2006) In vivo enhancer analysis of human conserved non-coding sequences. Nature 444:499–502PubMedCrossRefGoogle Scholar
  3. 3.
    Woolfe A et al (2005) Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol 3:e7PubMedCrossRefGoogle Scholar
  4. 4.
    Lettice LA et al (2003) A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet 12:1725–1735PubMedCrossRefGoogle Scholar
  5. 5.
    Sagai T et al (2004) Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog (Shh). Mamm Genome 15:23–34PubMedCrossRefGoogle Scholar
  6. 6.
    Benko S et al (2009) Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence. Nat Genet 41:359–364PubMedCrossRefGoogle Scholar
  7. 7.
    Fisher S et al (2006) Conservation of RET regulatory function from human to zebrafish without sequence similarity. Science 312:276–279PubMedCrossRefGoogle Scholar
  8. 8.
    Visel A et al (2009) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457:854–858PubMedCrossRefGoogle Scholar
  9. 9.
    Hellsten U et al (2010) The genome of the Western clawed frog Xenopus tropicalis. Science 328:633–636PubMedCrossRefGoogle Scholar
  10. 10.
    Kasahara M et al (2007) The medaka draft genome and insights into vertebrate genome evolution. Nature 447:714–719PubMedCrossRefGoogle Scholar
  11. 11.
    Canestro C, Yokoi H, Postlethwait JH (2007) Evolutionary developmental biology and genomics. Nat Rev Genet 8:932–942PubMedCrossRefGoogle Scholar
  12. 12.
    Ogino H, Fisher M, Grainger RM (2008) Convergence of a head-field selector Otx2 and Notch signaling: a mechanism for lens specification. Development 135:249–258PubMedCrossRefGoogle Scholar
  13. 13.
    Ovcharenko I et al (2004) ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Res 32:W280–W286PubMedCrossRefGoogle Scholar
  14. 14.
    Schwartz S et al (2003) MultiPipMaker and supporting tools: alignments and analysis of multiple genomic DNA sequences. Nucleic Acids Res 31:3518–3524PubMedCrossRefGoogle Scholar
  15. 15.
    Larkin MA et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  16. 16.
    Stathopoulos A, Levine M (2005) Genomic regulatory networks and animal development. Dev Cell 9:449–462PubMedCrossRefGoogle Scholar
  17. 17.
    Loots GG, Ovcharenko I (2004) rVISTA 2.0: evolutionary analysis of transcription factor binding sites. Nucleic Acids Res 32:W217–W221PubMedCrossRefGoogle Scholar
  18. 18.
    Sandelin A, Wasserman WW, Lenhard B (2004) ConSite: web-based prediction of regulatory elements using cross-species comparison. Nucleic Acids Res 32:W249–W252PubMedCrossRefGoogle Scholar
  19. 19.
    Portales-Casamar E et al (2010) JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles. Nucleic Acids Res 38:D105–D110PubMedCrossRefGoogle Scholar
  20. 20.
    Sive H, Grainger R, Harland R (2000) Early development of Xenopus laevis—a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  21. 21.
    Kost TA, Theodorakis N, Hughes SH (1983) The nucleotide sequence of the chick cytoplasmic β-actin gene. Nucleic Acids Res 11:8287–8301PubMedCrossRefGoogle Scholar
  22. 22.
    Navratilova P et al (2009) Systematic human/zebrafish comparative identification of cis-regulatory activity around vertebrate developmental transcription factor genes. Dev Biol 327:526–540PubMedCrossRefGoogle Scholar
  23. 23.
    Kleinjan DA et al (2006) Long-range downstream enhancers are essential for Pax6 expression. Dev Biol 299:563–581PubMedCrossRefGoogle Scholar
  24. 24.
    Xu PX et al (1999) Regulation of Pax6 expression is conserved between mice and flies. Development 126:383–395PubMedGoogle Scholar
  25. 25.
    Waterhouse AM et al (2009) Jalview version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191PubMedCrossRefGoogle Scholar
  26. 26.
    Rada-Iglesias A et al (2011) A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470:279–283PubMedCrossRefGoogle Scholar
  27. 27.
    Noyes MB et al (2008) Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites. Cell 133:1277–1289PubMedCrossRefGoogle Scholar
  28. 28.
    Lehman CW, Trautman JK, Carroll D (1994) Illegitimate recombination in Xenopus: characterization of end-joined junctions. Nucleic Acids Res 22:434–442PubMedCrossRefGoogle Scholar
  29. 29.
    Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  30. 30.
    Ogino H, Ochi H (2009) Resources and transgenesis techniques for functional genomics in Xenopus. Dev Growth Differ 51:387–401PubMedCrossRefGoogle Scholar
  31. 31.
    Ogino H, McConnell WB, Grainger RM (2006) High-throughput transgenesis in Xenopus using I-SceI meganuclease. Nat Protoc 1:1703–1710PubMedCrossRefGoogle Scholar
  32. 32.
    Kammandel B et al (1999) Distinct cis-essential modules direct the time-space pattern of the Pax6 gene activity. Dev Biol 205:79–97PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Hajime Ogino
    • 1
    Email author
  • Haruki Ochi
    • 1
  • Chihiro Uchiyama
    • 1
  • Sarah Louie
    • 2
  • Robert M. Grainger
    • 2
  1. 1.Graduate School of Biological SciencesNara Institute of Science and Technology (NAIST)Takayama, IkomaJapan
  2. 2.Department of BiologyUniversity of VirginiaCharlottesvilleUSA

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