Cracking the genome's second code: Enhancer detection by combined phylogenetic footprinting and transgenic fish and frog embryos

Miguel L. Allende, Miguel Manzanares, Juan J. Tena, Carmen G. Feijóo, José Luis Gómez-Skarmeta

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)

Abstract

Genes involved in vertebrate development are unusually enriched for highly conserved non-coding sequence elements. These regions are readily detected in silico, by genome-wide sequence comparisons between different vertebrates, from mammals to fish (phylogenetic footprinting). It follows that sequence conservation must be the result of positive selection for an essential physiological role. An obvious possibility is that these conserved sequences possess regulatory or structural functions important for gene expression and, thus, an in vivo assay becomes necessary. We have developed a rapid testing system using zebrafish and Xenopus laevis embryos that allows us to assign transcriptional regulatory functions to conserved non-coding sequence elements. The sequences are cloned into a vector containing a minimal promoter and the GFP reporter, and are assayed for their putative cis-regulatory activity in zebrafish or Xenopus transgenic experiments. Vectors used include plasmid DNA and the Tol2 transposon system in fish and X. laevis. We have followed this logic to detect and analyze conserved elements in an intergenic region present in the Iroquois (Irx) gene clusters of zebrafish, Xenopus tropicalis, Fugu rubripes and mouse. We have assayed ∼50 of these conserved elements and shown that the majority behave as modular positive regulatory elements (enhancers) that contribute to specific temporal and spatial domains that are part of the endogenous gene expression pattern. Moreover, comparison of the activity of cognate Irx enhancers from different organisms demonstrates that conservation of sequence is accompanied by in vivo functional conservation across species. Finally, for some of the most conserved elements, we have been able to identify a critical core sequence, essential for correct enhancer function.

Original languageEnglish
Pages (from-to)212-219
Number of pages8
JournalMethods
Volume39
Issue number3
DOIs
Publication statusPublished - Jul 2006

Keywords

  • Enhancer
  • Iroquois
  • Regulatory sequence
  • Tol2
  • Transgenic

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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