Conserved and non-conserved enhancers direct tissue specific transcription in ancient germ layer specific developmental control genes

BMC Dev Biol. 2011 Oct 20;11:63. doi: 10.1186/1471-213X-11-63.


Background: Identifying DNA sequences (enhancers) that direct the precise spatial and temporal expression of developmental control genes remains a significant challenge in the annotation of vertebrate genomes. Locating these sequences, which in many cases lie at a great distance from the transcription start site, has been a major obstacle in deciphering gene regulation. Coupling of comparative genomics with functional validation to locate such regulatory elements has been a successful method in locating many such regulatory elements. But most of these studies looked either at a single gene only or the whole genome without focusing on any particular process. The pressing need is to integrate the tools of comparative genomics with knowledge of developmental biology to validate enhancers for developmental transcription factors in greater detail

Results: Our results show that near four different genes (nkx3.2, pax9, otx1b and foxa2) in zebrafish, only 20-30% of highly conserved DNA sequences can act as developmental enhancers irrespective of the tissue the gene expresses in. We find that some genes also have multiple conserved enhancers expressing in the same tissue at the same or different time points in development. We also located non-conserved enhancers for two of the genes (pax9 and otx1b). Our modified Bacterial artificial chromosome (BACs) studies for these 4 genes revealed that many of these enhancers work in a synergistic fashion, which cannot be captured by individual DNA constructs and are not conserved at the sequence level. Our detailed biochemical and transgenic analysis revealed Foxa1 binds to the otx1b non-conserved enhancer to direct its activity in forebrain and otic vesicle of zebrafish at 24 hpf.

Conclusion: Our results clearly indicate that high level of functional conservation of genes is not necessarily associated with sequence conservation of its regulatory elements. Moreover certain non conserved DNA elements might have role in gene regulation. The need is to bring together multiple approaches to bear upon individual genes to decipher all its regulatory elements.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromosomes, Artificial, Bacterial / genetics
  • Conserved Sequence / genetics
  • Enhancer Elements, Genetic / genetics*
  • Gene Expression Regulation, Developmental*
  • Genes, Developmental
  • Genomics / methods
  • Germ Layers / metabolism*
  • Hepatocyte Nuclear Factor 3-alpha / genetics
  • Hepatocyte Nuclear Factor 3-alpha / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Morpholinos / genetics
  • Otx Transcription Factors / genetics
  • Otx Transcription Factors / metabolism
  • PAX9 Transcription Factor / genetics
  • PAX9 Transcription Factor / metabolism
  • Regulatory Sequences, Nucleic Acid / genetics*
  • Sequence Alignment
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcription, Genetic / genetics*
  • Zebrafish / embryology*
  • Zebrafish / genetics*
  • Zebrafish / metabolism
  • Zebrafish Proteins / genetics
  • Zebrafish Proteins / metabolism


  • Hepatocyte Nuclear Factor 3-alpha
  • Homeodomain Proteins
  • Morpholinos
  • Otx Transcription Factors
  • PAX9 Transcription Factor
  • Transcription Factors
  • Zebrafish Proteins
  • nkx3-2 protein, zebrafish
  • otx1 protein, zebrafish
  • pax9 protein, zebrafish