PUBLICATION

Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans

Authors
Shakes, L.A., Du, H., Wolf, H.M., Hatcher, C., Norford, D.C., Precht, P., Sen, R., and Chatterjee, P.K.
ID
ZDB-PUB-120907-2
Date
2012
Source
BMC Genomics   13(1): 451 (Journal)
Registered Authors
Chatterjee, Pradeep K., Shakes, Leighcraft
Keywords
none
MeSH Terms
  • Amyloid beta-Protein Precursor/genetics
  • Amyloid beta-Protein Precursor/metabolism*
  • Animals
  • Basic-Leucine Zipper Transcription Factors/genetics*
  • Binding Sites/genetics
  • Cell Line, Tumor
  • Chromatin Immunoprecipitation
  • Chromosomes, Artificial, Bacterial/genetics
  • DNA, Intergenic/genetics*
  • Forkhead Transcription Factors/metabolism
  • Gene Expression Regulation/genetics*
  • Gene Regulatory Networks/genetics*
  • Gene Transfer Techniques
  • Green Fluorescent Proteins/metabolism
  • Humans
  • Mutagenesis
  • Neurons/metabolism
  • Notochord/metabolism
  • Plasmids/genetics
  • Regulatory Sequences, Nucleic Acid/genetics*
  • Zebrafish
PubMed
22947103 Full text @ BMC Genomics
Abstract

Background

Non-coding DNA in and around the human Amyloid Precursor Protein (APP) gene that is central to Alzheimer's disease (AD) shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28--31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene.

Results

Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at -31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at -31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP) experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription factors.

Conclusion

The results suggest that the clock-regulated and immune system modulator transcription factor E4BP4/ NFIL3 likely regulates the expression of both appb in zebrafish and APP in humans. It suggests potential human APP gene regulatory pathways, not on the basis of comparing DNA primary sequences with zebrafish appb but on the model of conservation of transcription factors.

Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping