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

Leighcraft A. Shakes, Hansen Du, Hope M. Wolf, Charles Hatcher, Derek C. Norford, Patricia Precht, Ranjan Sen, Pradeep K. Chatterjee

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number451
JournalBMC Genomics
Volume13
Issue number1
DOIs
StatePublished - Sep 4 2012
Externally publishedYes

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Gene Transfer Techniques
Amyloid beta-Protein Precursor
Zebrafish
Transcription Factors
Introns
Genes
Regulator Genes
DNA
Zebrafish Proteins
Binding Sites
Gene Expression
Neurons
Chromatin Immunoprecipitation
Nucleic Acid Regulatory Sequences
Neuroblastoma
Transgenes
Chromatin
Immune System
Alzheimer Disease
Cell Line

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Shakes, L. A., Du, H., Wolf, H. M., Hatcher, C., Norford, D. C., Precht, P., ... Chatterjee, P. K. (2012). Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans. BMC Genomics, 13(1), [451]. https://doi.org/10.1186/1471-2164-13-451

Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans. / Shakes, Leighcraft A.; Du, Hansen; Wolf, Hope M.; Hatcher, Charles; Norford, Derek C.; Precht, Patricia; Sen, Ranjan; Chatterjee, Pradeep K.

In: BMC Genomics, Vol. 13, No. 1, 451, 04.09.2012.

Research output: Contribution to journalArticle

Shakes, LA, Du, H, Wolf, HM, Hatcher, C, Norford, DC, Precht, P, Sen, R & Chatterjee, PK 2012, 'Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans', BMC Genomics, vol. 13, no. 1, 451. https://doi.org/10.1186/1471-2164-13-451
Shakes, Leighcraft A. ; Du, Hansen ; Wolf, Hope M. ; Hatcher, Charles ; Norford, Derek C. ; Precht, Patricia ; Sen, Ranjan ; Chatterjee, Pradeep K. / Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans. In: BMC Genomics. 2012 ; Vol. 13, No. 1.
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AU - Precht, Patricia

AU - Sen, Ranjan

AU - Chatterjee, Pradeep K.

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N2 - 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.

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