During the 2010 fiscal year we established a collaboration with Dr. Pradeep Chatterjee (North Carolina Central University) to initiate this project. Dr. Chatterjee is interested in the methodology of transposon-tagging bacterial artificial chromosomes (BACs) to introduce specific mutations. The locus he has been mutating by this technology is the APP gene in zebrafish. The modified BACs are introduced into zebrafish embryos by microinjection and gene expression followed by GFP fluorescence. Since initiating the collaboration we have accomplished the following: 1) We identified two transcription factors whose binding sites are present in the cis-regulatory sequences identified by Dr. Chatterjee. 2) We cloned, over-expressed and purified the DNA binding domains of the zebrafish transcription factors predicted to bind to these sequence elements. Both transcription factors have easily identifiable mammalian orthologs. 3) We carried out electrophoretic mobility shift assays to demonstrate direct binding of these proteins to APP gene regulatory elements. While site-specific mutagenesis of the zebrafish sequence is ongoing in our collaborator's laboratory, we plan to switch directly to mammalian cells to determine if the mammalian orthologs interact with the murine or human APP locus using chromatin immunoprecipitation. Interestingly, one of the two transcription factors identified in this study is under circadian control. This may have implications in understanding the basis for regulation of Aβdeposition by the sleep-wake cycle.
| Shakes, Leighcraft A; Du, Hansen; Wolf, Hope M et al. (2012) Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans. BMC Genomics 13:451 |
