Working with a zebrafish group in North Carolina State University, we previously showed that regions of the zebrafish amyloid precursor protein (APP) gene involved in neuron-specific expression were especially enriched in binding sites for the transcription factors E4BP4 and FOXO. We cloned and expressed recombinant zebrafish E4BP4 and FOX0 DNA binding domains and showed that representative sites in the zebrafish APP gene bound these proteins. We then extended the analysis to the human APP gene to determine whether the genetic data from zebrafish could provide insight into human APP gene expression. During fiscal year 2011, we accomplished the following: 1. Initiated studies with a human neuroblastoma cell SH-SY5 cells. Quantitative RT-PCR demonstrated basal expression of the APP gene in these cells. We are currently differentiating these cells in culture to neurons using retinoic acid. We will determine whether APP gene expression changes upon differentiation. 2. We designed chromatin immunoprecipitation primers from several parts of the genomic locus and used these to screen anti-H3KH9ac immunoprecipitates. We found elevated histone acetylation in the 4th intron of the APP gene that coincides with a region of multiple E4BP4 binding sites. These observations suggest that E4BP4 may play a role in regulating the human APP gene. 3. We are attempting to knock-down expression of endogenous E4BP4 using siRNAs specific for this gene. Once E4BP4 down-regulation is achieved, we will examine the effects on APP gene expression before and after neuronal differentiation.
| 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 |
