Identification of an Enhancer Critical for the ephirn-A5 Gene Expression in the Posterior Region of the Mesencephalon
Eunjeong Park, Hyuna Noh, and Soochul Park*
Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, Korea
*Correspondence: scpark@sookmyung.ac.kr
Received March 29, 2017; Revised May 1, 2017; Accepted May 11, 2017.; Published online June 13, 2017.
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ABSTRACT
Ephrin-A5 has been implicated in the regulation of brain morphogenesis and axon pathfinding. In this study, we used bacterial homologous recombination to express a LacZ reporter in various ephrin-A5 BAC clones to identify elements that regulate ephrin-A5 gene expression during mesencephalon development. We found that there is mesencephalonspecific enhancer activity localized to a specific +25.0 kb to +30.5 kb genomic region in the first intron of ephrin-A5. Further comparative genomic analysis indicated that two evolutionary conserved regions, ECR1 and ECR2, were present within this 5.5 kb region. Deletion of ECR1 from the enhancer resulted in disrupted mesencephalon-specific enhancer activity in transgenic embryos. We also found a consensus binding site for basic helix-loop-helix (bHLH) transcription factors (TFs) in a highly conserved region at the 3'-end of ECR1. We further demonstrated that specific deletion of the bHLH TF binding site abrogated the mesencephalon-specific enhancer activity in transgenic embryos. Finally, both electrophoretic mobility shift assay and luciferase-based transactivation assays revealed that the transcription factor Ascl1 bound the bHLH consensus binding site in the mesencephalonspecific ephrin-A5 enhancer in vitro. Together, these results suggest that the bHLH TF binding site in ECR1 is involved in the positive regulation of ephrin-A5 gene expression during the development of the mesencephalon.
Keywords: bHLH transcription factor, EphA, ephrin-A5, mesencephalon


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31 May 2017 Volume 40,
Number 5, pp. 315~378

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