Identification of an Enhancer Critical for the ephirn-A5 Gene Expression in the Posterior Region of the Mesencephalon

• Published in: Molecules and cells Vol. 40; no. 6; pp. 426 - 433
• Main Authors: Park, Eunjeong, Noh, Hyuna, Park, Soochul
• Format: Journal Article
• Language: English
• Published: United States Korean Society for Molecular and Cellular Biology 01.06.2017; 한국분자세포생물학회
• Subjects: Animals; Base Sequence; Basic Helix-Loop-Helix Transcription Factors - genetics; Basic Helix-Loop-Helix Transcription Factors - metabolism; beta-Galactosidase - genetics; Binding Sites; Conserved Sequence - genetics; Electrophoretic Mobility Shift Assay; Enhancer Elements, Genetic; Ephrin-A5 - genetics; Gene Expression Regulation, Developmental; Genes, Reporter; HEK293 Cells; Humans; Introns; Mesencephalon - embryology; Mesencephalon - metabolism; Mice; Mice, Inbred C57BL; Mice, Transgenic; 생물학; ephrin-A5; EphA; bHLH transcription factor; mesencephalon
• ISSN: 1016-8478; 0219-1032
• DOI: 10.14348/molcells.2017.0052

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 BAC clones to identify elements that regulate gene expression during mesencephalon development. We found that there is mesencephalon-specific enhancer activity localized to a specific +25.0 kb to +30.5 kb genomic region in the first intron of . 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 assay revealed that the transcription factor Ascl1 bound the bHLH consensus binding site in the mesencephalon-specific enhancer . Together, these results suggest that the bHLH TF binding site in ECR1 is involved in the positive regulation of gene expression during the development of the mesencephalon.