DOI QR코드

DOI QR Code

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

  • Park, Eunjeong (Department of Biological Sciences, Sookmyung Women's University) ;
  • Noh, Hyuna (Department of Biological Sciences, Sookmyung Women's University) ;
  • Park, Soochul (Department of Biological Sciences, Sookmyung Women's University)
  • Received : 2017.03.29
  • Accepted : 2017.05.11
  • Published : 2017.06.30

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 mesencephalon-specific 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 assay revealed that the transcription factor Ascl1 bound the bHLH consensus binding site in the mesencephalon-specific 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

Acknowledgement

Supported by : National Research Foundation of Korea (NRF)

References

  1. Carreres, M.I., Escalante, A., Murillo, B., Chauvin, G., Gaspar, P., Vegar, C., and Herrera, E. (2011). Transcription factor Foxd1 is required for the specification of the temporal retina in mammals. J. Neurosci. 31, 5673-5681. https://doi.org/10.1523/JNEUROSCI.0394-11.2011
  2. Casarosa, S., Fode, C., and Guillemot, F. (1999). Mash1 regulates neurogenesis in the ventral telencephalon. Development 126, 525-534.
  3. Castro, D.S., Skowronska-Krawczyk, D., Armant, O., Donaldson, I.J., Parras, C., Hunt, C., Critchley, J.A., Nguyen, L., Gossler, A., Gottgens, B., et al. (2006). Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif. Dev. Cell 11, 831-844. https://doi.org/10.1016/j.devcel.2006.10.006
  4. Castro, D.S., Martynoga, B., Parras, C., Ramesh, V., Pacary, E., Johnston, C., Drechsel, D., Lebel-Potter, M., Garcia, L.G., Hunt, C., et al. (2011). A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets. Genes Dev. 25, 930-945. https://doi.org/10.1101/gad.627811
  5. Coulthard, M.G., Duffy, S., Down, M., Evans, B., Power, M., Smith, F., Stylianou, C., Kleikamp, S., Oates, A., Lackmann, M., et al. (2002). The role of the Eph-ephrin signalling system in the regulation of developmental patterning. Int. J. Dev. Biol. 46, 375-384.
  6. Depaepe, V., Suarez-Gonzalez, N., Dufour, A., Passante, L., Gorski, J.A., Jones, K.R., Ledent, C., and Vanderhaeghen, P. (2005). Ephrin signalling controls brain size by regulating apoptosis of neural progenitors. Nature 435, 1244-1250. https://doi.org/10.1038/nature03651
  7. Flanagan, J.G. (2006). Neural map specification by gradients. Curr. Opin. Neurobiol. 16, 59-66. https://doi.org/10.1016/j.conb.2006.01.010
  8. Flanagan, J.G., and Vanderhaeghen, P. (1998). The ephrins and Eph receptors in neural development. Annu. Rev. Neurosci. 21, 309-345. https://doi.org/10.1146/annurev.neuro.21.1.309
  9. Fulco, C.P., Munschauer, M., Anyoha, R., Munson, G., Grossman, S.R., Perez, E.M., Kane, M., Cleary, B., Lander, E.S., and Engreitz, J.M. (2016). Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769-773. https://doi.org/10.1126/science.aag2445
  10. Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J.A., et al. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442-451. https://doi.org/10.1016/j.cell.2013.06.044
  11. Holmberg, J., Clarke, D.L., and Frisen, J. (2000). Regulation of repulsion versus adhesion by different splice forms of an Eph receptor. Nature 408, 203-206. https://doi.org/10.1038/35041577
  12. Kim, Y., Song, E., Choi, S., and Park, S. (2007). Engineering lacZ Reporter gene into an ephA8 bacterial artificial chromosome using a highly efficient bacterial recombination system. J. Biochem. Mol. Biol. 40, 656-661.
  13. Klein, R. (2012). Eph/ephrin signalling during development. Development 139, 4105-4109. https://doi.org/10.1242/dev.074997
  14. Koshiba-Takeuchi, K., Takeuchi, J.K., Matsumoto, K., Momose, T., Uno, K., Hoepker, V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K., et al. (2000). Tbx5 and the retinotectum projection. Science 287, 134-137. https://doi.org/10.1126/science.287.5450.134
  15. Kullander, K., and Klein, R. (2002). Mechanisms and functions of Eph and ephrin signalling. Nat. Rev. Mol. Cell Biol. 3, 475-486. https://doi.org/10.1038/nrm856
  16. Lopes, R., Korkmaz, G., and Agami, R. (2016). Applying CRISPR-Cas9 tools to identify and characterize transcriptional enhancers. Nat. Rev. Mol. Cell Biol. 17, 597-604. https://doi.org/10.1038/nrm.2016.79
  17. Mui, S.H., Hindges, R., O'Leary, D.D., Lemke, G., and Bertuzzi, S. (2002). The homeodomain protein Vax2 patterns the dorsoventral and nasotemporal axes of the eye. Development 129, 797-804.
  18. Noh, H., Lee, H., Park, E., and Park, S. (2016). Proper closure of the optic fissure requires ephrin A5-EphB2-JNK signaling. Development 143, 461-472. https://doi.org/10.1242/dev.129478
  19. O'Leary, D.D., and Wilkinson, D.G. (1999). Eph receptors and ephrins in neural development. Curr. Opin. Neurobiol. 9, 65-73. https://doi.org/10.1016/S0959-4388(99)80008-7
  20. Park, S. (2013). Brain-region specific apoptosis triggered by Eph/ephrin signaling. Exp. Neurobiol. 22, 143-148. https://doi.org/10.5607/en.2013.22.3.143
  21. Park, E., Kim, Y., Noh, H., Lee, H., Yoo, S., and Park, S. (2013). EphA/ephrin-A signaling is critically involved in region-specific apoptosis during early brain development. Cell Death Differ. 20, 169-180. https://doi.org/10.1038/cdd.2012.121
  22. Pasquale, E.B. (2005). Eph receptor signalling casts a wide net on cell behaviour. Nat. Rev. Mol. Cell Biol. 6, 462-475. https://doi.org/10.1038/nrm1662
  23. Polleux, F., Ince-Dunn, G., and Ghosh, A. (2007). Transcriptional regulation of vertebrate axon guidance and synapse formation. Nat. Rev. Neurosci. 8, 331-340.
  24. Schulte, D., Furukawa, T., Peters, M.A., Kozak, C.A., and Cepko, C.L. (1999). Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map. Neuron 24, 541-553. https://doi.org/10.1016/S0896-6273(00)81111-3
  25. Takahashi, H., Shintani, T., Sakuta, H., and Noda, M. (2003). CBF1 controls the retinotectal topographical map along the anteroposterior axis through multiple mechanisms. Development 130, 5203-5215. https://doi.org/10.1242/dev.00724

Cited by

  1. Abnormal expression of ephrin-A5 affects brain development of congenital hypothyroidism rats vol.29, pp.11, 2018, https://doi.org/10.1097/WNR.0000000000001047