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D. C. Otteson, L. Pillai, K. Zhu; Msx1 and Msx2 are Repressors of the Mouse EphA3 Promoter in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4424. doi: https://doi.org/.
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Ephrin (Eph) receptors are expressed in nasal/temporal gradients across the developing retina and play a key role in establishing positional information in the retina. We have cloned the mouse EphA3 and identified consensus binding sites for the homeobox containing transcription factors Msx1 and Msx2. The purpose of this study is to determine whether Msx1 and/or Msx2 can regulate EphA3 promoter activity in vitro.
Rapid Amplification of cDNA Ends (GeneRacer; Invitrogen) used total RNA from E10-12 mouse. Full length cDNAs for mouse Msx1 and human Msx2 (OpenBiosystems) were subcloned into pcDNA3.1/V5-His-TOPO. Mouse EphA3 promoter fragments, extending -256, -376 and -573 bp upstream of the transcription start site, were subcloned in pGL2basic. After sequence verification, plasmids were cotransfected into QNR/D retinal cells using LipofectamineLTX. Promoter activity in total cell lysates, assayed using luciferase assays (Promega), was normalized to the mean luminance of pcDNA3.1 empty-vector co-transfected controls. Statistical anaysis used 2-tailed T-tests, with the Bonferroni correction for multiple comparisons.
The major transcription start site for mouse EphA3 was located 77 bp upstream of the first in-frame ATG and had a consensus initator sequence (PyPyAN(T/A)PyPy). In co-transfections with the three EphA3 promoter-luciferase reporter constructs, Msx1 and Msx2 each resulted in statistically significant reductions in promoter activity compared to pcDNA3.1 controls. Msx1 reduced luciferase expression by the -256, -376 and -573 bp EphA3 promoter constructs by 26% (p=0.006), 55% (p<0.001) and 56% (p=0.014) respectively. Msx2 showed stronger repressor activity in this assay, reducing luciferase expression by the -256, -376 and -573 bp EphA3 promoter constructs by 61% (p<.001) 79% (p<0.001) and 84% (p=0.002) respectively.
Msx1 and Msx2 function as negative regulators of the mouse EphA3 promoter in vitro. In the developing retina, Msx1 and Msx2 expression have reported in areas where EphA3 expression is low: the nasal half of the retinal neuroepithelium and the peripheral rim of the optic cup. This suggests a potential role for Msx1 and/or Msx2 in repressing repressing inappropriate EphA3 expression in the nasal retina and prospective ciliary body during retinal development. Additional homeobox transcription factors recognize the same core consensus binding site (TAATT), therefore further studies will address the role of additional homobox genes in regulating EphA3 promoter activity.
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