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E. Sorokina, S. S. Muheisen, N. A. Mlodik, N. S. Zinkevich, E. V. Semina; Analysis of PITX3 Pathway Identifies FOXE3 as a Potential Direct Downstream Target During Lens Development. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2911.
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PITX3 and FOXE3 are important transcriptional factors that control eye development. Alterations in these genes areimplicated in phenotypes associated with lens and anterior segment malformations in humans, mice and zebrafish. Shi and coauthors have recently demonstrated that foxe3 is acting downstream of pitx3 in zebrafish. However, it is unclear whether foxe3 represents a direct target of pitx3 or what aspects of foxe3 spatiotemporal expression pattern are sensitive to pitx3 dosage.
Expression studies were performed using in situ hybridization; transactivation was evaluated by luciferase reporter assays; DNA-binding was examined by EMSA.
We established overlap in zebrafish pitx3 and foxe3 expression patterns during early stages of lens development by double in situ hybridization. We also observed changes in foxe3 transcript level in pitx3 morphants. Analysis of human and mouse FOXE3/Foxe3 revealed a conservative region containing several bicoid sites ~ 3.7 kb upstream from FOXE3 transcriptional start site. Additional bicoid sites were found in ~600-bp region located immediately downstream of human FOXE3 gene. The luciferase reporter plasmid containing 3.7-kb-promoter of FOXE3 demonstrated more than 10 folds transactivation after co-transfection with PITX3 expression plasmid. Addition of the 3'-flanking sequence to this construct did not change transactivation level. Deletion analysis of the 3.7-kb region identified that a short 209-bp promoter fragment exhibits even higher level of transactivation (~20 folds) when co-transfected with PITX3. However, disruption of the only bicoid-like sequence found in this fragment by site-directed mutagenesis did not change the level of transactivation by PITX3. Therefore we believe that PITX3-mediated activation of FOXE3 promoter is likely to be executed either through its interaction with non-bicoid DNA sequences or by means of its association with other transcription factors or through activation of intermediate factors. Experiments to further delineate role of PITX3 in regulation of FOXE3 expression are currently in progress and involve EMSA and ChIP assays.
PITX3 and FOXE3 transcription factors demonstrate overlapping expression patterns during lens development and appear to be involved in the same pathway(s). Further delineation of their relationship will provide insight into genetic mechanisms of lens development and differentiation.
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