Gene Expression of the Mouse Corneal Crystallin Aldh3a1: Activation by Pax6, Oct1, and p300

• Published in: Investigative ophthalmology & visual science 2008-05, Vol.49 (5), p.1814-1826
• Main Authors: Davis, Janine, Davis, Dionne, Norman, Barbara, Piatigorsky, Joram
• Format: Article
• Language: English
• Subjects: Aldehyde Dehydrogenase - genetics; Animals; Blotting, Western; Cercopithecus aethiops; Cornea - growth & development; Cornea - metabolism; COS Cells; Crystallins - genetics; E1A-Associated p300 Protein - physiology; Electrophoretic Mobility Shift Assay; Eye Proteins - physiology; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Developmental - physiology; Homeodomain Proteins - physiology; In Situ Hybridization; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Organic Cation Transporter 1 - physiology; Paired Box Transcription Factors - physiology; PAX6 Transcription Factor; Polymerase Chain Reaction; Repressor Proteins - physiology; RNA, Messenger - metabolism; Transcriptional Activation; Transfection
• ISSN: 0146-0404; 1552-5783; 1552-5783
• DOI: 10.1167/iovs.07-1057

Aldehyde dehydrogenase 3a1 (Aldh3a1) represents approximately 50% of the water-soluble protein of the mouse corneal epithelial cells and thus, by analogy with the abundant lens crystallins, is considered a corneal crystallin. This study was conducted to examine the developmental pattern and transcriptional activation of Aldh3a1 gene expression in the mouse cornea. Aldh3a1 mRNA and protein were analyzed by quantitative (Q)-PCR and Western immunoblot analysis. Functional promoter analysis was examined by cotransfecting plasmids containing variable portions of the Aldh3a1 promoter fused to the luciferase reporter gene into COS-7 cells with selected transcription factors. Transcription factor binding sites were identified by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP). In situ hybridization and immunohistochemistry were used to assess expression of Aldh3a1, Pax6, and Oct1 in the cornea. Aldh3a1 expression is temporally regulated in the cornea beginning at birth and increasing 100-fold by 6 weeks of age. Pax6, Oct1, and p300 synergistically activate the Aldh3a1 promoter approximately 116-fold. One Pax6 and two Oct1 binding sites were identified in vitro and in vivo in the Aldh3a1 promoter fragment analyzed. Pax6 and Oct1 are both present in the nuclei of corneal epithelial cells of the 6-week-old mouse. Finally, a reduction of Aldh3a1 correlated with reduced Pax6 in the corneas of heterozygous Small eye Pax6(+/-) mice. Pax6, Oct1, and p300 activate gene expression of the corneal crystallin Aldh3a1 in the mouse. These transcription factors are also implicated in the high expression of crystallin genes in the lens, consistent with the "refracton hypothesis" unifying many aspects of the lens and cornea.