PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes.

Yuzuru Sasamoto,Satoshi Kawasaki,Andrew J Quantock,Kohji Nishida,Yutaka Suzuki,Ryuhei Hayashi,Sung-Joon Park,Mihoko Saito-Adachi,Kenta Nakai,Motokazu Tsujikawa

doi:10.1038/srep20807

Yuzuru Sasamoto, Satoshi Kawasaki + Show 8 more

Open Access

https://doi.org/10.1038/srep20807

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Abstract

PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.

Highlights

PAX6 is a key factor involved in the corneal epithelial phenotype
Co-immunoprecipitation followed by mass spectrometry showed that PAX6 did not form protein-protein complexes with the co-transduced factors OCT4 and KLF4 (Supplementary Table S2). These results suggest that PAX6 isoforms bind to their targets via both the PAI and RED domains without forming a complex with OCT4 and KLF4, and that the region upstream of the keratin 3 (KRT3) gene is a target of PAX6-a transduction
We found that eight transcription factors, including PAX6, contributed to the regulation of 424 PAX6-a- and PAX6-b-dependent differentially up-regulated genes (DUGs) (Supplementary Fig. S3e and Supplementary Table S5), which are not likely to be involved in keratinocyte development and differentiation (Supplementary Fig. S3c)

Summary

Introduction

Single-cell gene expression analysis revealed a positive correlation between both PAX6 isoforms and KRT3 and KRT12 in human limbal epithelial cells in vivo (Fig. 1c,d). We examined the induction of KRT3 expression and found that PAX6-a, PAX6-b, OCT4, and KLF4 transduction into OKF6/TERT-1 cells significantly increased KRT3 expression level (Fig. 2g). The combination of PAX6-b-OCT4-KLF4 (for KRT12) and either PAX6-a or PAX6-a-KLF4 (for KRT3) efficiently induced the corneal epithelium-specific keratin expression in OKF6/TERT-1 cells.

Results

Conclusion