Abstract
Purpose::
Basonuclin (Bnc1) is a novel type of transcription regulator, which can regulate both RNA polymerase I and II transcription. To understand Bnc1 function in corneal epithelium, we characterized Bnc1-null mouse corneal epithelium.
Methods::
All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Corneas of adult Bnc1 (-/-) mice and their wild-type littermates were analyzed. RNA and proteins were extracted from corneal epithelium and used for RT-PCR, quantitative PCR, and immunoblotting. Entire eyes were isolated for either paraffin- or cryo-sections, which were used for immunohistochemistry.
Results::
Immunohistochemistry showed that in Bnc1 (-/-) mice, Bnc1 was absent in corneal epithelium, confirming that the gene was knocked out. Compared with that of Bnc1 (+/+), Bnc1 (-/-) corneal epithelium was thinner, with a 17% reduction in cell number (n=6, P<0.05). The thinner epithelium in Bnc1(-/-) cornea was likely due to a reduction of proliferation, because the average number of Ki67-positive epithelial cells per section was reduced from 181±17 in Bnc1 (+/+) cornea to 135±25 in Bnc1 (-/-) (n=6, P<0.05). Consistent with the notion that Bnc1 regulates Pol I transcription of subsets of rDNA transcription units, RT-PCR analysis showed that Bnc1 null-mutation affected transcript level of some rDNA subtypes but not others. Pol II-transcribed Bnc1 target genes were also altered by its null mutation in corneal epithelium. Quantitative PCR, immunofluorescence or immunoblotting showed alteration of expression levels of Gli2, a DNA-binding protein/transcription factor, and connexin 43 and E-cadherin, which are cell-cell junction proteins. On the other hand, the amount of desmoglein 1, whose gene promoter was shown to interact with Bnc1 in epidermal keratinocytes, was not affected by Bnc1 null mutation in cornea epithelium.
Conclusions::
Bnc1 null mutation leads to morphological and molecular alterations in adult corneal epithelium. Our data support the notion that basonuclin regulates both Pol I and Pol II transcription. Bnc1 is unique among Pol I regulators, because its null mutation affects subsets of rDNA transcription. We conclude that basonuclin is a novel type of transcription regulator, which plays a key role in corneal epithelium development and maintenance.
Keywords: cornea: epithelium • transcription factors • transgenics/knock-outs