Cis-regulatory elements of the mouse Krt1.12 gene

Abstract

Purpose: Keratin 12 is a cornea epithelial cell-specific intermediate filament component. To better understand the regulatory mechanism of its expression, the cis-regulatory elements located between the transcription start site and 600 bp upstream of the Krt1.12 gene were determined.

Methods: The promoter activity of reporter gene constructs containing 0.6, 0.4, and 0.2 kb of DNA 5' upstream of Krt1.12 coupled to the lac Z gene were determined in rabbit corneas using Gene Gun technology. DNA foot printing and EMSA (electrophoresis mobility shift assay) were employed to identify putative cis-regulatory elements of the Krt1.12 gene using bovine corneal epithelial cell nuclear extracts.

Results: Enzyme activity assays and histochemical analysis of beta-galactosidase from the 0.6, 0.4, and 0.2 kb K12 promoter constructs indicated that the DNA elements between -0.2 and -0.6 kb 5' of the Krt1.12 gene contain cis-regulatory elements for its corneal epithelial cell-specific expression. Foot printing and EMSA showed that the sequences between -181 to -111 and -256 to -193 upstream of the Krt1.12 gene reacted to nuclear proteins isolated from bovine corneal epithelial cells. A Genbank search revealed that these two regions were potential binding sites for many transcription factors such as AP1, c/EBP, and KLF6. Immunofluorescent staining indicated the presence of c-jun and c/EBP transcription factors in the nuclei of corneal epithelial cells.

Conclusions: The data is consistent with the notion that the -182 to -111 and -256 to -193 fragments 5' of the Krt1.12 gene may serve as corneal epithelial cell-specific cis-regulatory elements, and the coordinated interactions of various transcription factors are required for cornea-specific expression of Krt1.12 gene.