Cis-regulatory elements of the mouse Krt1.12 gene.
OBJECTIVE
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.
Wang
I-Jong
IJ
Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA. Winston.Kao@uc.edu
Carlson
Eric C
EC
Liu
Chia-Yang
CY
Kao
Candace W C
CW
Hu
Fung-Rong
FR
Kao
Winston W Y
WW
D000818:Animals; D019470:Biolistics; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002417:Cattle; D004247:DNA / Q000032:analysis*; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D019573:Epithelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D007633:Keratins / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon; D051379:Mice; D016133:Polymerase Chain Reaction; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; C497250:Ddit3 protein, mouse; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D051742:Transcription Factor CHOP; D007633:Keratins; D004247:DNA; D001616:beta-Galactosidase