Analysis of mouse keratin 6a regulatory sequences in transgenic mice reveals constitutive, tissue-specific expression by a keratin 6a minigene.
The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta-galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.
Mahony
D
D
Department of Biochemistry and the Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.
Karunaratne
S
S
Cam
G
G
Rothnagel
J A
JA
D000818:Animals; D002478:Cells, Cultured; D019085:Fluorescent Antibody Technique, Direct; D015870:Gene Expression; D015603:Keratinocytes / Q000378:metabolism; D007633:Keratins / Q000235:genetics*; D007763:Lac Operon / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D014018:Tissue Distribution; D019076:Transgenes / Q000502:physiology
D007633:Keratins