Promoter analysis in the human SPRR gene family.
Protocols to study the regulation of a conserved multigene family (SPRR genes) during calcium-induced differentiation of cultured normal human keratinocytes (NHKs) are provided. Transfection of promoter-reporter (CAT or luciferase) constructs, combined with promoter truncation, can be used to study the expression of individual SPRR genes and to identify specific transcription factor binding sites. Interaction of regulatory factors with these control elements can be visualized and quantified by electro phoretic mobility shift analysis. Inclusion of specific antibodies in these experiments will identify the transcription factors involved in the observed mobility shift (supershift). A competitive electrophoretic mobility shift analysis, that is well suited to study the differential regulation of various SPRR members, is also described. These methods should be applicable to the study of other multigene families regulated during keratinocyte terminal differentiation.
Fischer
David F
DF
Netherlands Institute for Brain Research, Amsterdam, The Netherlands.
Backendorf
Claude
C
D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014157:Transcription Factors