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 electrophoretic 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.