Functional characterization of the promoter of the X-linked ectodermal dysplasia gene

Anhidrotic ectodermal dysplasia (EDA) is a disorder characterized by poor d evelopment of hair, teeth, and sweat glands, and results from lesions in th e X-linked EDA gene. We have cloned a 1,6-kilobase 5'-flanking region of th e human EDA gene and used it to analyze features of transcriptional regulat ion. Primer extension analysis located a single transcription initiation si te 264 base pairs (bp) upstream of the translation start site. When the int act cloned fragment or truncated derivatives were placed upstream of a repo rter luciferase gene and transfected into a series of cultured cells, expre ssion comparable with that conferred by an SV40 promoter-enhancer was obser ved. The region lacks a TATA box sequence, and basal transcription from the unique start site is dependent on two binding sites for the Spl transcript ion factor. One site lies 38 bp 5' to the transcription start site, in a 71 -bp sequence that is sufficient to support up to 35% of maximal transcripti on. The functional importance of the Sp1 sites was demonstrated when cotran sfection of an Sp1expression vector transactivated the EDA promoter in the SL2 Drosophila cell line that otherwise lacks endogenous Sp1. Also, both Sp 1 binding sites were active in footprinting and gel shift assays in the pre sence of either crude HeLa cell nuclear extract or purified Sp1 and lost ac tivity when the binding sites were mutated. A second region involved in pos itive control was localized to a 40-bp sequence between -673 and -633 bp, T his region activated an SV40 minimal promoter 4- to 5-fold in an orientatio n-independent manner and is thus inferred to contain an enhancer region.