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Identification of Regulatory Elements by Gene Family Footprinting and In Vivo Analysis

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Analytics of Protein–DNA Interactions

Abstract

Gene families of recently duplicated but subsequently diverged genes provide an unique opportunity for comparative analysis of regulatory elements. We have studied the human SPRR gene family of small proline rich proteins involved in barrier function of stratified squamous epithelia. These genes are all expressed in normal human keratinocytes, but respond differently to environmental insults. Comparisons of the functional promoter regions allows the rapid identification of both conserved and of novel regulatory elements that appeared after gene duplication. Competitive electrophoretic mobility shift assays can be used to confirm their presence.

Here we show the power of gene family footprinting by the identification of two novel elements in the SPRR3 promoter, not present in SPRR1A and SPRR2A. One of these elements binds a protein similar to GAAP-1, a pro-apoptotic activator of IRF-1 and p53. In vivo analysis shows that this element functions as an inhibitor of SPRR3 transcription. The second novel element functions as an activator of promoter activity and is characterized by its A/T rich sequence. The latter interacting protein indeed binds through contacts in the minor groove, and strikingly, depends on the presence of calcium for DNA interaction.

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Acknowledgments

We would like to thank Drs. R. Mantovani (Milan) and T. Taniguchi (Osaka) for the generous gift of antibodies and plasmids. This work was supported by the J.A. Cohen Institute, and by grants from NWO-SON and the EC.

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Correspondence to Claude Backendorf .

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Harald Seitz

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Fischer, D.F., Backendorf, C. (2006). Identification of Regulatory Elements by Gene Family Footprinting and In Vivo Analysis. In: Seitz, H. (eds) Analytics of Protein–DNA Interactions. Advances in Biochemical Engineering/Biotechnology, vol 104. Springer, Berlin, Heidelberg . https://doi.org/10.1007/10_027

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