An Autoregulatory Loop Directs the Tissue-Specific Expression of p63 through a Long-Range Evolutionarily Conserved Enhancer

Abstract

p63, a p53 family member, is essential for the development of various stratified epithelia and is one of the earliest markers of many ectodermal structures, including the epidermis, oral mucosa, apical ectodermal ridge, and mammary gland. Genetic regulatory mechanisms controlling p63 spatial expression during development have not yet been defined. Using a genomic approach, we identified an evolutionarily conserved cis-regulatory element, located 160 kb downstream of the first p63 exon, which functions as a keratinocyte-specific enhancer and is sufficient to recapitulate expression of the endogenous gene during mouse embryogenesis. Dissection of the p63 enhancer activity revealed a positive autoregulatory loop in which the p63 proteins directly bind to and are essential regulators of the enhancer. Accordingly, transactivating p63 isoforms induce endogenous p63 expression in cells that do not normally express this gene, whereas dominant negative isoforms suppress p63 expression in keratinocytes. In addition the transcription factor AP-2 also binds to the enhancer and cooperates with p63 to induce its activity. These results demonstrate that a long-range autoregulatory loop is involved in the regulation of p63 expression during embryonic development and in adult cells.

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We thank Andrea Ballabio, Graciana Diez-Roux, and Domenico Salvatore for their critical reading of the manuscript and Stefano Piccolo for helpful discussion. We are grateful to Ronald J. Weigel for CMV AP-2α and -γ constructs. We are indebted to Jean Gilder (Scientific Communication) for text editing.

T.D.P. is the recipient of an AIRC (Associazione Italiana per la Ricerca sul Cancro) fellowship. This work was supported by grants from the Italian Telethon Foundation (TCMP14TELB), from the Ministry of Instruction, University and Research (MIUR FIRB), from the European Union (LSHG-CT-2004-511990) to C.M., from AIRC to M.Z., and from the NIH (AR45284) to J.L.B. J.L.B. was also funded by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. agreement.