RESEARCH ARTICLE Molecular Reproduction & Development 79:337–345 (2012) Cloning the Uteroglobin Gene Promoter From the Relic Volcano Rabbit (Romerolagus diazi) Reveals an Ancient Estrogen-Response Element ADRIANA ACOSTA-MONTESDEOCA, 1 TERESA ZARIN ˜ A ´ N, 1 HE ´ CTOR MACI ´ AS, 1 MARCO A. PE ´ REZ-SOLI ´ S, 1 ALFREDO ULLOA-AGUIRRE, 2 AND RUBE ´ N GUTIE ´ RREZ-SAGAL 1 * 1 Research Unit in Reproductive Medicine, Hospital de Gineco Obstetricia, No. 4 ‘‘Dr. Luis Castelazo Ayala’’, Instituto Mexicano del Seguro Social, Tizap an San Angel, M exico, D.F., Mexico 2 Division of Reproductive Health, Research Center in Population Health, National Institute of Public Health, M exico, D.F., Mexico SUMMARY To gain further insight on the estrogen-dependent transcriptional regulation of the uteroglobin (UG) gene, we cloned the 5 0 -flanking region of the UG gene from the phylogenetically ancient volcano rabbit (Romerolagus diazi; Rd). The cloned region spans 812 base pairs (bp; 812/1) and contains a noncanonical TATA box (TACA). The translation start site is 48 bp downstream from the putative transcription initiation site (AGA), and is preceded by a consensus Kozak box. Comparison of the Rd-UG gene with that previously isolated from rabbits (Oryctolagus cuniculus) showed 93% in sequence identity as well as a number of conserved cis-acting elements, including the estrogen-response element (ERE; 265/251), which differs from the consensus by two nucleotides. In MCF-7 cells, 17b-estradiol (E 2 ) induced transcription of a lucifer- ase reporter driven by the Rd-UG promoter in a similar manner as in an equivalent rabbit UG reporter; the Rd-UG promoter was 30% more responsive to E 2 than the rabbit promoter. Mutagenesis studies on the Rd-ERE confirmed this cis-element as a target of E 2 as two luciferase mutant reporters of the Rd-promoter, one with the rabbit and the other with the consensus ERE, were more responsive to the hormone than the wild-type reporter. Gel shift and super-shift assays showed that estrogen receptor-a indeed binds to the imperfect palindromic sequence of the Rd-ERE. Mol. Reprod. Dev. 79: 337–345, 2012. ß 2012 Wiley Periodicals, Inc. Received 4 November 2011; Accepted 2 February 2012 * Corresponding author: Research Unit in Reproductive Medicine Instituto Mexicano del Seguro Social (IMSS) Apartado Postal 99-065 Unidad Independencia C.P. 10101, M exico, D.F., Mexico. E-mail: ruben.gutierrezs@imss.gob.mx Grant sponsor: Consejo Nacional de Ciencia y Tecnolog  ıa (CONACyT, M exico); Grant number: 57397; Grant sponsor: Fondo de Investigaci on en Salud, Instituto Mexicano del Seguro Social; Grant numbers: FIS/IMSS/ PROT/038, FIS/IMSS/PROT/525 Published online 14 February 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22030 INTRODUCTION Uteroglobin (UG), also known as Clara Cell 10-kDa Protein (CC10) or Clara Cell Secretory Protein (CCSP), is a homodimeric secretory protein of low molecular weight (16 kDa) discovered in the uterine secretion of rabbits at early pregnancy (Krishnan and Daniel, 1967; Beier, 1968). Although the actual physiological function of UG is not completely clear, it is believed that this protein is involved in blastocysts development and implantation (Krishnan and Abbreviations: E 2 , 17b-estradiol; ER, estrogen receptor; ERE, estrogen- response element; UG, uteroglobin; Rd, Romerolagus diazi. ß 2012 WILEY PERIODICALS, INC.