Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins.

Cloning, Characterization, and Steroid-Dependent Posttranscriptional Processing of RUSH-la and p, Two Uteroglobin Promoter-Binding Proteins Amanda Hayward-Lester*, Aveline Hewetson*, Elmus G. Beale, Peter J. Oefner, Peter A. Doris, and Beverly S. Chilton Department of Cell Biology-_ & Biochemistry _ (A.H-L., PAD., B.S.C.) Texas Tech University Health Sciences Center Lubbock, Texas 79430 Department of Biochemistry Stanford University Stanford, California 94305 A.H., (P.J.O.), We previously used gel shift assays, Southwestern blots, and UV cross-linking to identify four proteins that bind to the 203-bp 5’-flanking region (-194/ +9) of the rabbit uteroglobin gene. Here we report cloning, by recognition site screening, the cDNAs for two of the uteroglobin promoter-binding proteins (95 kDa and 113 kDa). Their presumptive nucleotide-binding motifs share 61% identity with the SWl2YSNF2 helicase superfamily, and each protein has the novel C,HC, (RING) zinc-finger signature near its C terminus. RUSH-l (Y,the 113-kDa protein, is the rabbit homolog of human HIP116, a protein that binds to the human immunodeficiency virus-l promoter. RUSH-16 is a 95kDa truncated version of RUSH-1 (Ythat results from alternative splicing of a 57-bp exon as confirmed by genomic cloning. Northern analysis showed mRNA expression (5.2 kb) was induced by progesterone + PRL and antagonized by estrogen. However, because the two proteins result from alternative splicing of a 57-bp exon, the small difference in their mRNA sizes could not be detected by Northern analysis. Therefore, competitive RT-PCR and HPLC were used to quantify differences in the ratios of their mRNAs. Progesterone 2 PRL treatment increased (P < 0.005) the ratio of message for RUSH-1 LYcompared with RUSH-16. Western analysis showed the RUSH-la protein is increased in response to progesterone f PRL and decreased in response to estrogen. The antiserum used for immunoblotting specifically supershifts uteroglobin promoter-protein complexes in gel shift experiments. Because RUSH-la and 6 messages were detected in lung, 0888-8609/96/$3.00/O Molecular Endocrinology Copyright 0 1996 by The Endocrine E.G.B., liver, and HRE-H9 cells, these proteins may regulate genes in numerous cell types. (Molecular Endocrinology 10: 1335-1349, 1996) INTRODUCTION Uteroglobin (UG)/CClO is an emerging family of secretory proteins produced by nonciliated epithelial cells from several organ systems. Rabbit blastokinin (1) or UG (2), the original member of the family, was first reported in uterine secretions during the preimplantation period of pregnancy and thought to be uterine specific in its expression. However, UG is expressed in oviduct and male genital tract, as well as in some nonreproductive tract tissues such as esophagus and lung (for reviews see Refs. 3 and 4). UG has become an excellent candidate for studies of tissue-specific gene expression because the UG gene is subjected to multihormonal regulation. In rabbit uterus, progesterone and estradiol regulate transcription of the UG gene (57), and PRL enhances the progesterone-dependent increase in gene expression (8). Testosterone and glucocorticoids are the inducing steroids in the epididymis (9) and the lung (lo), respectively. UG was initially found in the uterine endometrium of rabbits, and later in hare and pica (1 l), all members of the order Lagomorpha. Then came reports of a UGlike antigen in tracheobronchial washings (12) and in human uterine washings (13). Ultimately, molecular cloning strategies were used to demonstrate that Clara cell 1 0-kDa protein (CC1 0), the major secretory product of the nonciliated cells of the tracheobronchial epithelium, is the human counterpart of UG (14-16). CC1 0 is also expressed in human endometrium where, like rabbit UG, the highest level of expression occurs in Society 1335 MOL 1336 END0 1996 Vol IO No. 11 response to progesterone dominance. UG/CClO expression has been studied in rats and mice. Analysis of gene sequences indicates that the rabbit and human UG genes are more closely related to each other than to the rat gene (15). Biochemical studies have demonstrated that UG binds progesterone (17) and certain methylsulfonyl metabolites of polychlorinated biphenyls (18) and has immunosuppressive and antiinflammatory properties through the inhibition of phospholipase A2 (19, 20). The highest levels of UG expression in the rabbit (1, 2) and in the human (21) endometrium coincide with the events of embryo implantation. The recent localization of a high-affinity UG-binding protein on human trophoblast cells (22) suggests that UG/CClO may play a pivotal role in regulating cellular invasiveness. Endometrial expression of UG is regulated by the combinatorial interaction of transcription factors with their promoter or enhancer elements. Progesterone-dependent transcription of the UG gene (3) is probably mediated by two strong and two weak progesterone receptor-binding sites that are located between positions -2.7 kb and -2.3 kb and correspond to deoxyribonuclease I (DNase I)-hypersensitive regions previously identified in chromatin from endometrium of hormonally manipulated animals (23). Estrogen alters the accumulation of UG mRNA (3), and an estrogen response element has been identified (24) in the proximal promoter region (-263/-251). Promoter deletion and linker scanning analysis have been used to show that OCT I, Spl , and Sp3 bind to the UG promoter (25-28). Other regions of the promoter include a motif that is 92% identical to the GT-I motif in the SV40 enhancer (-258/-220), and a 7-bp inverted repeat (CAGlllC) that is found in the long terminal repeat (-171/-148) of all murine leukemia viruses and proviruses (15). We previously used gel shift assays, Southwestern blots, and UV cross-linking to identify four proteins that bind to the 203-bp 5’-flanking region (-194/+9) of the rabbit UG gene (29). In this paper, we report cloning the cDNAs for two of the UG promoter-binding proteins (95-kDa and 113-kDa). RUSH-l LYis the 113kDa rabbit homolog of human HIP1 16, a protein that binds to the human immunodeficiency virus (HIV-l) promoter (30) and is related to the yeast SNF2/SWl2 family of transcription factors. Members of this family are required for transcriptional activation by several steroid hormone receptors (31-33). RUSH-16 is the 95-kDa truncated version of RUSH-la that results from alternative splicing of a 57-bp exon. Competitive RT-PCR and HPLC were used to demonstrate that RUSH-l (Y is the progesterone-dependent isoform. (-194/+9), a region of the UG promoter (Fig. 1) previously shown to participate in hormone-dependent, sequence-specific binding of nuclear proteins (29). This region of the UG promoter includes the transcription initiation site, a noncanonical TATA box motif (TACA box), and degenerated octamer motifs, which are bound by Ott I in vitro (25, 27). Approximately 5.5 x IO5 plaques were screened to obtain a single phage clone (1.5- (...truncated)