Rabbit Uteroglobin Research Articles

Cloning the uteroglobin gene promoter from the relic volcano rabbit (Romerolagus diazi) reveals an ancient estrogen‐response element

To gain further insight on the estrogen-dependent transcriptional regulation of the uteroglobin (UG) gene, we cloned the 5'-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, 17β-estradiol (E(2)) induced transcription of a luciferase 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-α indeed binds to the imperfect palindromic sequence of the Rd-ERE.

Uteroglobin induces the development and cellular proliferation of the mouse early embryo

Two-cell mouse embryos cultured in vitro in the presence of either purified rabbit uteroglobin (UG) or recombinant human UG developed and proliferated faster than controls cultured in the absence of this protein. Both the percentage of embryos developing to the blastocyst stage and the number of cells per embryo were increased. Treatment with UG for 3 hr was enough to trigger this response. The effect of UG was blocked by genistein, an inhibitor of tyrosine protein kinases, suggesting the involvement of these kinases in the stimulation of the embryo by UG. To further support this suggestion, embryos were metabolically labeled in vitro with [32P] and the phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine. Analysis of the immunoprecipitates by SDS-PAGE showed that UG induced the phosphorylation of several proteins of M(r) between 200 and 37 kDa. This induction was observed after 1 hr of stimulation with UG and further increased after 3 hr of treatment. Since UG is synthesized and secreted in the uterus and the oviduct, these results suggest a physiological role of this protein in the correct development of the embryo in vivo.

Coupedu RoiBisection of Proteins. Spontaneous Tetramerization of Two Peptides That Span the Sequence of the Rabbit Uteroglobin Monomer

The study of dividing objects into isometric segments has yielded novel approaches to the synthesis of high-symmetry organic compounds. Reported herein is the first application of this concept to a protein, rabbit uteroglobin (UG). Bisection of UG into two identical homochiral segments led to the design of the heterodimeric 70mer peptide alpha(1,2)-S-S-alpha(3,4) that spans the sequence of the native UG monomer. The ability of this compound to form a globular 140mer tetramer consisting of two noncovalently bound heterodimers was assessed by ultracentrifugation at sedimentation equilibrium and by fluorescent spectroscopy. On the other hand, the monomeric peptides alpha(1,2)-SH and alpha(3,4)-SH were shown to selectively form the alpha(1,2)-S-S-alpha(3,4) heterodimer via spontaneous air oxidation in phosphate buffer at neutral pH.

Prolactin signals through RUSH/SMARCA3 in the absence of a physical association with Stat5a.

Jak/Stat-mediated prolactin signal transduction culminates in the sequence-selective binding of Stat5a. However, in the absence of Stat-binding sites, a RUSH-binding element mediates the prolactin signal in the rabbit uteroglobin promoter. Speculation about the existence of a Jak/RUSH pathway prompted this series of experiments to examine potential interactions between RUSH and Stat5a. Profiles of Jak/Stat pathway-specific genes by RT-PCR showed that mRNA for Jak2 and Stat5a is expressed in the endometrium of estrous, progesterone-treated, and 5-day pseudopregnant rabbits. Interspecies microarrays showed that transcripts for Stat5a were present at equal concentrations in the endometrium regardless of hormone treatment. The absence of a physical interaction between RUSH and individual Stat proteins bound to enhancer sites was demonstrated with transcription factor interaction arrays. These studies confirm that transmission of the prolactin signal through RUSH occurs in the absence of a physical association with Stat5a. Although a strong physical interaction between RUSH and Egr-1 was identified with the same arrays, no Egr-1 consensus sites were found in the region of the uteroglobin promoter (-175/-80) that contains the authentic RUSH site. Because the major transducer molecules (Jak2, Stat5a) are activated by tyrosine phosphorylation, Western analysis of immunoprecipitated samples, and gel shift assays were used to show that tyrosine phosphorylation is required for RUSH-DNA binding. The precise role for Jak2 in this process remains undefined. By comparison, serine-threonine-specific protein phosphorylation had no effect on RUSH-DNA binding.

Identification of the RUSH consensus-binding site by cyclic amplification and selection of targets: demonstration that RUSH mediates the ability of prolactin to augment progesterone-dependent gene expression.

RUSH-1alpha(beta) transcription factors were cloned by recognition site screening with an 85-bp region (-170/-85) of the rabbit uteroglobin gene. Deletion analysis showed this region was essential to prolactin (PRL) action, but conclusions were limited by the complexity of the large deletion. Cyclic amplification and selection of targets (CASTing) was used to identify the RUSH-binding site (-126/-121). Endometrial nuclear proteins were incubated with a pool of degenerate oligonucleotides and immunoprecipitated with RUSH-1alpha(beta) antibodies. Bound DNA was amplified by PCR. The consensus motif (MCWTDK) was identified after five rounds of CASTing, authenticated by CASTing with RUSH-1alpha-specific antibodies and recombinant protein, and refined with EMSA. Dissociation rate constants (K(d) = 0.1-1.0 nM; r = 0.99) revealed high-affinity binding. Chromatin immunoprecipitation confirmed in vivo binding of RUSH to the transcriptionally active uteroglobin promoter. CASTing also revealed RUSH-GATA transcription factor interactions. Endometrial GATA-4 expression is progesterone dependent (Northern analysis) and preferentially localized in the epithelium (in situ hybridization). Although physically affiliated with RUSH, uterine forms of GATA-4 were not required for RUSH-DNA binding. Site-directed mutagenesis and transient transfection assays showed the RUSH motif mediates the ability of PRL to augment progesterone-dependent uteroglobin transcription. RUSH is central to the mechanism whereby PRL augments progesterone-dependent gene transcription.

All Human Genes of the Uteroglobin Family Are Localized on Chromosome 11q12.2 and Form a Dense Cluster

Rabbit uteroglobin is the founder member of a family of mammalian proteins that has expanded to more than 20 members within the last few years. All members are small, secretory, rarely glycosylated dimeric proteins with unclear physiological functions and are mainly expressed in mucosal tissues. A phylogenetic analysis shows that the family can be grouped into five subfamilies, A to E. Subfamily A contains rabbit uteroglobin and its orthologues from various species; most of these have been described to form antiparallel homodimers via two intermolecular disulfide bonds. All other subfamily members contain a third conserved cysteine and, from existing biochemical data, it can be predicted that a member of subfamily B or C will likely form heterodimers with a partner from subfamily E or D, respectively. Besides the mentioned cysteines, only one central lysine is conserved in all family members. In the known uteroglobin structures, this lysine forms an exposed salt bridge with an aspartate side chain, which is conserved in almost all sequences. Using radiation hybrid mapping and P1 clone analysis and utilizing data from the human genome project, we show that all known five human family members (Clara cell 10-kDa protein, lipophilins A and B, lacryglobin, mammaglobin) and a new member, we call lymphoglobin, are localized on chromosome 11q12.2 in a dense cluster spanning not more than approximately 400 kbp.

Uteroglobin gene transcription: what's the RUSH?

Prolactin enhances progesterone-dependent transcription of the rabbit uteroglobin gene. RUSH transcription factors are implicated in the signal transduction pathway. The RUSH acronym identifies key features of these nuclear phosphoproteins, that is, RING-finger motif, binds the uteroglobin promoter, structurally related to the SWI/SNF family of transcription factors, and helicase-like. Cloned by recognition site screening, RUSH proteins bind to an 85-bp region (-170/-85) of the uteroglobin promoter that was subsequently identified as a novel prolactin-responsive region by promoter deletion analysis. Gel shift and linker-scanning assays further reduced the RUSH target site to -160/-110. A hexameric core of MCWTDK was identified as the RUSH-specific DNA-binding site (-126/-121) by CASTing. This site overlaps authentic HNF3 beta and OCT-1 binding sites. A unique Type IV P-type ATPase that is embedded in the inner nuclear membrane binds the RING domain of RUSH. The conformationally flexible loop portion of this RING-finger binding protein (RFBP) extends into the nucleoplasm to contact euchromatin. The physical association of RFBP with transcriptionally active chromatin supports the speculation that RFBP targets RUSH transcription factors to the active uteroglobin promoter.

The discovery of uteroglobin and its significance for reproductive biology and endocrinology.

The discovery of uteroglobin resulted from investigations on the biochemical composition of oviductal and uterine secretions of the rabbit and other mammals. These determinations about physiological composition were urgently requested to prepare culture media for research on early mammalian development in vitro. Discovery of significant proteins during the sixties reflected the laboratory skills of that time. Protein characterization was achieved by isolation via Sephadex gels, electrophoresis on polyacrylamide gels, and finally immunoprecipitation using classical polyclonal antibodies. The molecular biology was not yet established. Uteroglobin could be found as the major protein component of rabbit uterine secretion. From the beginning, it was already identified as an unusually small, spheric uterine secretory molecule without any carbohydrates--hence its name. Uteroglobin was the first mammalian protein that turned out to be progesterone-regulated and, at the same time, released in mg amounts actually in one organ compartment. Moreover, uteroglobin and its gene proved to be a reliable model for the description of progesterone/progesterone receptor complex action at the DNA level. After its original observation in the uterus, however, uteroglobin was detected also in several other organs, for example, the epididymis, the seminal vesicle, and the lung. Initially, it could not be found in the blood, which challenged the hypothesis that uteroglobin specifically should operate by local activation rather than by a humoral or endocrine effect. Later, though, the human uteroglobin molecule, isolated from blood filtrate, was used for detailed structural analyses. The rabbit uteroglobin model certainly was beneficial for reproductive biological research. Experimental interference with steroid hormone regulation during preimplantation presented surprising effects, which led to the discovery of the transposition of the implantation window. The uterine secretion protein patterns, in particular the uteroglobin fraction and the beta-glycoprotein fraction, served as decisive marker profiles to identify the biological stage of the intrauterine microenvironment during preimplantation. This diagnostic procedure, using only protein parameters, enabled us to precisely predict the receptive stage of the endometrium for donated blastocysts to achieve implantation successfully.

Expression of RUSH Transcription Factors in Developing and Adult Rabbit Gonads1

The RUSH transcription factors 1alpha and 1beta bind to the Rabbit Uteroglobin promoter and are members of the SWI/SNF complex that facilitates transcription by remodeling chromatin (Helicase). To characterize gonadal expression of RUSH, a cRNA probe that recognizes both isoforms was used for in situ hybridization studies. We found RUSH mRNA to be abundant in Sertoli cells from embryonic, neonatal, prepubertal, and pubertal rabbit testes. In adults, RUSH mRNA was detected in tubules with preleptotene spermatocytes and mature spermatids lining the lumen. However, RUSH was undetectable in tubules that contained leptotene spermatocytes and that lacked mature spermatids. In females, RUSH was expressed in presumptive granulosa cells of embryonic and neonatal ovaries before follicle organization. Abundant RUSH mRNA was detected in granulosa and theca cells surrounding preantral follicles of prepubertal and adult ovaries. Expression of RUSH remained high in granulosa cells of antral follicles in mature ovaries but was negligible in late-stage atretic follicles and in corpora lutea. Western blot analysis confirmed the RUSH-1alpha isoform predominated in both testicular and ovarian tissues. The expression pattern of RUSH indicates transcriptional activity in Sertoli cells and during multiple stages of differentiating granulosa cells, especially those of primordial follicles, which heretofore were considered to be dormant.

Expression of uteroglobin in the human endometrium.

Uteroglobin is a progesterone binding protein, a member of the antiflammin gene family and possibly a novel cytokine. Initially, uteroglobin was identified as the major protein of rabbit uterine secretion during the phase of preimplantation. Counterparts of the rabbit uteroglobin or its gene are described in rat, mouse, hamster, hare, pig, horse and human. While uteroglobin appears as one of the most extensively studied proteins, particularly its physico-chemical properties, including its crystal structure and its gene, the true physiological role of this protein still remains to be unravelled. Essential to understanding the significance of human uteroglobin in reproductive organs, particularly in the endometrium, is a knowledge of the spatial and chronological expression of this secretory protein. Our studies on 115 volunteers combined reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and quantitative assessment by an enzyme-linked immunosorbent assay for uteroglobin. The expression, localization and release of uteroglobulin in the human endometrium are presented. Secretory uteroglobin is found in endometrial tissue homogenates in highest levels of expression during the early luteal phase (days 15-19, 340 pg/mg total protein). In turn, uteroglobin is released into the uterine lumen in peak amounts during the receptive phase of the menstrual cycle (mid-luteal phase, days 20-23, secretion level 833.4 pg/mg total protein). Our immunohistochemical studies match with these results, as uteroglobin is located during the early and mid-luteal phase in the apical compartments of endometrial gland cells. These observations strongly suggest an involvement of uteroglobin in endometrial preparations for implantation.

Application of the disulfide trapping approach to explain the antiparallel assembly of dimeric rabbit uteroglobin: A preliminary study using short peptide models

The spontaneous air oxidation of the peptides H-GICPRFAHVIENLL-NH2 and Ac-LPQTTRENIMKLTEKIVKSPLCM-OH, whose sequences correspond to the helicoidal fragments 1–14 and 48–70 of the monomer of rabbit uteroglobin, was studied. While no oxidation products were observed in the absence of trifluoroethanol, the heterodimer was formed selectively in the presence of the structuring agent. Head-to-head and tail-to-tail homodimers were not detected under these conditions. These results suggest that the N- and C-terminal extremes of the monomer of uteroglobin might contain enough structural information to govern dimerization with a head-tail topology, through a selective helix-to-helix recognition process.

Hormone-dependent Recruitment of NF-Y to the Uteroglobin Gene Enhancer Associated with Chromatin Remodeling in Rabbit Endometrial Epithelium

Expression of the rabbit uteroglobin gene is hormonally induced in cells of the endometrial epithelium during the preimplantation phase of pregnancy. Here we show that progesterone activation of the gene is mediated by two clusters of hormone responsive elements located between 2.4 and 2.7 kilobase pairs upstream of the transcriptional start site. Between these two clusters, genomic footprinting studies in the intact endometrial epithelium reveal the hormone-inducible occupancy of several cis-acting elements. One of the protected elements shows sequence homology to the consensus binding site of the transcription factor NF-Y, which binds to the element in gel shift experiments. This uteroglobin Y box is essential for enhancer activity in transient transfection experiments with endometrial and non-endometrial cell lines, in accordance with the ubiquitous expression of NF-Y. To understand why binding of this ubiquitous factor to the uteroglobin Y box in endometrium depends on hormone induction, we examined the chromatin structure of the relevant gene region. In the uninduced state, the enhancer region appears to be organized into positioned nucleosomes. Upon hormone induction, this nucleosomal pattern is lost and the enhancer region becomes hypersensitive to nucleases, suggesting that a hormone-induced change in the local chromatin structure unmasks previously unaccessible binding sites for transcription factors. Our results emphasize the limitations of using transient transfection assays for the functional analysis of cis-acting elements and underline the need for including the native chromatin organization in this kind of studies.

Solution structure of the recombinant oxidized rabbit uteroglobin using homonuclear and heteronuclear multidimensional NMR.

Rabbit uteroglobin (rab-UG) is a 16-kDa homodimeric secretory protein with potent anti-inflammatory/immunomodulatory properties. Its physiological role is still unclear, although it was observed that several small hydrophobic molecules bind to the oxidized and the reduced uteroglobin. It is suggested that the formation and/or disruption of the two disulphide bridges not only regulates this binding itself, but also the affinity to the ligand. The determination of the solution structure has been started with the assignment of 1H, 15N and 13C resonances of the oxidized rabbit uteroglobin, based on several two-dimensional and three-dimensional homonuclear and heteronuclear double and triple resonance experiments. The assignment was possible with the overproduction of the wild-type as well as of uniformly 15N-labeled and 15N/13C-labeled samples of the recombinant protein. A complete assignment of 1H, 15N and 13C resonances, the secondary-structure elements and the tertiary structure in solution is presented. The tertiary solution structure was found to be in good agreement with the previously determined crystal structure of rab-UG and with the solution structure of human uteroglobin (h-UG). h-UG and rab-UG are extremely stable proteins within a wide range of pH and temperatures. Some of the binding characteristics of ligands of rab-UG and a mutant with all cysteine residues exchanged to serine residues are discussed.

Identification of Mammaglobin B, a Novel Member of the Uteroglobin Gene Family

In this report, we have identified, sequenced, and characterized the expression pattern of a novel human gene, mammaglobin B. Mammaglobin B (MGB2) is highly homologous to mammaglobin (MGB1), a previously characterized human gene whose expression is limited to the mammary epithelium and frequently up-regulated in human breast cancer cells. Based upon amino acid sequence similarities, both mammaglobin and mammaglobin B may be considered members of a larger, mammalian multigene family that includes rabbit uteroglobin, human Clara Cell 10-kDa protein (CC10), and the multimeric rat prostatein protein. Together with the human CC10 gene, mammaglobin and mammaglobin B are closely linked on human chromosome 11q13. However, despite their primary sequence similarity and close chromosomal proximity, the expression of mammaglobin and mammaglobin B is nonconcordant in both nonmalignant and neoplastic tissue.

Zinc Finger Proteins RUSH in Where Others Fear to Tread1

The uteroglobin (UG) gene family encodes a fascinating group of secreted proteins that includes rat prostatic steroid-binding protein subunit C3, human Clara cell 10-kDa protein, human mammaglobin, and rabbit UG. In the rabbit, UG is a progesterone-dependent preimplantation uterine protein with peak availability on Day 5 of pregnancy. Progesterone stimulates UG synthesis in estrous rabbits and in short-term (3 day to 4 wk) ovariectomized rabbits. However, with increased time after ovariectomy, the uterus becomes increasingly refractory to progesterone challenge, such that after 12 weeks, normal levels of UG synthesis could not be measured. The treatment of these long-term ovariectomized rabbits with either prolactin or progesterone resulted in a dramatic increase in the receptor for the other hormone. Sequential treatment with prolactin plus progesterone increased the endometrial UG mRNA content and stimulated UG production to a concentration equal to that found on the fifth day of pregnancy. Because the increase in UG mRNA could result from an increased rate of transcription, gel shift assays, Southwestern blots, and UV cross-linking were used to show that prolactin augments the binding of four progesterone-dependent proteins to an 85-base pair (bp) 5'-flanking region (-170/-85) of the UG gene. The cDNAs for two of these UG promoter-binding proteins, RUSH-1alpha (113 kDa) and -1beta (95 kDa), were cloned by recognition site screening and identified as new members of the SWI/SNF superfamily of nuclear receptor coactivators. RUSH-1alpha and -1beta result from alternative splicing of a 57-bp exon, and each phosphoprotein has the novel RING-finger motif near its C terminus. Competitive reverse transcription-polymerase chain reaction and HPLC analysis showed that RUSH-1alpha is the progesterone-dependent splice variant. When an endometrial cell line (HRE-H9) was transfected with either the full-length UG construct, pUG3.1-LUC, or the deletion mutant, pUG3.1 deltaRUSH-LUC, progesterone increased the transcriptional activity of pUG3.1. Transcription of pUG3.1-LUC was further increased when cells were treated with prolactin plus progesterone. Prolactin alone had no effect. Progesterone also increased the transcriptional activity of pUG3.1 deltaRUSH-LUC. However, deletion of the proximal promoter region (pUG3.1 deltaRUSH-LUC) eliminated the prolactin effect and implicated RUSH proteins as prolactin signal transducers.

Hormone-induced Recruitment of Sp1 Mediates Estrogen Activation of the Rabbit Uteroglobin Gene in Endometrial Epithelium

Steroid hormones activate gene expression by interaction of their receptors with hormone-responsive DNA elements and tissue-specific or ubiquitous factors. To monitor the molecular changes on the promoter of the rabbit uteroglobin gene during early pseudopregnancy in vivo, we have applied the genomic footprinting methodology to endometrial tissue. Estrogen induction results in the simultaneous occupancy of an estrogen-responsive element and an adjacent GC/GT box in the promoter. DNA binding assays demonstrate that the corresponding regulatory factors are the ligand-induced estrogen receptor and the ubiquitous transcription factor Sp1. Both factors functionally synergize in primary endometrial cells, showing that the GC/GT box is an essential part of a composite estrogen-responsive unit. However, the estrogen receptor and Sp1 do not bind cooperatively to their sites in vitro, suggesting that other mechanisms might be responsible for the hormone-dependent binding of Sp1 in vivo. Since hormone treatment leads to the appearance of a distinct DNase I-hypersensitive site over the promoter chromatin, an estrogen-induced change in the local chromatin structure could facilitate binding of Sp1 in vivo.

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

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 SWI2/SNF2 helicase superfamily, and each protein has the novel C3HC4 (RING) zinc-finger signature near its C terminus. RUSH-1 alpha, the 113-kDa protein, is the rabbit homolog of human HIP116, a protein that binds to the human immunodeficiency virus-1 promoter. RUSH-1 beta is a 95-kDa truncated version of RUSH-1 alpha that 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 +/- PRL treatment increased (P < 0.005) the ratio of message for RUSH-1 alpha compared with RUSH-1 beta. Western analysis showed the RUSH-1 alpha protein is increased in response to progesterone +/- PRL and decreased in response to estrogen. The antiserum used for immunoblotting specifically supershifts uteroglobin promoter-protein complexes in gel shift experiments. Because RUSH-1 alpha and beta messages were detected in lung, liver, and HRE-H9 cells, these proteins may regulate genes in numerous cell types.

Binding of YY1 to a site overlapping a weak TATA box is essential for transcription from the uteroglobin promoter in endometrial cells.

The gene for rabbit uteroglobin codes for a small calcium-, steroid-, and biphenyl metabolite-binding homodimeric protein which is expressed in a variety of epithelial cell types such as Clara cells (lung) and the glandular and luminal cells of the endometrium. One important region mediating its efficient transcription in a human endometrium-derived cell line, Ishikawa, is centered around a noncanonical TATA box. Two factors, TATA core factor (TCF), expressed in cell lines derived from uteroglobin-expressing tissues, and the ubiquitously expressed TATA palindrome factor, bind to the DNA major groove at two adjacent sites within this region. Here, we report the identification of the TATA palindrome factor as the transcription/initiation factor YY1 by microsequencing of the biochemically purified factor from HeLa cells. The binding site for YY1 within the uteroglobin gene is unique in its sequence and its location overlapping a weak TATA box (TACA). Binding of YY1 was required for efficient transcription in TCF-positive Ishikawa cells, which responded only weakly to a change of TACA to TATA, although in vitro binding affinity for the TATA-box-binding protein increased by 1 order of magnitude. In contrast, in CV-1 cells, lacking TCF, binding of YY1 was not required for transcription in the context of a wild-type TACA box, whereas a change from TACA to TATA led to significantly increased reporter gene expression. DNA binding data exclude a role of YY1 in stabilizing the interaction of the TATA-box-binding protein with the uteroglobin promoter. We conclude that cell lines derived from uteroglobin-expressing tissues overcome the weak TATA box with the help of auxiliary factors, one of them being YY1.

Expression of human Krev-1 gene in lungs of transgenic mice and subsequent reduction in multiplicity of ethyl carbamate-induced lung adenomas.

Mice of the A/J strain are useful models of lung cancer because they develop tumors spontaneously or after treatment with ethyl carbamate. These tumors are thought to arise from either Clara cells (papillary tumors) or alveolar type 2 cells (alveolar tumors); like many human lung adenocarcinomas, the mouse tumors involve Kiras activation. Transformation with Ki-ras can be reversed by coexpression of the Krev-1 gene in tissue culture. To test the tumor suppressor activity of Krev-1 in vivo, we produced transgenic A/J mice expressing Krev-1 under the control of the rabbit uteroglobin promoter, which directs expression of heterologous genes to the lung Clara cells. Krev-1 was expressed specifically in the lungs of transgenic mice. Sixty-six mice (35 transgenic and 31 nontransgenic) from three lines were given ethyl carbamate, and the numbers of resulting lung tumors were compared between transgenic and nontransgenic animals. The mean number (+/-standard deviation) of ethyl carbamate-induced lung tumors was 21.7 +/- 1.3 in transgenic mice and 26.9 +/- 1.3 in their nontransgenic littermates (P < 0.01). Sequencing of polymerase chain reaction-amplified ras DNA from 15 transgenic mouse tumors and 16 nontransgenic mouse tumors (controls) detected mutations in codon 61 in 13 tumors from the transgenic group and 11 tumors in the control group, whereas mutations in codon 12 were detected in only one tumor in the transgenic group and in four tumors in the controls. Together, these data demonstrate for the first time the tumor suppressor activity of Krev-1 in vivo and suggest that Krev-1 tumor suppressor activity may be specific for cells harboring mutations in codon 12 of ras.

Interactions of Progesterone-Dependent Endometrial Nuclear Factors with the Promoter of the Rabbit Uteroglobin Gene

We have studied thein vitrointeractions of a promoter fragment of the rabbit uteroglobin (UG) gene with endometrial nuclear factors from either control rabbits or progesterone-treated animals, a treatment that induces the transcription of the gene in the endometrium. Nuclear factors from liver, which does not express UG, were also compared. Using DNase I footprinting, several protected zones were observed on the promoter; some of these were common to all three nuclear extracts, whereas others seemed to be specific to progesterone treated endometrium. One of these footprints was over the TATA box region. A 28-bp synthetic oligonucleotide encompassing the sequence of that region was used as a probe in electrophoretic mobility shift assays (EMSA) and UV-crosslinking assays. In EMSA experiments, endometrial nuclear extracts, but not liver extracts, generated a major retarded complex that was strongly increased following progesterone stimulation. Competition experiments with unlabeled mutated versions of the probe showed that sequences 3′ downstream from the TATA box (but not this motif) were responsible for the formation of the complex. UV-crosslinking experiments indicated that the probe interacted with two progesterone-dependent nuclear proteins of 55 and 60 kDa, different from the TATA box-binding protein. The results suggest that progesterone induction of the UG gene in the endometrium might be mediated by both general and tissue-specific nuclear factors.