Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under control of the rabbit uteroglobin promoter in transgenic mice.

Transgenic mice bearing two fragments of the rabbit uteroglobin 5'-flanking region fused to the new reporter gene (pac) encoding puromycin N-acetyltransferase (PAC) showed a different pattern of expression. Transgenic lines (C0.4) harboring a 404-bp fragment (-396/+8) had a uterus-specific expression slightly inducible by estrogen, lacking detectable expression in other tissues where the uteroglobin-encoding gene is naturally expressed in rabbit. Transgenic lines (C3.2) bearing a longer fragment of 3.2-kb (-3254/+8) showed hormonally regulated expression in the uterus and the male genital tract, and detectable expression in the lung. In addition, the nonstimulated uterine expression of the transgene was higher in C0.4 lines than in C3.2 lines. It could be concluded that all sequences required for uterus-specific expression should be present within the 404-bp fragment, and that other upstream (-396) sequences are responsible for expression in the lung and male genital tract, as well as for a possible down modulation of expression in the uterus.     

Tissue-specific expression, hormonal regulation and 5''-flanking gene region of the rat Clara cell 10 kDa protein: comparison to rabbit uteroglobin.

The amino acid sequence of rat Clara Cell 10 kDa secretory protein (CC10) shows 55% identity to rabbit uteroglobin. In order to define the relationship between rat CC10 and rabbit uteroglobin in detail, the tissue-specific expression and hormonal regulation of rat CC10 mRNA was analyzed. We report that like rabbit uteroglobin, rat CC10 mRNA is expressed in lung and esophagus, as well as in uteri of estrogen- and progesterone-treated females. Expression of CC10 mRNA in lung is regulated by glucocorticoids. The similarity in expression pattern of rat CC10 mRNA and rabbit uteroglobin mRNA is reflected by a striking similarity in the 5'-flanking regions of the two genes. Despite this overall similarity, two regions of 0.3 kb and 2.1 kb are absent in the rat CC10 upstream gene region. The larger region includes a cluster of hormone receptor binding sites, believed to be responsible for differential regulation of rabbit uteroglobin by glucocorticoids and progesterone. Thus, while the sequence identities in the coding and 5'-flanking regions point towards a common ancestor for the uteroglobin and CC10 gene, later events (deletions/insertions) might have caused species-specific differences in their regulation.     

Expression and regulation of the rabbit uteroglobin gene in transgenic mice

The rabbit uteroglobin (UG) gene, with varying lengths of 5' flanking sequence, was introduced into the mouse genome to investigate the DNA sequences required for tissue-specific expression and regulation by steroid hormones. The pattern of expression and steroid hormone regulation of the transgene was compared to the expression and regulation of the endogenous mouse UG-like gene. In the rabbit, UG is induced in the uterus by progesterone and is expressed constitutively in the lungs, where it is weakly regulated by glucocorticoids. Genomic DNA fragments containing the complete UG-coding sequence with 4.0 (UG4.0), 3.0 (UG3.0), 2.3 (UG2.3), or 0.6 (UG0.6) kilobases of 5' flanking sequence were used to establish lines of transgenic mice. Expression of UG mRNA was observed in the lungs of UG4.0 (2/4 lines), UG3.0 (4/4 lines), UG2.3 (1/2 lines), and UG0.6 (4/4 lines) mice. Uterine expression was observed in UG3.0 (3/4 lines), UG2.3 (1/2 lines), and UG0.6 (2/4 lines). In the lungs of UG3.0 and UG2.3 mice, RNA expression was stimulated by treatment with dexamethasone. In the one line of UG3.0 mice examined, UG was regulated by ovarian steroids in the uterus. The endogenous mouse UG-like gene showed the major site of expression to be in the lung. Unlike the transgene, the endogenous gene was more strongly stimulated by glucocorticoids. Thus, we conclude that the cis elements needed for pulmonary expression of UG are contained within the UG2.3 fragment used to generate transgenic mice, but that other elements are required for full glucocorticoid regulation. Also, the transgene did not show the full uterine expression observed in the rabbit, but regulation by the ovarian steroids was observed.     

The uteroglobin promoter contains a noncanonical estrogen responsive element

Uteroglobin is expressed in various tissues of the rabbit under complex hormonal control. In the endometrium the uteroglobin gene is transcribed only in epithelial cells after administration of ovarian hormones. In this paper we demonstrate that within the promoter region of the rabbit uteroglobin gene, there is a functional estrogen-responsive element (ERE) located between -265 and -252. Hybrid constructions containing sequences of the uteroglobin promoter up to -299, linked to the chloramphenicol acetyltransferase gene of E. coli respond to estrogens in gene transfer experiments, whereas a deletion that removes half of the ERE does not. A synthetic oligonucleotide corresponding to the putative ERE is able to confer estrogen inducibility to an otherwise unresponsive promoter. Binding experiments with purified estrogen receptor from calf uterus reveal a DNase-I footprint over the ERE. Within this protected region six guanine residues that have been shown to be contacted by the receptor in other EREs are protected against methylation by dimethylsulfate in the presence of the estrogen receptor. We compare this ERE with the vitellogenin A2 ERE from Xenopus and find that the relative affinity of the uteroglobin ERE is slightly lower than that of the vitellogenin ERE. Thus, this uteroglobin ERE could be involved in physiological regulation of uteroglobin expression in the genital tract.     

Cloning of rabbit genomic fragments containing the uteroglobin gene

Hybrid phages containing the uteroglobin gene were isolated from a library of Charon 4A lambda phages carrying fragments of rabbit genome. Two phages which contain overlapping rabbit DNA covering approximately 35 kilobase pairs (kb) on both sides of the uteroglobin gene have been analyzed. One of the phages contains the complete gene. Digestion of rabbit liver DNA by restriction endonucleases and Southern hybridization showed a pattern identical with that obtained with the DNA of recombinant phages suggesting that no rearrangement has taken place during cloning. These experiments also showed the presence of a single gene for uteroglobin in the rabbit genome. Isolation of this gene is of interest for the study of the mechanism of action of progesterone.     

Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4.

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.     

The mitochondrial uncoupling protein gene in brown fat: correlation between DNase I hypersensitivity and expression in transgenic mice.

The mitochondrial uncoupling protein gene is rapidly induced in mouse brown fat following cold exposure. To identify cis-regulatory elements, approximately 50 kb of chromatin surrounding the uncoupling protein gene was examined for its hypersensitivity to DNase I. Seven DNase I-hypersensitive sites were identified in the 5'-flanking DNA, and one site was identified in the 3'-flanking DNA. Transgenic mice with an uncoupling protein minigene were generated by microinjection of fertilized eggs with a transgene containing 3 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. Expression of the transgene is restricted to brown fat and is cold inducible. Four additional transgenic lines were generated with a second transgene containing a 1.8-kb deletion in the 5'-flanking DNA, and expression of this minigene is absent in all tissues analyzed. A DNase I-hypersensitive site located in the 1.8-kb deletion contains a cyclic AMP response element that binds a brown fat tumor enriched nuclear factor. On the basis of these observations, we propose that a cis-acting regulatory sequence between -3 and -1.2 kb of the 5'-flanking region, possibly at a DNase I-hypersensitive site, is required for controlling uncoupling protein expression in vivo.     

Primary structure of rabbit lung uteroglobin as deduced from the nucleotide sequence of a cDNA

Double-stranded cDNA was synthesized from partially purified uteroglobin mRNA from rabbit lung. A cDNA coding for lung uteroglobin was then cloned in the plasmid pUC18 and both the nucleotide sequence and the derived amino acid sequence were determined. This allowed us to demonstrate unequivocally that uteroglobins from lung and uterus are identical proteins.     

Isolation and characterization of uteroglobin from the lung of the hare (Lepus capensis)

Uteroglobin has been purified from hare lung by gel filtration and chromatography on carboxymethyl-cellulose. Hare uteroglobin appears homogeneous by electrophoresis under both denaturing and nondenaturing conditions. Its chemical and immunological properties as well as its ability to bind progesterone are compared to those of rabbit uteroglobin. The two proteins have the same N-terminal residue (glycine) and both lack tryptophan but differ in amino acid composition. Sodium dodecyl sulfate-gel electrophoresis shows that hare uteroglobin is composed of two subunits of identical Mr (about 7000) held together by disulfide bridges. The amino acid composition indicates a subunit composed of 65-67 residues, which is compatible with the apparent Mr observed. Thus, hare uteroglobin appears to be slightly smaller than the rabbit protein. Hare uteroglobin partially reacts with anti-rabbit uteroglobin in a radioimmunoassay and also binds progesterone, although this binding is relatively unaffected by dithiothreitol. The synthesis of hare uteroglobin in the uterus appears to be rather insensitive to ovarian steroid hormones.     

Pem homeobox gene regulatory sequences that direct androgen-dependent developmentally regulated gene expression in different subregions of the epididymis

The epididymis is a useful model system to understand the mechanisms that govern region-specific gene expression, as many gene products display spatially restricted expression within this organ. However, surprisingly little is known about how this regulation is achieved. Here, we report regulatory sequences from the Pem homeobox gene that drive expression in different subregions of the mouse epididymis in vivo. We found that the 0.3-kb 5'-flanking sequence (region I) from the Pem proximal promoter (Pem Pp) was sufficient to confer androgen-dependent and developmentally regulated expression in the caput region of the epididymis. Expression was restricted to the normal regions of expression of Pem in the caput (segments 2-4), but there was also aberrant expression in the corpus region. This corpus misexpression was extinguished when 0.6 kb of Pem Pp 5'-flanking sequence was included in the transgene, indicating that one or more negative regulatory elements exist between 0.6 and 0.3 kb upstream of the Pem Pp start site (region II). When heterologous sequences were introduced upstream of the Pem Pp, expression was further restricted, mainly to caput segment 3, implying that the Pem Pp has segment-specific regulatory elements. To our knowledge, the regulatory regions we have identified are the shortest so far defined that dictate regionally localized expression in the epididymis in vivo. They may be useful for identifying the factors that regulate region-specific expression in the epididymis, for expressing and conditionally knocking out genes in different subregions of the epididymis, for treating male infertility, and for generating novel methods of male contraception.