A tale of three fingers: the family of mammalian Sp/XKLF transcription factors.

One of the most common regulatory elements is the GC box and the related GT/CACC box, which are widely distributed in promoters, enhancers and locus control regions of housekeeping as well as tissue-specific genes. For long it was generally thought that Sp1 is the major factor acting through these motifs. Recent discoveries have shown that Sp1 is only one of many transcription factors binding and acting through these elements. Sp1 simply represents the first identified and cloned protein of a family of transcription factors characterised by a highly conserved DNA-binding domain consisting of three zinc fingers. Currently this new family of transcription factors has at least 16 different mammalian members. Here, we will summarise and discuss recent advances that have been directed towards understanding the biological role of these proteins.

Transcription factor Sp3 is regulated by acetylation.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Previous analyses showed that, unlike Sp1, Sp3 fails to activate transcription in certain promoter settings. This is due to the presence of an inhibitory domain located between the second glutamine-rich activation domain and the DNA-binding domain. To further analyze the transcriptional properties of Sp3, we have expressed and purified recombinant Sp3 and Sp1 as epitope-tagged proteins from stable transfected insect cells. We found that Sp3 does act as a strong activator similar to Sp1 in an in vitro transcription assay using Sp1/Sp3-depleted HeLa nuclear extract. However, on the same promoter Sp3 is almost inactive when transfected into cells. Mutational studies demonstrate that a single lysine residue is responsible for the low transcriptional activity of Sp3 in vivo. We show that Sp3, but not a mutant of Sp3 that lacks this lysine residue, is highly acetylated in vivo. Our results strongly suggest that the transcriptional activity of Sp3 is regulated by acetylation. The consequences of acetylation for the activity of Sp3 are discussed.

Differential transcriptional regulation of c-myc promoter through the same DNA binding sites targeted by Sp1-like proteins.

Sp1 and Sp3 are closely related members of a gene family encoding proteins with very similar structural features. The zinc finger DNA binding domains of Sp1 and Sp3 are highly conserved and they bind to GC and GT box with comparable affinities. To begin to delineate the specific roles of these two members of the Sp1-like gene family, here we have analysed the DNA binding specificity and their effects on activation of human c-myc promoter. We found that both proteins bind to the same sites of c-myc promoter, upstream to both the P1 and P2 initiation sites. Cotransfection experiments, in mammalian and insect cells, indicated that Sp1 trans-activate c-myc promoter, whereas Sp3 did not. In addition, enforced expression of Sp3 repressed Sp1-mediated activation of c-myc. Finally, we found that Sp1 and E2F-1/DP-1 cooperatively trans-activate c-myc promoter. In contrast enforced expression of Sp3 fails to repress E2F-1/DP-1-mediated activation.

The uteroglobin promoter targets expression of the SV40 T antigen to a variety of secretory epithelial cells in transgenic mice.

Adenocarcinomas derived from the lining epithelia of various organs are the most common malignant tumors in human pathology and about 50% are hormone dependent. The tissue-specific and hormally regulated expression of the rabbit uteroglobin gene is secretory epithelial cells could provide a means of establishing in vivo models for a variety of human tumors originating from such tissues. We have generated trangenic mice inheriting a hybrid gene containing 4.7 kb of the rabbit uteroglobin 5'-flanking sequences fused to the SV40 T antigen encoding region. All transgenic founders examined exhibited bronchio-alveolar adenocarcinomas, probably due to expression of the transgene in Clara cells. Most founders also developed tumors of the submandibular salivary gland, and adenocarcinomas of the stomach. Adenocarcinomas and dysplasias in epithelial cells of the male and female genital tract were found in single founders. Immunohistochemical analysis showed that T antigen expression interfered with stable maintenance of the differentiated phenotype as documented by expression of the endogenous uteroglobin gene. One founder gave rise to a mouse line, UT7.1. Transgenic descendants of UT7.1 developed lung adenocarcinomas and, depending on the genetic background, exhibited tumors of the stomach, the salivary gland and the pancreas. Sporadically male descendants developed prostatic adenocarcinoma whereas females developed dysplasias and adenocarcinomas of the uterus and the oviduct. Thus, the UT7.1 mouse line could be a useful model for several epithelial neoplasias.

Impaired ossification in mice lacking the transcription factor Sp3.

Sp3 is a ubiquitously expressed member of the Sp family of transcription factors. Recently, the mouse Sp3 gene has been disrupted by homologous recombination. Sp3 null mice die immediately after birth due to respiratory failure. In addition, these mice show a pronounced defect in late tooth formation. Here we show that Sp3 is also required for proper skeletal ossification. Both endochondral and intramembranous ossification are impaired in E18.5 Sp3-/- embryos. The delay in ossification is reflected by reduced expression of the osteoblast-specific marker gene osteocalcin. The transcription factor - core binding factor 1 (Cbfa1)--that is essential for bone formation, however, is expressed at normal levels. In vitro differentiation studies using Sp3-/- ES cells further support the conclusion that Sp3 is needed for correct bone formation. The capacity of Sp3-/- cells to undergo osteogenic differentiation in vitro is reduced and osteocalcin expression is significantly diminished. Our studies establish Sp3 as an essential transcription factor for late bone development.

Cell-specific, developmentally and hormonally regulated expression of the rabbit uteroglobin transgene and the endogenous mouse uteroglobin gene in transgenic mice.

We have generated a transgenic mouse line by introducing the rabbit uteroglobin gene with 4 kb of 5'-flanking DNA and 1 kb of 3'-flanking DNA into the mouse germ line via microinjection into fertilized oocytes. Expression of the rabbit uteroglobin transgene was examined and compared with the endogenous mouse uteroglobin gene. Both genes are expressed in the lung, male genital tract and uterus. In the lung, mRNA expression is enhanced by glucocorticoids and restricted to the Clara cells that line terminal and respiratory bronchioli. During embryonic lung development, transcripts are first detected at day 17. Expression in the uterus is restricted to the glandular epithelium and can be induced by sequential treatment with estrogens and progesterone. In the uterus of these pseudopregnant mice the level of rabbit uteroglobin transcripts is higher than that of the mouse endogenous uteroglobin transcripts. In the male genital tract, expression of both genes is restricted to the epithelial layers of the vesicular gland, vas deferens and epididymis. Our results indicate that the rabbit uteroglobin gene together with 4 kb of 5'-flanking DNA and 1 kb of 3'-flanking DNA contains the information required for cell type-specific, developmentally, and hormonally regulated expression.

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.

The Sp-family of transcription factors.

GC-boxes and related motifs are frequently occurring DNA-elements present in many promoters and enhancers. In contrast to other elements it was generally thought that the transcription factor Sp1 is the only factor acting through these motifs. The cloning of paralogous genes of the Sp1 factor uncovered the existence of a small protein family consisting of Sp1, Sp2, Sp3 and Sp4. All four proteins exhibit very similar structural features. They contain a highly conserved DNA-binding domain composed of three zinc fingers close the C-terminus and serine/threonine- and glutamine-rich domains in their N-terminal regions. The high degree of structural conservation between these four proteins suggested that they do exert similar functions. Molecular, genetic and biochemical analyses, however, demonstrated that Sp2, Sp3 and Sp4 are not simply functional equivalents of Sp1. Here, I will summarize and discuss recent advances which have been made towards understanding the mode of action and biological function of individual family members.

Characterization and promoter analysis of the mouse gene for transcription factor Sp4.

Transcription factor Sp4 is a member of the Sp1 family. It functions differently from other members of this family, such as Sp1 and Sp3, and the gene for Sp4 is transcribed in a tissue-specific manner. Recent studies in mice suggest that Sp4 might play an important role in growth, viability, and male fertility. We report here the isolation and characterization of the gene for Sp4 from a mouse genomic library. The mouse gene for Sp4 was about 80 kb in length and it consisted of six exons and five introns. The promoter was found in a CpG island and had a high G+C content. The proximal promoter contained multiple putative binding sites for the transcription factors Sp1 and MAZ but lacked a consensus TATA box. Multiple sites for the initiation of transcription were mapped in a GC-rich region from 286 bp to 211 bp upstream of the ATG triplet at the site of initiation of translation, and all of the sites were either C or G. Transfection experiments and deletion analysis allowed us to localize the promoter to a region that was no more than 93 bp upstream from the first site of initiation of transcription. We also found that ectopic expression of Sp1 and of MAZ, but not of Sp3, suppressed expression of the Sp4 promoter in a dose-dependent manner.

Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized form MDCK cells and mainly basolaterally from Caco-2 cells.

A complete cDNA encoding rabbit uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete uteroglobin mainly to the basolateral side. Both MDCK and Caco-2 cells thus secrete uteroglobin in a non-sorted manner. It has, however, previously been shown that uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type.

RAP1-like binding activity in islet cells corresponds to members of the Sp1 family of transcription factors.

Deletion and mutational analyses of the gastrin promoter have identified a binding site for the yeast transcription factor RAP1 relevant for transcriptional activation in islet cells. We here report that the mammalian transcription factors binding to this site in islet cells are the Sp transcription factor members Sp1 and Sp3. Furthermore, functional analyses revealed Sp1- and Sp3-mediated transcriptional activation of gastrin. These data reveal that the zinc finger proteins Sp1 and Sp3 do have similar binding specificities as the multifunctional yeast RAP1 protein.

Identification of residues essential for progesterone binding to uteroglobin by site-directed mutagenesis.

In order to identify amino acids directly involved in progesterone binding to rabbit uteroglobin we have mutated Phe 6, Tyr 21 and Thr 60 by site-directed mutagenesis of the uteroglobin cDNA. These residues have been postulated previously to participate in progesterone binding. High-level expression of the mutated uteroglobin cDNAs in Escherichia coli yields recombinant protein mutants that, like natural uteroglobin, form stable dimers, suggesting that the tertiary structure of the protein has not been altered. Substitution of Phe 6 by Ser or Ala does not change the progesterone binding characteristics. In contrast, replacement of Tyr 21 by Phe or Ala, drastically decreases progesterone binding. In addition, replacement of Thr 60 by Ala reduces the affinity for progesterone by a factor of three. These data suggest a direct interaction of progesterone with these two amino acids and support the idea of direct hydrogen bonding of the carbonyl (C3 and C20) of progesterone with the hydroxyl groups of Tyr 21 and Thr 60, respectively.

Regulation of the Clara cell secretory protein/uteroglobin promoter in lung.

Clara cell secretory protein/uteroglobin (CCSP/UG) is specifically expressed in the conducting airway epithelium of the lung in a differentiation-dependent manner. The proximal promoter region of the rodent CCSP/UG gene directs Clara cell specificity. Previously, it was shown that the forkhead transcription factors HNF-3 alpha and beta and the homeodomain factor TTF-1 are important transcription factors acting through this region, suggesting that they contribute to cell specificity of the CCSP/UG gene. Members of the C/EBP family of transcription factors can also interact with elements of the proximal rat and mouse CCSP/UG promoters. The onset of C/EBP alpha expression in Clara cells correlates with the strong increase of CCSP/UG expression. Thus, C/EBP alpha may play a crucial role for differentiation-dependent CCSP/UG expression. Transfection studies demonstrate that C/EBP alpha and TTF-1 can synergistically activate the murine CCSP/UG promoter. Altogether, these results suggest that C/EBP alpha, TTF-1, and HNF-3 determine the Clara cell-specific, differentiation-dependent expression of the CCSP/UG gene in murine lung. The relative importance of these three transcription factors, however, differs in rabbits and humans.

Expression of the uteroglobin promoter in epithelial cell lines from endometrium.

To understand the molecular mechanism of endometrial differentiation we have initiated an analysis of the uteroglobin promoter. Uteroglobin is normally expressed in endometrial tissues under the control of ovarian hormones. In gene transfer experiments with the Ishikawa cell line, derived from a human endometrial adenocarcinoma, we have identified several regions in the promoter of the uteroglobin gene that are responsible for its endometrium-specific expression. To evaluate the generality of these findings, we have begun cloning the promoter regions of potential endometrial markers, including the rat, mouse, and human uteroglobin gene. In the rat, expression of the uteroglobin-like gene, CC10, is dominant in the lung but is also observed in the endometrium of progesterone treated animals. A comparison of the 5'-flanking sequence of the rat and rabbit uteroglobin gene resulted in the detection of similarities and differences that could explain their differential expression in vivo. To substantiate these findings we have established several cell lines from rat endometrium using murine retroviral vectors containing a positive selection marker and various viral oncogenes, such as SV40 large T antigen, adenovirus E1A, and Ha-ras. Cell lines immortalized by SV40 T-antigen were subsequently transformed with the Ha-ras oncogene. Several cell lines exhibit properties of epithelial endometrial cells. Two cell lines generated with a temperature sensitive mutant of the SV40 large T-antigen grow as transformed cells at the permissive temperature, but differentiate upon shifting to the non-permissive temperature. These rat endometrial cell lines should be useful for the analysis of endometrium-specific gene expression and as model systems for endometrial carcinoma.

Elements of the rabbit uteroglobin promoter mediating its transcription in epithelial cells from the endometrium and lung.

The rabbit uteroglobin gene is specifically expressed in certain epithelial cells of ontogenetically unrelated origin. In the endometrium, expression is restricted to the glandular and luminal epithelium and is inducible by progesterone and estradiol. In the lung, Clara cells lining the bronchiolar epithelium show constitutive expression of uteroglobin, which is modulated by glucocorticoids. To explore the molecular basis for this cell type specificity, we have transiently transfected the uteroglobin promoter region fused to the chloramphenicol acetyl transferase gene (CAT gene) in the endometrial cell line Ishikawa; in the human lung cell line NCI-H441, which shows morphological Clara cell characteristics; in HeLa cells; and in three fibroblast cell lines. The uteroglobin promoter efficiently drives expression of the CAT gene in Ishikawa and NCI-H441 cells, but not in HeLa and fibroblast cells. To identify the responsible elements we have analyzed progressive promoter 5'-deletion mutants and randomly generated linker scanning mutants spanning the sequence from -258 to -14 of the uteroglobin promoter. Transfection experiments reveal seven mutation-sensitive regions located around -30, -70, -95, -130, -190, -230, and -255. Several mutants display strong cell type-specific phenotypes. Most significantly, the integrity of the region around -190 is essential for full CAT gene expression in Ishikawa cells, but not in NCI-H441 cells.

A comparison of mouse and rabbit embryos for the production of transgenic animals by pronuclear microinjection.

Procedures for the production of transgenic animals have low overall efficiency. To evaluate factors responsible for low efficiency, zygotes of two species, varying intensities of microscope light, different qualities of injection pipettes, and six different genes were tested for their influence on the efficiency of pronuclear gene injection for the production of transgenic rabbits and mice. Rabbit zygotes were less sensitive to mechanical manipulation during injection than mouse zygotes. Exposing zygotes to a microscope light intensity of 5550 lx significantly reduced their cleavage rate, while a lower intensity (2280 lx) did not. Using pipettes with a filament for pronuclear gene injection of mouse zygotes resulted in a higher cleavage rate of zygotes after injection than when pipettes were used without filament (30.3 vs 20.6%). Implantation rates varied between 2.9% (HB72CAT) and 23.1% (ts 58-2) depending on the gene used. No transgenic animals were obtained after injection of uteroglobin-CAT-hybrid genes (B2B3UGCAT, HB72CAT), while all other genes used (UG 11.8, UGTAg, RSV lacZ, ts 58-2) resulted in transgenic embryos, fetuses, and newborn animals.

Combinatorial action of HNF3 and Sp family transcription factors in the activation of the rabbit uteroglobin/CC10 promoter.

It has been reported that respiratory epithelium-specific transcription is mediated by thyroid transcription factor 1 and members of the HNF3/forkhead family of transcription factors. Here, we show that the uteroglobin/Clara cell 10-kDa promoters from rabbit and man are regulated by HNF3alpha and HNF3beta but not by HFH-4 and TTF-1. We have identified two HNF3-responsive elements in the rabbit uteroglobin/CC10 promoter located around 95 and 130 base pairs upstream of the transcriptional start site. Both elements contribute to promoter activity in H441 cells expressing uteroglobin/CC10 and HNF3alpha. Gene transfer experiments into Drosophila Schneider cells that lack many mammalian transcription factor homologs revealed that HNF3alpha and HNF3beta on their own cannot activate the uteroglobin/CC10 promoter. However, HNF3alpha and HNF3beta strongly enhanced Sp1-mediated promoter activation. Synergistic activation by HNF3alpha and Sp1 was absolutely dependent on the integrity of two Sp1 sites located at around -65 and -230. We show further that multiple activation domains of Sp1 are required for cooperativity with HNF3alpha. These studies demonstrate that transcription from the rabbit uteroglobin/CC10 promoter in lung epithelium is controlled by the combinatorial action of the cell-specific factor HNF3alpha and the ubiquitous factor Sp1.