Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice.

Fas is an apoptosis-signalling cell surface antigen that has been shown to trigger cell death upon specific ligand or antibody binding. Treatment of mice with an anti-Fas antibody causes fulminant hepatic failure due to massive apoptosis. To test a putative protective effect of the anti-apoptotic Bcl-2 protein, transgenic mice were generated to express the human bcl-2 gene product in hepatocytes. Early onset of massive hepatic apoptosis leading to death was observed in all nontransgenic mice treated with an anti-Fas antibody. By contrast, hepatic apoptosis was delayed and dramatically reduced in transgenic animals, yielding a 93% survival rate. These results demonstrate that Bcl-2 is able to protect from in vivo Fas-mediated cytotoxicity, and could be of significance for preventing fulminant hepatic failure due to viral hepatitis in humans.

Fast-muscle-specific expression of human aldolase A transgenes.

The expression of the human aldolase A gene is controlled by three alternative promoters. In transgenic mice, pN and pH are active in all tissues whereas pM is activated specifically in adult muscles composed mainly of fast, glycolytic fibers. To detect potential regulatory regions involved in the fast-muscle-specific activation of pM, we analyzed DNase I hypersensitivity in a 4.3-kbp fragment from the 5' end of the human aldolase A gene. Five hypersensitive sites were located near the transcription initiation site of each promoter in those transgenic-mouse tissues in which the corresponding promoter was active. Only one muscle-specific hypersensitive site was detected, mapping near pM. To functionally delimit the elements required for muscle-specific activity of pM, we performed a deletion analysis of the aldolase A 5' region in transgenic mice. Our results show that a 280-bp fragment containing 235 bp of pM proximal upstream sequences together with the noncoding M exon is sufficient for tissue-specific expression of pM. When a putative MEF-2-binding site residing in this proximal pM region is mutated, pM is still active and no change in its tissue specificity is detected. Furthermore, we observed a modulation of pM activity by elements lying further upstream and downstream from pM. Interestingly, pM was expressed in a tissue-specific way in all transgenic mice in which the 280-bp region was present (32 lines and six founder animals). This observation led us to suggest that the proximal pM region contains elements that are able to override to some extent the effects of the surrounding chromatin.

Paradoxical inhibition of c-myc-induced carcinogenesis by Bcl-2 in transgenic mice.

Here, we investigated changes in apoptosis during tumor progression by analyzing the effect of coexpressing various antiapoptotic genes on the multistage process of c-myc-induced hepatocarcinogenesis in transgenic mice. Whereas continuous c-myc gene overexpression in the liver led to cellular hepatocarcinoma, the coexpression of the bcl-2 gene inhibited the emergence of liver tumors, by inhibiting a pretumoral phase characterized by increased proliferation and apoptosis. This antioncogenic effect was specific to Bcl-2 and was not shared by other antiapoptotic genes such as bcl-xL and a dominant negative form of p53. Thus, we have shown that Bcl-2 can have a tumor suppressor effect in vivo on c-myc-induced hepatocarcinogenesis during the emergence of neoplastic foci.

Intestinal dysplasia and adenoma in transgenic mice after overexpression of an activated beta-catenin.

Mutations in the adenomatous polyposis coli gene or activating mutations in the beta-catenin gene itself are thought to be responsible for the excessive beta-catenin signaling involved in intestinal carcinogenesis. We generated transgenic mice that expressed large amounts of a NH2-terminally truncated mutant beta-catenin (deltaN131beta-catenin) in the intestine. These mice had multifocal dysplastic lesions in the small intestine, reminiscent of the early lesions observed in the mouse models of familial adenomatous polyposis. The number of apoptotic cells in the villi of these transgenic mice was 3-4-fold higher than in nontransgenic mice. Expression of the truncated beta-catenin mutant in the kidney led to the development of severe polycystic kidney disease. Our findings support the concept that deregulation of the beta-catenin signaling pathway is the major oncogenic consequence of adenomatous polyposis coli mutations in intestinal neoplasia.

Molecular cloning and characterization of a rat intestinal sucrase-isomaltase cDNA. Regulation of sucrase-isomaltase gene expression by sucrose feeding.

To investigate the regulation of expression of intestinal sucrase-isomaltase (SI) complex in response to sucrose feeding, we isolated a cDNA (RPSI1) encoding partially the pro-SI of rat intestinal mucosa. The clone consists of 1929 mRNA-derived nucleotides, which covered the region including the C-terminal part of the isomaltase and the N-terminal part of the sucrase in the final SI complex. Nucleotide and amino-acid sequences of RPSI1 were compared with their corresponding regions in rabbit pro-SI. A greater similarity was found in sucrase parts than in isomaltase parts of the two species. Northern blot analysis revealed that the mRNA levels of SI increased rapidly after sucrose force feeding, while those of rats fed a carbohydrate-free diet did not. These changes in mRNA levels correlated with the corresponding enzyme activities. The results demonstrate that the induction of SI activities is directly associated with an increase in SI mRNA levels. Our results also suggest that circadian modulation of SI transcription may occur in basic SI gene expression.

Transgenic analysis of the response of the rat calbindin-D 9k gene to vitamin D.

The promoter of the calbindin-D 9k (CaBP9k) gene, previously analyzed in transgenic mice, contains all of the information necessary for expression of a transgene similar to the endogenous gene and also for an appropriate response to vitamin D. In the present study we first investigated the role of a putative vitamin D-responsive element (9k/VDRE), located at nucleotides -489 to -445 on the rat CaBP9k promoter gene, using transgenic mice. As expected, the pattern of transgene expression in mice carrying this putative VDRE mutated in its whole promoter context was similar to that in mice bearing the wild-type sequence. These transgenic mice also responded to 1,25-dihydroxyvitamin D3 in the same way as those bearing the wild-type transgene and as those carrying a transgene with a large deletion (from -2894 to -117) eliminating the putative 9k/VDRE. Thus, the putative 9k/VDRE is not required for the control of rat CaBP9k gene expression by vitamin D in vivo. We also found that responsiveness to 1,25-dihydroxyvitamin D3 depends on the site at which the transgene is integrated into the host genome, in a tissue-specific manner. These data together with the fact that vitamin D-responsive sequences are present in a two-module region (from -3731 to -2894 and/or -117 to +365) and that this region does not contain any classical VDRE show that the CaBP9k gene is submitted to a non-conventional control by vitamin D.

Bidirectional activity and orientation-dependent specificity of the rat aldolase C promoter in transgenic mice.

We previously reported that the rat aldolase C 115 bp promoter is sufficient to ensure the brain specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice. We identify in a further reduced 84 bp promoter several putative binding sites for the transcriptional factors Sp1, USF, AP1, and AP2. Deletion or mutation of these partially overlapping binding sites results in inactivation of the cognate transgenes. Moreover, we show that the 115 bp sequence is able to direct bidirectional transcription in vivo but, surprisingly, transcriptional activity in the opposite direction is no more brain specific.

Regulated expression of mature human insulin in the liver of transgenic mice.

Transgenic mice expressing either human proinsulin cDNA or mutated proinsulin cDNA in the liver were created. The human proinsulin cDNA was mutated to generate a protein cleavable by the ubiquitous prohormone convertase furin, thus leading to mature insulin peptide. All transgenic lines expressed human C-peptide in the blood, whose level varied according to nutritional conditions. High performance liquid chromatography fractionation of mouse serum revealed that mutant proinsulin was effectively processed into mature insulin in vivo. This transgenic mouse model provides a useful tool for further prospects of gene therapy of insulin-dependent diabetes mellitus.

A mouse Sertoli cell line expressing anti-Müllerian hormone and its type II receptor.

Anti-Müllerian hormone (AMH) induces the regression of Müllerian ducts in the male foetus; it is secreted by prepubertal testicular Sertoli cells and repressed at puberty. Using an AMH promoter/Simian virus 40 (SV40) oncogene fusion gene, we generated transgenic mouse lines exhibiting heritable Sertoli cell tumorigenesis. One cell line, derived from an adult male, expressed mRNAs characteristic of mature Sertoli cells, but no AMH. Two other cell lines were obtained from pretumoral testes at 6.5 days. One was cloned to yield SMAT1, whose expression pattern was characteristic of prepubertal Sertoli cells, namely no transferrin and high SF-I and AMH expression. SMAT1 also secretes AMH protein into the culture medium and expresses the AMH receptor. To the best of our knowledge, this is the first Sertoli cell line stably expressing AMH and its receptor. Our results show that, in targeted oncogenesis, the timing of cell line derivation plays a critical role even when using a developmentally regulated promoter.

Tissue specificity of L-pyruvate kinase transgenes results from the combinatorial effect of proximal promoter and distal activator regions.

The L-type pyruvate kinase (L-PK) gene is regulated by diet and hormones and expressed at high levels in the hepatocytes, enterocytes, and proximal tubular cells of the kidney and at low levels in the endocrine pancreatic cells. Two regulatory regions have been shown to be important in transgenic mice to confer on a reporter gene a similar tissue-specific and diet-responsive expression: a proximal promoter fragment, with binding sites for the tissue-specific hepatocyte nuclear factors 1 and 4, and presence of the glucose-response element (GIRE) and a distal activator corresponding to a liver-specific hypersensitive site at -3000 bp with respect to the cap site. Although the proximal promoter is able to confer by itself tissue-specific expression on a reporter gene, its activity in vivo is strongly stimulated by the distal activator. To determine the possible role of the distal region on diet responsiveness and tissue specificity of the L-PK gene expression, we have created lines of transgenic mice in which the gene for SV40 T antigen (Tag) was directed by composite regulatory sequences consisting of the L-PK promoter and different enhancers: either the SV40 early enhancer (SV) or the H enhancer of the aldolase A gene (H). The induction of the composite H-PK/Tag and SV-PK/Tag transgenes by a carbohydrate-rich diet in the liver was similar to that of the endogenous L-PK gene. This suggests that in fasted mice the L-PK promoter, and especially the GIRE, is able to silence the activating influence of a strong viral enhancer such as the SV40 enhancer. The H-PK/Tag mice expressed the transgene similarly to the endogenous gene, except in the pancreas, where expression was practically undetectable. Consistently, whereas L-PK/Tag mice develop insulinomas, H-PK/Tag mice develop only hepatomas. In contrast, the transgene expression was partly aberrant in SV-PK/Tag mice. In addition to a normal activation of the transgene in the liver, a strong expression was also detected in the kidney medulla, whereas the transgene was practically silent in enterocytes. Finally, the effect of the distal region (-2070 to -3200) on an ubiquitous promoter was tested by ligating the distal L-PK gene fragment in front of a thymidine kinase/CAT transgene. Such a transgene was constantly expressed in the pancreas and, strikingly, in the brain. It appears, therefore, that the L-PK distal activator exhibits, by itself, a certain neuropancreatic specificity required in combination with the proximal promoter for L-PK gene expression in pancreas endocrine cells.

Exploration of a liver-specific, glucose/insulin-responsive promoter in transgenic mice.

The functional role of the different sites binding transcriptional factors on the tissue-specific, glucose-responsive promoter of the L type pyruvate kinase gene (L-PK) has been investigated in transgenic mice. These sites are able to bind, from 3' to 5', HNF1, NF1, HNF4, and MLTF/USF, respectively. We have compared the level of chloramphenicol acetyltransferase reporter transgene expression when driven by a L-PK promoter fragment of either -96 base pairs (bp) (containing only the HNF1 binding site) or -150 bp (lacking the MLTF/USF binding site) or driven by a -183-bp L-PK promoter fragment with or without the NF1 binding site. Our results demonstrate that: 1) HNF1 alone is not sufficient to promote an efficient L-PK gene transcription in vivo; 2) with only binding sites for HNF1, NF1, and HNF4, though the tissue-specific pattern of expression is respected, the level of the gene transcription is low and the hormonal control is lost; 3) the MLTF/USF binding site is the target of the hormonal control, required for both positive response to carbohydrates and negative response to glucagon; 4) the role of NF1 in the promoter activity could be to negatively modulate the L-PK gene expression in the different tissues, without interfering with the glucose and hormone responsiveness.

Identification and functional characterization of an erythroid-specific enhancer in the L-type pyruvate kinase gene.

The rat L-type pyruvate kinase gene is transcribed either from promoter L in the liver or promoter L' in erythroid cells. We have now cloned and functionally characterized an erythroid-specific enhancer, mapped in the fetal liver as hypersensitive site B (HSSB) at 3.7 kilobases upstream from the promoter L'. Protein-DNA interactions were examined in the 200-base pair core of the site by in vivo footprinting experiments. In the fetal liver, footprints were revealed at multiple GATA and CACC/GT motifs, whose association is the hallmark of erythroid-specific regulatory sequences. Functional analysis of the HSSB element in transgenic mice revealed properties of a cell-restricted enhancer. Indeed, this element was able to activate the linked ubiquitous herpes simplex virus thymidine kinase promoter in erythroid tissues. The activation was also observed in a variety of nonerythroid tissues known to synthesize GATA-binding factors. In the context of L'-PK transgenes, HSSB was not needed for an erythroid-specific activation of the L' promoter, while it was required to stimulate the L' promoter activity to a proper level. Finally, HSSB cannot be replaced by strong ubiquitous viral or cellular enhancers, suggesting a preferential interaction of the HSSB region with the L' promoter.

Characterization of the aldolase B intronic enhancer.

The aldolase B gene is transcribed at a high level in the liver, kidney, and small intestine. This high level of gene expression results from cooperation between a weak but liver-specific promoter and an intronic activator. A deletional study of this activator present in the first intron allowed us to ascribe the maximal enhancer function to a 400-base pair (bp) fragment (+1916 to + 2329). This enhancer is highly liver-specific and enhances the activity of heterologous minimal promoters in a position and distance-independent fashion in transiently transfected Hep G2 hepatoma cells. The aldolase B enhancer is composed of two domains, a 200-bp module (Ba) inactive by itself but which synergizes with another 200-bp module (Bb) that alone retains 25% of the total enhancer activity. The Bb sequence is 76% homologous between human and rat genes and contains several binding sites for liver-enriched nuclear factors. By electrophoretic mobility shift assays, we demonstrated that elements 5 and 7 bind hepatic nuclear factor 1 (HNF1), whereas element 2 binds hepatic nuclear factor 4 (HNF4). A functional analysis of the enhancer whose elements have been mutated demonstrated that mutation of any of the HNF1 sites totally suppressed enhancer activity, whereas mutation of the HNF4-binding site reduced it by 80%.

The human pH aldolase A promoter directs widespread but muscle-predominant expression in transgenic mice.

In order to identify regulatory elements that direct widespread in vivo expression of a linked gene, we have examined one of the human aldolase A alternative promoters, the ubiquitous pH promoter, which is active in most foetal and adult tissues. We have used the pH promoter region to drive expression of an heterologous CAT reporter gene in transgenic mice. We show that a short 820 bp pH promoter fragment is able to confer a ubiquitous and reproducible activity pattern on the CAT reporter gene in most of the transgenic lines analysed, with a particularly high level of expression in adult skeletal muscle. Activity of this transgene was detected from early embryonic stages. Therefore, this pH promoter region appears to be a powerful tool to direct ubiquitous and early expression of a transgene in vivo. Deletion analysis revealed that: (i) the region between -651 and -369 bp relative to the pH promoter transcription start site includes DNA elements capable of overriding effects of the surrounding chromatin at the integration site, (ii) the region between -285 and -211 bp is involved in pH promoter tissue-specific expression pattern in skeletal muscle and/or nervous tissues, (iii) the region located between -211 and -108 bp is necessary for its ubiquitous and muscle-predominant activity and (iv) the most proximal region downstream from -108 bp is still sufficient to confer an activity in brain and lung.

Bcl-2 targeted overexpression into the erythroid lineage of transgenic mice delays but does not prevent the apoptosis of erythropoietin-deprived erythroid progenitors.

Erythropoietin (Epo) is known to control the erythroid developmental program through various biologic activities including maintenance of viability, cell proliferation, and/or cell maturation. In vitro experiments showed massive apoptosis in cultures of Epo-deprived colony-forming unit-erythroid (CFU-E) progenitors, demonstrating the Epo requirement of late-stage erythroid progenitors for survival. Based on these data, a model has been proposed whereby from CFU-E to proerythroblast stages, Epo acts rather as a survival factor than a proliferating factor. To investigate the relationship between Epo dependence and apoptotic mechanisms, we generated transgenic mice expressing the antiapoptotic human bcl-2 gene product in erythroid progenitors. Transgenic animals developed without any evidence of erythropoietic disorders. In vitro studies showed that overexpression of bcl-2 in erythroid progenitors delayed, but did not prevent the loss of CFU-E from Epo-deprivation. By measuring burst-forming unit-erythroid (BFU-E) and CFU-E-derived colonies, an enhanced sensitivity to low levels of Epo was demonstrated in adult bone marrow of transgenic mice with respect to nontransgenic animals. No spontaneous erythroid colonies were, however, observed in vitro in the absence of the cytokine, indicating that overexpression of bcl-2 is not sufficient to induce by itself a complete erythroid differentiation. Taken together, our data indicate that targeted erythroid overexpression of bcl-2 fails to alter the normal erythropoietic development in vivo and that erythroid progenitors remain strictly dependent on Epo for their survival.

An overlapping CArG/octamer element is required for regulation of desmin gene transcription in arterial smooth muscle cells.

The desmin gene encodes an intermediate filament protein that is present in skeletal, cardiac, and smooth muscle cells. This study shows that the 4-kb upstream region of the murine desmin promoter directs expression of a lacZ reporter gene throughout the heart from E7.5 and in skeletal muscle and vascular smooth muscle cells from E9. 5. The distal fragment (-4005/-2495) is active in arterial smooth muscle cells but not in venous smooth muscle cells or in the heart in vivo. It contains a CArG/octamer overlapping element (designated CArG4) that can bind the serum response factor (SRF) and an Oct-like factor. The desmin distal fragment can replace a SM22alpha regulatory region (-445/-126) that contains two CArG boxes, to cis-activate a minimal (-125/+65) SM22alpha promoter fragment in arterial smooth muscle cells of transgenic embryos. lacZ expression was abolished when mutations were introduced into the desmin CArG4 element that abolished the binding of SRF and/or Oct-like factor. These data suggest that a new type of combined CArG/octamer element plays a prominent role in the regulation of the desmin gene in arterial smooth muscle cells, and SRF and Oct-like factor could cooperate to drive specific expression in these cells.

Differential protective effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice.

Fas ligand (CD95L) and tumor necrosis factor-alpha (TNF-alpha) are pivotal inducers of hepatocyte apoptosis. Uncontrolled activation of these two systems is involved in several forms of liver injury. Although the broad antiapoptotic action of Bcl-2 and Bcl-xL has been clearly established in various apoptotic pathways, their ability to inhibit the Fas/CD95- and TNF-alpha-mediated apoptotic signal has remained controversial. We have demonstrated that the expression of BCL-2 in hepatocytes protects them against Fas-induced fulminant hepatitis in transgenic mice. The present study shows that transgenic mice overexpressing BCL-XL in hepatocytes are also protected from Fas-induced apoptosis in a dose-dependent manner. Bcl-xL and Bcl-2 were protective without any change in the level of endogenous Bcl-xL or Bax and inhibited hepatic caspase-3-like activity. In vivo injection of TNF-alpha caused massive apoptosis and death only when transcription was inhibited. Under these conditions, PK-BCL-XL mice were partially protected from liver injury and death but PK-BCL-2 mice were not. A similar differential protective effect of Bcl-xL and Bcl-2 transgenes was observed when Fas/CD95 was activated and transcription blocked. These results suggest that apoptosis triggered by activation of both Fas/CD95 and TNF-alpha receptors is to some extent counteracted by the transcription-dependent protective effects, which are essential for the antiapoptotic activity of Bcl-2 but not of Bcl-xL. Therefore, Bcl-xL and Bcl-2 appear to have different antiapoptotic effects in the liver whose characterization could facilitate their use to prevent the uncontrolled apoptosis of hepatocytes.

Expression of myogenin during embryogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site.

Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a beta-galactosidase transgene during embryogenesis. We show here that mutation of one of the DNA motifs present in this region, the MEF3 motif, abolished correct expression of this beta-galactosidase transgene. We have found that the proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin.

Upstream elements involved in vivo in activation of the brain-specific rat aldolase C gene. Role of binding sites for POU and winged helix proteins.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a 115-base pair (bp) promoter fragment was able to ensure the brain-specific expression of the chloramphenicol acetyltransferase (CAT) reporter gene in transgenic mice, but only at a low level (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). Here we show that in vivo activation of this promoter at a high level requires cooperation between an upstream 0.6-kilobase pair (kb) fragment and far upstream sequences. In the 0.6-kb region, a 28-bp DNA element is shown to include overlapping in vitro binding sites for POU domain regulatory proteins and for the Winged Helix hepatocyte nuclear factor-3beta factor. An hepatocyte nuclear factor-3beta-binding site previously described in the short proximal promoter fragment is also shown to interact in vitro with POU proteins, although with a lower affinity than the 28-bp motif. Additional binding sites for POU factors were detected in the upstream 0.6-kb sequences. Progressive deletion in this region resulted in decreased expression levels of the transgenes in mice, suggesting synergistic interactions between these multiple POU-binding sites. We propose that DNA elements characterized by a dual binding specificity for both POU domain and Winged Helix transcription factors could play an essential role in the brain-specific expression of the aldolase C gene and other neuronal genes.

Intestinal expression of the calbindin-D9K gene in transgenic mice. Requirement for a Cdx2-binding site in a distal activator region.

The calbindin-D9K gene encodes a vitamin D-induced calcium-binding protein that is expressed as a marker of small intestine differentiation. We have shown that 4580 base pairs of its 5' DNA regulatory region can target reporter transgene expression in the intestine and cause this transgene to respond like the endogenous gene to vitamin D active metabolite and that the homeoprotein Cdx2 is bound to the TATA box in the intestine. We now show that the 4580 base pairs construct confers a differentiated pattern of reporter transgene expression in the intestine and that cooperation between the proximal promoter and a distal element located in an opened chromatin structure is responsible for the intestinal expression and vitamin D responsiveness of the transgene. Gel shift and footprinting assays using duodenal nuclear extracts indicate that this distal element contains a Cdx2-binding site. Finally, a mutation in this distal Cdx2-binding site dramatically decreases intestinal expression in transgenic mice. This report, using an in vivo approach, demonstrates the crucial role of Cdx2 for the transcription of an intestinal gene.