Biological effects of FoxJ2 over-expression.

As reported previously, we have extensively studied FoxJ2, a member of the Fork Head transcription factors family. While the biochemical and functional structures of this transcription factor are well understood, its biological function remains unknown. Here, we present data that address this point using transgenic mouse technology. We found that the birth rate and the number of transgenic animals obtained when transferring embryos over-expressing the FoxJ2 protein were lower than those obtained with embryos over-expressing a control protein, suggesting FoxJ2 overexpression has a negative effect on embryonic development. Transient FoxJ2 transgenesis experiments have confirmed that FoxJ2 over-expression has a lethal effect on embryonic development from E10.5. Moreover, in vitro culture of FoxJ2-microinjected embryos demonstrated a significant developmental blockage, indicating that FoxJ2 could also have an effect on pre-implantation stages. Most probably, these negative effects of FoxJ2 over-expression during development also explain the low percentage of adult transgenic mice obtained. Furthermore, most of the transgenic mice that lived to adulthood did not show transgene expression. In fact, the only two adult transgenic animals (one male and one female) in which FoxJ2 transgene expression was detected showed a mosaic expression and died prematurely as a result of cardio-respiratory failure. Postmortem analysis of these animals revealed a hypertrophic heart and abnormal testes in the male. In order to identify genes regulated by FoxJ2 consistent with the phenotypes observed for FoxJ2 transgenic mice, EMSA assays and co-transfection experiments were carried out. Our data indicate that the genes coding for the gap junction protein Connexin-43 and the cell-cell contact protein E-Cadherin, may be good candidates for FoxJ2-regulated genes. Interestingly, Connexin-43 and E-Cadherin show expression patterns similar to FoxJ2, and the phenotypes of Connexin-43 and E-Cadherin mutants resemble those of our FoxJ2 transgenic animals. These data suggest that the lethal effect on embryonic development of FoxJ2 overexpression, as well as the alterations observed in the heart and testes of adult transgenic mice, could be determined by changes in the transcription of genes such as Connexin-43 and/or E-Cadherin.

Fhx (Foxj2) expression is activated during spermatogenesis and very early in embryonic development.

FHX (FOXJ2) is a recently characterized human fork head transcriptional activator that binds DNA with a dual sequence specificity. We have cloned the cDNA for the mouse orthologue Foxj2 and characterized its expression in the gonads and along the early pre-implantation development of the mouse. In the testis, Foxj2 is expressed from pachytene spermatocytes to round spermatids, but not in spermatogonia. In addition to the germ lineage, only Sertoli cells of the testis showed expression of Foxj2. In the ovary, only granulosa cells of the follicles express the factor. Neither mature spermatozoa nor oocytes showed expression of Foxj2. Foxj2 expression is early activated in zygotic development, being detected since as early as 8-cell stage embryos. Both cell layers of the blastocyst: the trophectoderm (TE) and the inner cell mass (ICM), express Foxj2.

FHX.L and FHX.S, two isoforms of the human fork-head factor FHX (FOXJ2) with differential activity.

Many biological phenomena are dependent on mechanisms that fine-tune the expression levels of particular genes. This can be achieved by altering the relative activity of a single transcription factor, by post-translational modifications or by interaction with regulatory molecules. An alternative mechanism is based on competition between two or more differently active isoforms of the same transcription factor. We found that FHX, a recently characterized human fork-head transcriptional activator, may show such a mechanism for balancing its activity by expressing two differently sized isoforms, FHX.S and FHX.L, encoded by a single gene located on human chromosome 12. FHX. L and FHX.S showed different transcriptional capacities, the larger form, FHX.L, behaving as the more potent transactivator. A transactivation domain of the acidic type present only in FHX.L would account for this functional difference. The relative concentrations of these two FHX isoforms appear to vary in a number of cell types, a circumstance that may regulate the final activity of this transcription factor.

Collagen alpha1(I) gene contains an element responsive to tumor necrosis factor-alpha located in the 5' untranslated region of its first exon.

The aims of the present study were to identify the cis-acting element through which tumor necrosis factor-alpha (TNFalpha) inhibits collagen alpha1(I) gene transcription and the trans-acting factors involved in this effect in cultured hepatic stellate cells. Deletion analysis of the collagen alpha1(I) promoter demonstrated that TNFalpha inhibited gene expression through an element located between -59 and + 116 bp relative to the transcription start site. DNase I protection assays revealed a footprint between +68 and +86 bp of the collagen first exon, the intensity of which decreased when the DNA probe was incubated with nuclear protein from TNFalpha-treated hepatic stellate cells. This footprint contained a G+C-rich box. Transfection experiments demonstrated that mutations in this G+C-rich element abrogated the inhibitory effect of TNFalpha on the collagen alpha1(I) promoter. Gel retardation experiments using a radiolabeled oligonucleotide containing sequences of this region confirmed that TNFalpha treatment decreased the formation of two complexes between nuclear proteins and DNA. These complexes were efficiently blocked with an oligonucleotide containing an Spl-binding site and were supershifted with specific Spl and Sp3 antibodies. These results suggest that TNFalpha inhibits collagen alpha1(I) gene expression by decreasing the binding of Spl to a G+C-rich box in the 5' untranslated region of its first exon.

FHX, a novel fork head factor with a dual DNA binding specificity.

The HNF3/fork head family includes a large number of transcription factors that share a structurally related DNA binding domain. Fork head factors have been shown to play important roles both during development and in the adult. We now describe the cloning of a novel mammalian fork head factor that we have named FHX (fork head homologous X (FHX), which is expressed in many adult tissues. In the embryo, FHX expression showed a very early onset during the cleavage stages of preimplantation development. Polymerase chain reaction-assisted site selection experiments showed that FHX bound DNA with a dual sequence specificity. Sites recognized by FHX could be classified into two different types according to their sequences. Binding of FHX to sequences of each type appeared to occur independently. Our data suggest that either different regions of the fork head domain or different molecular forms of this domain could be involved in binding of FHX to each type of site. In transfection assays, FHX was capable of activating transcription from promoters containing FHX sites of either type.

Transcriptional induction of endothelial nitric oxide gene by cyclosporine A. A role for activator protein-1.

We have previously shown that the immunosuppressant cyclosporine A (CsA) increases the activity, the protein level, and the steady-state levels of the mRNA of the endothelial nitric-oxide synthase (eNOS) gene in bovine aortic endothelial cells (BAEC). We have now investigated the mechanisms responsible for these effects. Preincubation with an inhibitor of RNA polymerase II abolished CsA-induced eNOS up-regulation. Nuclear run-on experiments demonstrated a 1.6-fold increase in the induction of eNOS gene by CsA. In agreement with these results, transient transfections showed that CsA augmented the transactivation of the eNOS promoter. Electrophoretic mobility shift assays showed an increase in the activator protein-1 (AP-1) DNA binding activity in BAEC treated with CsA. An increase in the level of c-fos mRNA and in the nuclear content of c-Fos protein was detected in BAEC treated with CsA. Site-directed mutagenesis of the AP-1 cis-regulatory element in the context of the human eNOS promoter resulted in the abrogation of the induction mediated by CsA. Hence, up-regulation of eNOS mRNA by CsA is a transcriptional phenomenon involving the proximal AP-1 site in the 5'-regulatory region of the human eNOS gene. Furthermore, our data exemplify how immunosuppressive drugs may result in the regulation of specific genes involved in the homeostasis of endothelial function, such as eNOS.

A set of polyclonal and monoclonal antibodies reveals major differences in post-translational modification of the rat HNF1 and vHNF1 homeoproteins.

The related homeodomain-containing transcription factors HNF1 (HNF1 alpha) and vHNF1 (HNF1 beta) recognise common target DNA sequences in the regulatory regions of many genes and are expressed in several parenchymal cell types, predominantly in liver, kidney, intestine and pancreas. HNF1-null mutant mice, with a wild-type vHNF1 gene, develop normally, but die within a few weeks of birth with severe liver and kidney failure. Humans with a mutation in the HNF1 alpha gene develop non-insulin dependent diabetes on maturity (MODY 3). To determine distinctive roles for each of these proteins we produced a set of polyclonal sera and monoclonal antibodies, directed against different parts of the rat HNF1 and vHNF1 proteins. These antibodies reveal that HNF1 is present in vivo as a heterogeneous mixture of 92-98 kDa molecular mass polypeptides, a mass higher than that expected from its amino acid sequence. vHNF1 is present in the form of two isoforms of roughly the expected molecular masses, 65 and 68 kDa. In addition, some antibodies prepared against bacterially-produced HNF1 recognise vHNF1 but not HNF1, in liver and kidney extracts. Hence, we present the first evidence for differential post-translational modification of HNF1 and vHNF1 proteins.

Expression of the human gene coding for the alpha-chain of C4b-binding protein, C4BPA, is controlled by an HNF1-dependent hepatic-specific promoter.

C4b-binding protein (C4BP) is an abundant oligomeric plasma glycoprotein which controls the activation of the complement cascade through the classical pathway. In humans, the majority form of C4BP is composed of seven alpha-chains and one beta-chain, covalently linked by their C-termini. C4BP is mainly expressed in the liver. We have previously cloned and characterized the structure of the genes encoding the alpha and beta chains, C4BPA and C4BPB, respectively. Here we addressed the characterization of the mechanisms controlling the hepatic restricted expression of the C4BPA gene. We found that the C4BPA promoter is contained within the first 369 bp upstream of the transcription start site. The activity of this promoter is restricted to hepatic cells in transfection experiments. The hepatic transcription factor HNF1 interacts with a region of this promoter at -38 bp. This region is absolutely required for the activity of this promoter, suggesting that HNF1 is essential for the hepatic activity of the C4BPA promoter. We speculate that this extreme requirement of HNF1 for the activity of the human C4BPA promoter is related to the fact that this promoter lacks a TATA box.

The gene coding for the beta-chain of C4b-binding protein (C4BPB) has become a pseudogene in the mouse.

C4BP beta is one of the two polypeptides that in humans compose the plasma glycoprotein C4b-binding protein (C4BP). C4BP beta binds the anticoagulant vitamin K-dependent protein S. Two, nonmutually exclusive, roles have been proposed for the C4BP-protein S interaction. It has been suggested to play a role in the control of the protein C anticoagulatory pathway. In addition, it may serve an important role in localizing C4BP to the surface of injured or activated cells. While the physiological significance of C4BP-protein S interaction is unclear, it has clinical relevance because elevated plasma levels of C4BP are associated with increased risk for thromboembolic disorders in humans, due to an inactivation of the protein C anticoagulatory pathway. Using a human C4BP beta cDNA probe, we have isolated and characterized a genomic DNA fragment that includes the murine C4BPB gene. Murine C4BPB is a single-copy gene that maps close to the C4BPA gene in chromosome 1. It contains two exons homologous to the exons coding for the SCR-1 and SCR-2 repeats of the human C4BP beta polypeptide chain. Sequence analysis of the C4BPB exons in the Mus musculus inbred strains CBA, Balb/c, and C57BL/6, in pen-bred Swiss mice, and in Mus spretus demonstrated the presence of two in-phase stop codons that are incompatible with the expression of a functional C4BP beta polypeptide. Thus, the characterization of the murine C4BPB gene documents the peculiar situation of a single-copy gene that is functional in humans but has become a pseudogene in the mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

The transactivation potential of variant hepatocyte nuclear factor 1 is modified by alternative splicing.

Two forms of the transcription factor vHNF1 (HNF1 beta or LFB3) have been previously described, derived by alternative splicing from a common premessenger RNA, and have been called vHNF1-A and vHNF1-B. vHNF1 proteins share a homologous homeo-related DNA-binding domain with the HNF1 protein, initially characterized as a liver-restricted transcription factor, and bind to a similar sequence motif. Here we demonstrate that vHNF1-A is a stronger transactivator than vHNF1-B when assayed in transient transfections using two different promoters. vHNF1-A also binds DNA with a higher affinity suggesting that a region of the protein located immediately upstream of the homeodomain can modulate the protein/DNA interaction and transactivation. Both vHNF1 transcripts were found at a constant ratio in every tissue where vHNF1 expression could be detected, using a quantitative reverse transcriptase-polymerase chain reaction.

C4BPAL1, a member of the human regulator of complement activation (RCA) gene cluster that resulted from the duplication of the gene coding for the alpha-chain of C4b-binding protein.

The regulator of complement activation (RCA) gene cluster evolved by multiple gene duplications to produce a family of genes coding for proteins that collectively control the activation of the complement system. We report here the characterization of C4BPAL1, a member of the human RCA gene cluster that arose from the duplication of the C4BPA gene after the separation of the rodent and primate lineages. C4BPAL1 maps 20 kb downstream of the C4BPA gene and is the same 5' to 3' orientation found for all RCA genes characterized thus far. It includes nine exon-like regions homologous to exons 2-8, 11, and 12 of the C4BPA gene. Analysis of the C4BPAL1 sequence suggests that it is currently a pseudogene in humans. However, comparisons between C4BPAL1 and the human and murine C4BPA genes show sequence conservation, which strongly suggests that, for a long period of time, C4BPAL1 has been a functional gene coding for a protein with structural requirements similar to those of the alpha-chain of C4b-binding protein.

vHNF1 is expressed in epithelial cells of distinct embryonic origin during development and precedes HNF1 expression.

HNF1 (Hepatic Nuclear Factor 1) and vHNF1 are transcriptional regulators containing a highly divergent homeodomain. The first was initially found in liver nuclear extracts and is crucial for the transcription of albumin and many other hepatocyte specific genes, while the second was found in dedifferentiated hepatoma cells. Both recognize the same DNA binding site and can form homo and heterodimers in vitro and in vivo. In situ hybridization analyses have been performed to delineate the spatial and temporal pattern of expression of vHNF1 relative to HNF1 during mouse embryogenesis. The results show that accumulation of vHNF1 mRNAs expression is detected in several tissues of the embryo of both endodermal and mesodermal origin. Expression occurs in the yolk sac, the primitive gut, the liver primordium, and at different stages of kidney development in polarized epithelial structures and usually precedes that of HNF1. vHNF1 expression seems particularly prevalent with morphogenetic events in the kidney and may be a marker for certain polarized epithelium.

vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1.

vHNF1 and HNF1 are two nuclear proteins that bind to an essential element in the promoter proximal sequences of albumin and of many other liver-specific genes. HNF1 predominates in hepatocytes but is absent in dedifferentiated hepatoma cells. These cells contain vHNF1 but fail to express most of the liver traits. In the present work we have isolated cDNA clones for vHNF1 and found that it is a homeoprotein homologous to HNF1 in regions important for DNA binding. Unexpectedly, vHNF1 transactivated the albumin promoter in transfection experiments. Like the HNF1 mRNA, the vHNF1 message was found in kidney, liver and intestine although in different proportions. The fact that vHNF1 and HNF1 readily form heterodimers in vitro and the biochemical characterization of vHNF1/HNF1 heterodimers in nuclear extracts of kidney, liver and several cell lines, strongly argue that such heterodimers exist in vivo. Our results raise the possibility that heterodimerization between homeoproteins could be a common phenomenon in higher eukaryotes, which may have implications in the regulatory network sustained between these factors.

Structure of the gene coding for the alpha polypeptide chain of the human complement component C4b-binding protein.

The human gene coding for the 70-kD polypeptide of the complement regulatory component C4b-binding protein (C4BP alpha) spans over 40 kb of DNA and is composed of twelve exons. Upon transcription in liver, or in Hep-G2 cells, this gene produces a single transcript of 2,262 nucleotides, excepting the poly A tail, that presents an unusually long 5' untranslated region (5' UTR) of 223 nucleotides. The C4BP alpha gene is organized as follows: the first exon codes for the first 198 nucleotides of the 5' UTR. It is separated by a large intron from the second exon including the remaining of the 5' UTR and the coding region for the signal peptide. Each of the eight 60-amino acid repeats (short consensus repeats [SCRs]) that compose the C4BP alpha polypeptide chain is encoded by a single exon, except for the second SCR, which is split in two exons. At the 3' end of the C4BP alpha gene, the twelfth exon codes for the COOH-terminal 57 amino acids of the mature protein, which have no similarities to the SCRs, and the 245 nucleotides of the 3' UTR. Examination of the nucleotide sequence of the first exon revealed an interesting characteristic, strongly suggesting that this exon may specify a functional domain of the C4BP alpha transcript. It includes two in-phase ATG codons, in a different frame respect to that coding the C4BP alpha polypeptide, followed by an in-frame termination codon, also within the first exon. Comparison between mouse and human C4BP alpha transcripts indicates conservation of this structure within the 5' UTR. C4BP is expressed in the liver and is an acute phase protein. A computer search of the genomic sequences upstream the transcription start site demonstrates the presence of potential cis-acting regulatory elements similar to those found in the promoters of other liver-expressed and/or acute phase genes.

Human genes for the alpha and beta chains of complement C4b-binding protein are closely linked in a head-to-tail arrangement.

C4b-binding protein (C4BP) is an important component in the regulation of the complement system and also binds the anticoagulant vitamin K-dependent protein S. These activities are performed by distinct, although structurally related, polypeptides of 70 kDa (alpha chain) and 45 kDa (beta chain), respectively. In this report we have investigated the genetic relationships between these polypeptides. Using pulsed field gel electrophoresis analysis we demonstrate that the genes coding for the alpha (C4BP alpha) and beta (C4BP beta) chains are closely linked within the regulator of complement activation gene cluster. In addition, we have determined that the 3' end of the C4BP beta gene lies 3.5-5 kilobases from the 5' end of the C4BP alpha gene. These findings support the concept that the C4BP alpha and C4BP beta genes are the result of a gene duplication event.

Definition of IDDM-associated HLA DQ and DX RFLPs by segregation analysis of multiplex sibships.

Genetic variation of the DQ alpha and beta and of the DX alpha genes, detectable as RFLP in genomic DNA digests, has been suggested to improve the identification of individuals at high risk for insulin-dependent diabetes mellitus (IDDM). DNA from all members of 32 IDDM multiplex families was digested with six restriction endonucleases and the resulting fragments analyzed in Southern blots for hybridization with labeled cDNA probes for those genes. A computerized segregation analysis procedure was then used to assign fragments to haplotypes. Associations among fragments and between fragments and haplotypes characterized serologically and biochemically for their class II genes and IDDM-carrier status were calculated. The results indicate that the alleles of the DX alpha polymorphism maintain linkage disequilibrium with those of the DQ beta genes responsible for the well-known DQ beta 3.2-IDDM association, so that IDDM-carrier haplotypes carry disproportionally often both DQ beta 3.2 and DX alpha-TaqI-2.2kb. Thus, these RFLPs identify a DR-DQ-DX haplotype in high linkage disequilibrium, rather than the locus or loci that account for their high relative risk. However, four DR4-DQ beta 3.2 haplotypes that lack DX alpha-TaqI-2.2kb were encountered, two of which are "affected." These haplotypes suggest that the identification of the "disease locus" can be facilitated by the study of unusual haplotypes in which distinct IDDM-associated alleles occur separated from their neighbors of the standard genetic configurations.

A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP.

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster.