Involvement of histone H4 gene transcription factor 1 in downregulation of vimentin gene expression during skeletal muscle differentiation.

Upon in vitro myogenesis, the intermediate filament protein vimentin is replaced by desmin, the switch in gene expression occurring essentially at the transcriptional level. Trying to elucidate the molecular mechanisms of this genetic control, we show here that the vimentin promoter is specifically recognized and activated by a protein most probably identical to H4TF-1, and that this factor is present in proliferating myoblasts but disappears upon fusion of these cells into multinucleated myotubes. Our results suggest that H4TF-1 is a differentiation stage-specific factor involved in the downregulation of vimentin gene expression during myogenesis.

NF-kappa B is developmentally regulated during spermatogenesis in mice.

To analyze NF-kappa B activity in the testis, we used murine transgenic lines carrying a LacZ reporter gene under the control of a NF-kappa B-responsive promoter (Schmidt-Ullrich et al. [1996] Dev 122:2117-2128). We constructed three independent lines containing the promoter of the gene encoding p105, the precursor of the p50 subunit. This promoter contains three NF-kappa B-binding sites in its proximal part. Our results show that in adult mice, the beta-galactosidase activity which reflects nuclear NF-kappa B activity, is first detected in spermatocytes at the pachytene stage and remains activated in the following steps of germ cell differentiation and maturation. Using transgenic mice carrying a p105nlslacZ construct with the 3 NF-kappa B sites mutated in the p105 promoter, we found a significant reduction in the transgene activity, confirming the important role of NF-kappa B in the activation of the transgene. To confirm the stage of induction during spermatogenesis, we analysed the beta-galactosidase activity in the testes from prepuberal mice in which cells synchrouneously enter meiosis. We detected the transgene activity at 18 days after birth, corresponding to the pachytene stage in spermatocytes. In nuclear extracts prepared from prepuberal mice, we found a peak of NF-kappa B DNA-binding activity made of p50 and p65 subunits at day 18 after birth, which remains high in the later stages. Further analysis showed that I kappa B alpha and beta, but not epsilon are expressed in the testes. Altogether, these data suggest that NF-kappa B factors are stage specifically controlled and may play a role during the development of sperm cells.

In vivo identification of lymphocyte subsets exhibiting transcriptionally active NF-kappaB/Rel complexes.

To analyze the NF-kappaB/Rel activity pattern in a living organism, we previously generated transgenic mice carrying a kappaB-dependent lacZ gene. In situ analysis of both primary and secondary lymphoid organs revealed a strong NF-kappaB transcriptional activity in antigen-presenting cells, some endothelial cells and sinus lining cells of the lymph node capsula with very little activity in lymphocytes and thymocytes. Using fluorescein-di-beta-D-galactopyranoside (FDG) as a vital substrate for the beta-galactosidase, we re-examined by flow cytometry the NF-kappaB/Rel transcriptional activity in our mouse model. We report here that such constitutive NF-kappaB/Rel activity was significantly detected in thymocytes at the CD44+CD25(-) stage. This constitutive activity extended with CD25 expression to the majority of the CD44(-)CD25(+) thymocytes and was then restricted to a few mature T cells. In the spleen, constitutive NF-kappaB/Rel activity was found in most B cells, unlike T cells which were largely negative. Virgin IgD(+) B cells expressed higher levels of NF-kappaB transcriptional activity than other B cell types. Altogether, these results suggest that NF-kappaB/Rel complexes are key players in the in vivo differentiation of IgD(+) B lymphocytes and possibly CD25(+) thymocytes.

Listeriolysin O-dependent activation of endothelial cells during infection with Listeria monocytogenes: activation of NF-kappa B and upregulation of adhesion molecules and chemokines.

The facultative intracellular bacterium Listeria monocytogenes is an invasive pathogen that crosses the vascular endothelium and disseminates to the placenta and the central nervous system. Its interaction with endothelial cells is crucial for the pathogenesis of listeriosis. By infecting in vitro human umbilical vein endothelial cells (HUVEC) with L. monocytogenes, we found that wild-type bacteria induced the expression of the adhesion molecules (ICAM-1 and E-selectin), chemokine secretion (IL-8 and monocyte chemotactic protein-1) and NF-kappa B nuclear translocation. The activation of HUVEC required viable bacteria and was abolished in prfA-deficient mutants of L. monocytogenes, suggesting that virulence genes are associated with endothelial cell activation. Using a genetic approach with mutants of virulence genes, we found that listeriolysin O (LLO)-deficient mutants inactivated in the hly gene did not induce HUVEC activation, as opposed to mutants inactivated in the other virulence genes. Adhesion molecule expression, chemokine secretion and NF-kappa B activation were fully restored by a strain of Listeria innocua transformed with the hly gene encoding LLO. The relevance in vivo of endothelial cell activation for listerial pathogenesis was investigated in transgenic mice carrying an NF-kappa B-responsive lacZ reporter gene. NF-kappa B activation was visualized by a strong lacZ expression in endothelial cells of capillaries of mice infected with a virulent haemolytic strain, but was not seen in those infected with a non-haemolytic isogenic mutant. Direct evidence that LLO is involved in NF-kappa B activation in transgenic mice was provided by injecting intravenously purified LLO, thus inducing stimulation of NF-kappa B in endothelial cells of blood capillaries. Our results demonstrate that functional listeriolysin O secreted by bacteria contributes as a potent inflammatory stimulus to inducing endothelial cell activation during the infectious process.

Cadherin function is required for axon outgrowth in retinal ganglion cells in vivo.

The cell-cell adhesion molecule N-cadherin strongly promotes neurite outgrowth in cultured retinal neurons. To test whether cadherins regulate process outgrowth in retinal neurons in vivo, we have blocked cadherin function in single cells by expression of a dominant negative N-cadherin mutant. We report that when cadherin function is inhibited, axon and dendrite outgrowth are severely impaired, particularly in retinal ganglion cells. Laminar migration and cell type specification, by contrast, appear unaffected. Further, expression of the catenin-binding domain of N-cadherin, which blocks cadherin-mediated adhesion in early embryos, does not affect axon outgrowth, suggesting that outgrowth and adhesion are mediated by distinct regions of the cytoplasmic domain. These findings indicate that cadherins play an essential role in the initiation and extension of axons from retinal ganglion cells in vivo.

NF-kappaB activity in transgenic mice: developmental regulation and tissue specificity.

The transcription factor family NF-kappaB/Rel is responsible for the regulation of a large number of cellular genes and some viruses. Since there is a strong similarity between the NF-kappaB/Rel family members and the Drosophila melanogaster protein DORSAL, which is activated early during embryogenesis, we were interested in determining the pattern of NF-kappaB activity during mouse development. Two lacZ reporter constructs, each driven by promoter elements that are dependent on the presence of nuclear NF-kappaB/Rel activity, were used to produce transgenic mice. The analysis of these mice did not identify nuclear NF-kappaB/Rel activity in early development prior to implantation or during the gastrulation processes. Earliest expression of the lacZ transgene was detected on day E12.5. Before birth lacZ expression was seen in discrete regions of the rhombencephalon of the developing brain, in the spinal medulla, in some of the blood vessels and in the thymus. After birth, the NF-kappaB/Rel activity in the thymus remained but nuclear activity was also found in the bone marrow, in the spleen and in the capsule of the lymph nodes. In the central nervous system, drastic changes in NF-kappaB/Rel activity could be observed in the first 3 weeks after birth, when the cortex and the cerebellum reach functional and morphological maturity. Considering the results of the p50, p65, relB and c-rel knock-out mice and our present findings, we believe that the NF-kappaB/Rel proteins known so far are probably not implicated in processes of early development and differentiation of the different tissues, but rather in maintaining their function once matured.

A 28-bp negative element with multiple factor-binding activity controls expression of the vimentin-encoding gene.

The promoter of the human vimentin-encoding gene (VIM) contains two enhancers separated by a negative region. The distal and proximal enhancers bind the transcription factors, AP-1 and NK-kappaB, respectively, which contribute to serum induction of Vim synthesis. We were interested in looking for particular regulatory elements that might be responsible for tissue-specific extinction and culture-dependent activation of human VIM. We have identified a 48-bp sequence in the distal enhancer which had not been reported before. This sequence includes a negative element, NE2, which confers transcriptional repression in transfection experiments and binds at least two factors in vitro. NE2 may participate in the differentiation-stage-specific control of VIM expression which involves multiple regulatory sequences and several positive and negative trans-acting factors.

Interplay between a new HNF3 and the HNF1 transcriptional factors in the duck hepatitis B virus enhancer.

We identified a new hepatocyte nuclear factor 3 (HNF3) binding site in the DHBV enhancer. This site is close to the hepatocyte nuclear factor 1 (HNF1) binding site, responsible for most of the enhancing activity. No differences in the migrating properties were found between this new site and the two other HNF3 sites recently described in this enhancer. Factor HNF1 strongly inhibits binding of the HNF3 factor in this newly characterized site. The two factors were never detected simultaneously on the DNA fragment, even when their respective concentrations were modified. Competition persisted after enlarging by 5 and 10 nucleotides the space between the two sites. On the contrary, when the HNF3 binding site was changed into the perfect consensus site, binding of the HNF3 factor was not inhibited any longer by HNF1 and a supershift, corresponding to the binding of both factors, was observed. Thus a limited mismatching appears to modulate the interaction between transcriptional proteins and DNA and allows a second transcriptional protein to interplay with the former one.

Chimeric integrins expressed in retinal ganglion cells impair process outgrowth in vivo.

Integrin function in retinal ganglion cell (RGC) development was examined in vivo by transfecting genes encoding various dominant forms of the chicken beta 1 integrin subunit into intact eye primordia of Xenopus embryos. RGCs expressing the chimeric chicken/Xenopus integrin receptors exhibited a marked reduction in process outgrowth with only 27% extending an axon and 41% bearing dendrites compared to control levels of 85-88%. None of the integrin constructs impaired the ability of RGC axons to pathfind appropriately or of retinal precursors to migrate to different laminar positions. Chimeric integrin expression also impaired process outgrowth in cells of the inner nuclear layer, although to a lesser extent than RGCs. Transfected diencephalic neurons, by contrast, showed normal levels of process outgrowth. These findings show that beta 1 integrins play an important role in regulating the outgrowth of axons and dendrites from RGCs in the retina but that chimeric integrins do not impair growth cone steering in general.

Binding of nuclear factors to functional domains of the duck hepatitis B virus enhancer.

We have analyzed the structures, relative organization, and activities of binding sites for nuclear factors in the duck hepatitis B virus (duck HBV) enhancer. DNase I footprinting analysis and mobility shift assays demonstrate that this enhancer of 192 bp contains at least three binding sites for transcription factors: one for hepatocyte-adipocyte C/EBP, a second for the liver-specific transactivator hepatocyte nuclear factor 1 HNF-1, and a third for a factor, called F3, which binds to a DNA sequence bearing some resemblance to that for the ubiquitous factor EF-C. Analysis of transcriptional activity reveals that oligonucleotides corresponding to the individual binding sites, inserted upstream from a heterologous promoter, display very weak enhancer activity, whereas the enhancer encompassing these three sites displays very high activity. Analysis of duck HBV enhancer mutants indicates that the deletion of any of these sites leads to a modification of transcriptional enhancer activity. The hepatocyte nuclear factor 1 binding site is crucial, since an internal deletion of 14 bp abolishes the activity. The C/EBP site can act as repressor, and the F3 site is required for full activity. Comparative analysis reveals that the nuclear factors are similar to those bound to the human HBV enhancer but that the organization of their binding sites in the duck HBV enhancer is different.

Organization and expression of intermediate filaments in epithelial cells expressing the HTLV-I Tax protein.

Intermediate filaments (IF) represent major components of the cytoskeletal network. These proteins which are differentially expressed according to the cell type, constitute a dynamic structure which not only contributes to the cell architecture but also defines its state of differentiation. Furthermore, numerous observations have shown that the IF network is altered in cells transformed by tumorigenic viruses. We have previously demonstrated that HTLV-I (human T-cell leukemia virus type I) transformed T cells were characterized by a high level of vimentin transcripts and that the HTLV-I Tax regulatory protein was able to transactivate the vimentin promoter transfected into Jurkat and HeLa cells. To enlarge the scope of this study, we investigated the effects of the Tax protein on the expression and organization of IF of epithelial cells in which the IF network is composed of vimentin and cytokeratin. To this aim, we have developed a model of epithelial cells (HeLa) stably expressing the tax sequences which were introduced by using retrovirus-mediated gene transfer. Half of the Tax expressing HeLa clones were loosely adherent to the culture surface and were displaying remarkable morphological alterations, as ascertained by the presence of round-shaped or spindle-shaped cells. In these cells, expression of this viral protein correlated to a pronounced disruption in the distribution of both the vimentin and the cytokeratin networks, as shown by immunofluorescence and ultrastructural analysis. Indeed, vimentin filaments appeared to be concentrated in discrete spots throughout the cytoplasm, while the cytokeratin filaments appeared to form a dense ring around the nucleus. More importantly, mRNA and protein analysis indicate an enhanced expression of the cytokeratin 7 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the human vimentin gene by the Tax human T-cell leukemia virus. I. Mechanisms of regulation by the NF-kappa B transcription factor.

The molecular basis for transactivation of the human vimentin gene by the Tax protein from the human T-cell leukemia virus (HTLV-1) is analyzed in this report. We first demonstrate that the factor NF-kappa B binds to the vimentin promoter. Using gel retardation assays, we found the putative NF-kappa B protein. Specific antibodies and competition experiments between the NF-kappa B-binding site in the interleukin-2R alpha and HIV-1 promoters and the vimentin promoter show that the three sites have identical affinity for the factor. We further show that the mechanisms of activation of NF-kappa B by the Tax protein involve a cellular inducer. Nuclear extract from lymphoid cells expressing Tax can induce in vitro a NF-kappa B binding activity in nonlymphoid cytosolic extract. This inducer, if preexisting in an inactivate state in T-cells which are not expressing Tax, cannot be switched to an active state by addition of partially purified Tax protein. While found in the nucleus of Tax-expressing cells in our experiments, this inducer might be cytoplasmic as well. In a first attempt to identify and characterize the inducer, we present the results of fractionation assays of nuclear extract.

Identification of a negative element in the human vimentin promoter: modulation by the human T-cell leukemia virus type I Tax protein.

The vimentin gene is a member of the intermediate filament multigene family and encodes a protein expressed, in vivo, in all mesenchymal derivatives and, in vitro, in cell types of various origin. We have previously demonstrated that the expression of this growth-regulated gene could be trans activated by the 40-kDa Tax protein of HTLV-I (human T-cell leukemia virus type I) and that responsiveness to this viral protein was mediated by the presence of an NF-kappa B binding site located between -241 and -210 bp upstream of the mRNA cap site (A. Lilienbaum, M. Duc Dodon, C. Alexandre, L. Gazzolo, and D. Paulin, J. Virol. 64:256-263, 1990). These previous assays, performed with deletion mutants of the vimentin promoter linked to the chloramphenicol acetyltransferase gene, also revealed the presence of an upstream negative region between -529 and -241 bp. Interestingly, the inhibitory activity exerted by this negative region was overcome after cotransfection of a Tax-expressing plasmid. In this study, we further characterize the vimentin negative element and define the effect of the Tax protein on the inhibitory activity of this element. We first demonstrate that a 187-bp domain (-424 to -237 bp) behaves as a negative region when placed upstream either of the NF-kappa B binding site of vimentin or of a heterologous enhancer such as that present in the desmin gene promoter. The negative effect can be further assigned to a 32-bp element which is indeed shown to repress the basal or induced activity of the NF-kappa B binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a strong enhancer element upstream from the pregenomic RNA start site of the duck hepatitis B virus genome.

The genome of the duck hepatitis B virus (DHBV) contains an enhancer element. This sequence, of 192 bp, is located in the 3'-terminal coding region of the DNA polymerase gene (nucleotides 2159 to 2351), upstream from the pregenomic RNA start site. This enhancer potentiates a marked increased activity from the heterologous thymidine kinase promoter in an orientation-independent manner and at a proximal, as well as a distal, location. The DHBV enhancer activates transcription in a relatively cell-type-independent manner. Sequence homologies with the nuclear factor EF-C binding site are located in the DHBV enhancer. By using the HepG2 nuclear extracts and the DHBV enhancer as probes, a complex was observed in mobility shift assays.

Genes occupy a fixed and symmetrical position on sister chromatids.

A high-resolution fluorescence methodology for nonisotopic in situ hybridization was applied to determine the positions occupied by several single-copy genes, DNA sequences, and integrated viral genomes on sister chromatids. The lateral and longitudinal mapping of the probes was performed on prometaphase and metaphase chromosomes. A fixed lateral position, exterior or median in relation to the longitudinal axis of the chromatids, was observed for a given probe, with a symmetrical position of the double fluorescent spots. This position appears to be independent of chromosome condensation stage from prometaphase to metaphase. These observations suggest an opposite helical-handedness conformation of DNA on both chromatids with a mirror symmetry. They support the model of chromosome packaging recently proposed by Boy de la Tour and Laemmli. Moreover, our results indicate that the last stages of chromosome condensation occur by packing down the coils without further coiling.

Effect of human T-cell leukemia virus type I tax protein on activation of the human vimentin gene.

We report that the expression of the vimentin gene, a cytoskeletal growth-regulated gene, is activated in trans by the Tax (p40x) transactivator protein encoded by the human T-cell leukemia virus type I. Expression of the Tax protein activates a number of cellular genes, such as those coding for the alpha chain of the high-affinity interleukin-2 receptor and interleukin-2. These findings indicate that the Tax protein is involved in the unregulated T-cell growth associated with human T-cell leukemia virus type I infection. Higher levels of vimentin mRNA were expressed in two human T-cell leukemia virus type I-transformed T cell lines, C91/PL and C81-66/45, when compared with that in Jurkat T cells. We demonstrate that this activation is conferred by the vimentin upstream flanking sequences. Indeed, enhanced activity was detected when constructs with the vimentin promoter linked to the chloramphenicol acetyltransferase gene were transfected in HeLa cells and in two cell lines of hematopoietic origin (Jurkat T lymphoblastoid cells and U937 promonocytic cells) together with a Tax expression plasmid. By introducing a series of deletions in the vimentin promoter, we further restrict these sequences to 30 base pairs, located between 241 and 210 base pairs upstream of the mRNA cap site. A 40-base-pair oligonucleotide containing this regulatory region proved sufficient to confer Tax inducibility upon a heterologous promoter linked to chloramphenicol acetyltransferase. Importantly, this segment includes an 11-base-pair promoter segment that has homology with the binding site for the NF-kappa B transactivating factor. Our findings indicate that constitutive expression of the vimentin gene under the control of the Tax protein may be relevant in understanding the progression of the lymphoproliferative process associated with human T-cell leukemia virus type I infection.

Expression of desmin gene in skeletal and smooth muscle by in situ hybridization using a human desmin gene probe.

We have used a probe encoding for the human desmin gene to study the expression of the desmin gene in skeletal and smooth muscle by in situ hybridization. In human skeletal muscle, the results showed a strong and homogeneous level of desmin mRNA contrasting with the faintly immunostaining of the desmin protein. In smooth muscle cells of colon and uterus, in situ hybridization and immunofluorescence staining suggests that there are some cells which do not contain desmin. The optimal condition of desmin mRNA detection was in cryostat sections fixed with paraformaldehyde and in paraffin embedded tissue with the same fixative. The human desmin probe can be used as a marker of cell differentiation and a way to study the regulation of the expression of the desmin gene in pathological events.

Regulatory elements of the human vimentin gene: activation during proliferation.

We investigated the constitution of the vimentin regulatory region through the use of cloned deletion mutants and the nucleotide sequence analysis in order to determine the elements which are implicated in the various physiological stimulations. We report that the vimentin promoter is constituted of a juxtaposition of at least 20 different putative regulatory elements illustrating the molecular tinkering theory. Fifty-eight motifs were found, representing 20 different sequences. Each of these mini-elements displays a consensus sequence homologous to or closely related to that found in regulatory regions of different genes correlated with processes of cell activation and proliferation.

Chromosomal localization of the mouse gene coding for vimentin.

The chromosomal location of the mouse gene coding for vimentin, one of the intermediate filament subunits, was determined by in situ hybridization using specific H3-labelled DNA probes. There is only one copy of the vimentin gene and it is located on chromosome 2 region A2.