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V Mezger

Publications and source records attributed to V Mezger.

16 recordsLinked to original sources
PubMed1999

Genomic structure and chromosomal localization of the mouse Hsf2 gene and promoter sequences.

The mouse heat shock factor 2 (HSF2) cDNA was previously cloned by homology to HSF1, the heat shock factor involved in the cellular response to stress [Sarge, K.D., Zimarino, V., Holm, K., Wu, C., Morimoto, R.I., Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. Genes Dev. 5 (1991) 1902-1911]. HSF2 is active in restricted cell types during pre- and post-implantation stages of development, and only in male germ cells of adult mice. However, the function of this factor remains elusive. We report here the cloning of the mouse Hsf2 gene and its genomic structure. We show that the gene is composed of 13 exons of variable sizes spanning at least 43kb in the genome. The transcription start site has been determined, and upstream sequences with promoter activity have been identified by their ability to direct the expression of a luciferase reporter gene in transfected cells. A preliminary analysis of the proximal promoter sequence determined that the TATA box is absent, but that a GC-rich region with several potential binding sites for transcription factors is present. The gene has been mapped to mouse chromosome 10 by in-situ hybridization on metaphase chromosomes.

Animals↗
PubMed1997

Function and regulation of heat shock factor 2 during mouse embryogenesis.

The spontaneous expression of heat shock genes during development is well documented in many animal species, but the mechanisms responsible for this developmental regulation are only poorly understood. In vertebrates, additional heat shock transcription factors, distinct from the heat shock factor 1 (HSF1) involved in the stress response, were suggested to be involved in this developmental control. In particular, the mouse HSF2 has been found to be active in testis and during preimplantation development. However, the role of HSF2 and its mechanism of activation have remained elusive due to the paucity of data on its expression during development. In this study, we have examined HSF2 expression during the postimplantation phase of mouse development. Our data show a developmental regulation of HSF2, which is expressed at least until 15.5 days of embryogenesis. It becomes restricted to the central nervous system during the second half of gestation. It is expressed in the ventricular layer of the neural tube which contains mitotically active cells but not in postmitotic neurons. Parallel results were obtained for mRNA, protein, and activity levels, demonstrating that the main level of control was transcriptional. The detailed analysis of the activity of a luciferase reporter gene under the control of the hsp70.1 promoter, as well as the description of the protein expression patterns of the major heat shock proteins in the central nervous system, show that HSF2 and heat shock protein expression domains do not coincide. This result suggests that HFS2 might be involved in other regulatory developmental pathways and paves the way to new functional approaches.

Animals↗
PubMed1997

HSP gene expression and HSF2 in mouse development.

During the pre-implantation phase of development, the mouse embryo synthesizes HSC70, and HSP90 alpha and beta at a very high rate. After implantation, the expression of HSPs appears non-coordinated and is not uniform in the different tissues. The expression of inducible HSPs appears later in development than that of constitutive members of the family. HSP25 is highly expressed early in heart and muscle development, but also in some structure of the central nervous system. HSC70 and HSP90 beta are expressed ubiquitously, but their expression reaches very high levels in the nervous system (neural tracks) and during bone morphogenesis (in the hypertrophic chondrocytes). The mechanisms involved in HSP expression during mouse embryogenesis are probably diverse, involving tissue-specific sequences. Although the DNA-binding activity and expression of the second heat shock transcription factor, HSF2, seems to be developmentally regulated, becoming detectable at the blastocyst stage and reaching a peak at day 10 of development, there is no obvious correlation between the level of this factor and the expression of HSPs. HSF2 might be involved in the onset of expression of HSPs, regulate (inhibit) their expression, or control the expression of other developmental genes yet to be discovered.

Animals↗
PubMed1997

Evidence for the involvement of mouse heat shock factor 1 in the atypical expression of the HSP70.1 heat shock gene during mouse zygotic genome activation.

The mouse HSP70.1 gene, which codes for a heat shock protein (hsp70), is highly transcribed at the onset of zygotic genome activation (ZGA). This expression, which occurs in the absence of stress, is then repressed. It has been claimed that this gene does not exhibit a stress response until the blastocyst stage. The promoter of HSP70.1 contains four heat shock element (HSE) boxes which are the binding sites of heat shock transcription factors (HSF). We have been studying the presence and localization of the mouse HSFs, mHSF1 and mHSF2, at different stages of embryo development. We show that mHSF1 is already present at the one-cell stage and concentrated in the nucleus. Moreover, by mutagenizing HSE sequences and performing competition experiments (in transgenic embryos with the HSP70.1 promoter inserted before a reporter gene), we show that, in contrast with previous findings, HSE boxes are involved in this spontaneous activation. Therefore, we suggest that HSF1 and HSE are important in this transient expression at the two-cell stage and that the absence of typical inducibility at this early stage of development results mainly from the high level of spontaneous transcription of this gene during the ZGA.

Animals↗
PubMed1994

Detection of heat shock element-binding activities by gel shift assay during mouse preimplantation development.

Heat shock gene expression is regulated by highly conserved sequence elements (HSE for "heat shock elements"). Some of heat shock genes display an atypical expression during preimplantation mouse development. We have examined the profile of HSE-binding activities (HSE-BA) in matured ovulated oocytes and during the preimplantation development by gel shift assay and quantified the data by PhosphorImager. In each of our experiments, the F9 embryonal carcinoma cell line that contains both constitutive and heat-induced activity has been used as a control. We determine the number of oocytes or embryos required to get reproducible signals and accurate quantification by PhosphorImager. Oocytes, one-cell, and two-cell embryos respond to heat shock by inducing a strong HSE-BA. At the four-cell stage, no HSE-BA can be induced by heat shock, which suggests that noninducibility of heat shock genes at this stage (when the general mechanism of transcription is well established) might result from a defect in HSF or in the mechanism of HSF activation. A progressive reappearance of the ability to induce HSE-BA by stress is observed between the eight-cell stage and the blastocyst stage, and this parallels the appearance of heat shock gene inducibility. Matured ovulated oocytes and the first cleavage stages of embryos do not contain any HSE-BA at normal temperature but we observed a HSE-BA at normal temperature at the morula stage, which is increased at the blastocyst stage. These data, which, to our knowledge, for the first time describe the profile of a DNA-binding activity during the mouse preimplantation development, could serve as a basis for the study of other transcription factors during early embryogenesis.

Animals↗
PubMed1994

Heat shock factor 2-like activity in mouse blastocysts.

The expression of heat shock genes is induced in all living cells by a series of proteotoxic treatments. Heat shock genes are also activated spontaneously during different phases of embryonic development. HSP89 alpha and HSC70 are expressed at a high level in the mouse blastocyst. A family of factors, called HSFs, are able to bind to the promoters of heat shock genes on upstream conserved elements (HSEs). HSF1 is unable to bind to HSE sequences in absence of stress. It is activated after a stress by post-translational modifications and conformational change. HSF2 shows common structural domains with HSF1; however, it is active at normal temperatures. Recently we showed the presence of an abundant HSE-binding activity in nonshocked blastocysts. We demonstrate here by using polyclonal antibodies that HSF2 is the major constituent of this constitutive HSE-binding activity. HSF2 might be involved in the control of heat shock gene expression during early mammalian embryogenesis.

Animals↗
PubMed1992

Mammalian heat shock protein families. Expression and functions.

When prokaryotic or eukaryotic cells are submitted to a transient rise in temperature or to other proteotoxic treatments, the synthesis of a set of proteins called the heat shock proteins (hsp) is induced. The structure of these proteins has been highly conserved during evolution. The signal leading to the transcriptional activation of the corresponding genes is the accumulation of denatured and/or aggregated proteins inside the cells after stressful treatment. The expression of a subset of hsp is also induced during early embryogenesis and many differentiation processes. Two different functions have been ascribed to hsp: a molecular chaperone function: chaperones mediate the folding, assembly or translocation across the intracellular membranes of other polypeptides, and a role in protein degradation: some of the essential components of the cytoplasmic ubiquitin-dependent degradative pathway are hsp. These functions of hsp are essential in every living cell. They are required for repairing the damage resulting from stress.

Animals↗
PubMed1991

On the mechanism of action of H2O2 in the cellular stress.

We propose a hypothesis according to which the reactive and reduced species of oxygen could be the intracellular inducers of the stress (or "heat-shock") response. This hypothesis is based on the following observations on Drosophila cells: a) the return to normoxia after 24 h anaerobiosis is sufficient to induce the synthesis of the "heat shock" proteins without elevation of temperature together with a rapid increase of O2 consumption; b) hydrogen peroxide introduced in the culture medium induces the early transcriptional activation of the "heat shock" genes (maximal after 5 minutes); c) hydrogen peroxide added to cellular extracts in vitro (thus acting as an intracellular metabolite) activates instantaneously the binding capacity of a "heat shock" factor to a DNA "heat shock" regulatory element. Thus, hydrogen peroxide, and possibly other reactive reduced species of oxygen, could trigger the onset of the stress (or "heat shock") response.

Animals↗
PubMed1990

Hydrogen peroxide activates immediate binding of a Drosophila factor to DNA heat-shock regulatory element in vivo and in vitro.

The synthesis of heat-shock proteins via activation of heat-shock genes occurs in response to heat and various physical or chemical stressing agents. Transcriptional activation of heat-shock genes requires a heat-shock regulatory element in their promoter, to which a heat-shock specific transcription factor binds. In Drosophila cells, the heat-shock factor already exists in unstressed cells in an inactive form and acquires the capacity to bind to the heat-shock element following stress. The mechanism of this activation is not known: neither is it known whether the different stressing agents induce the heat-shock response through a common mechanism. We previously proposed that many agents known to induce the heat-shock response (substances interfering with respiratory metabolism, agents reacting with sulphydryl groups, metals, recovery from anaerobiosis and ischemia) might act via accumulation of reactive oxygen species, i.e. superoxide ion or H2O2. We show here that H2O2, introduced either in Drosophila cell cultures or in cell extracts, was able to activate heat-shock-element binding. Activation was rapid and H2O2 concentration dependent, with a threshold of 1 microM. These results were confirmed with mouse fibroblast cells. This very rapid activation, in vivo or in vitro, suggests a direct effect of H2O2 either on the heat-shock factor itself or on its activator.

Amino Acid Sequence↗
PubMed1989

High constitutive transcription of HSP86 gene in murine embryonal carcinoma cells.

In order to investigate HSP86 heat-shock gene expression in embryonal carcinoma cell lines (EC), a partial mouse HSP86 cDNA clone was isolated and characterized. As observed for the corresponding protein, HSP86 RNA is shown to be constitutively more abundant in PCC4 and undifferentiated F9 EC cells than in fibroblasts, while its amount decreases upon F9 differentiation. Although mRNA stabilization is suggested to account in part of the high constitutive expression of the heat-shock-like protein HSC73 in F9 cells, HSP86 RNA appears as stable in fibroblasts as in F9 cells. Using run-on experiments we have established that high HSP86 expression in undifferentiated F9 cells in mainly due to enhanced transcription of the gene. Possible mechanisms responsible for this high level of transcription are discussed.

Animals↗
PubMed1989

Unusual levels of heat shock element-binding activity in embryonal carcinoma cells.

In contrast to differentiated somatic cells, mouse embryonal carcinoma (EC) cell lines spontaneously express high levels of major members of the heat shock protein (HSP) family. In addition, some EC cell lines (noninducible) are not able to induce HSP gene transcription and HSP synthesis after a stress. However, after in vitro differentiation, constitutive HSP expression decreases and the differentiated derivatives become able to induce HSP gene transcription after a stress. These cells were tested by gel shift assays for the presence of an activity able to bind the heat shock element (HSE) before and after a stress. Control fibroblasts grown at 37 degrees C did not contain significant levels of HSE-binding activity, but heat shock dramatically increased the level of HSE-binding activity. In contrast to control fibroblasts, all EC cells contained significant levels of HSE-binding activity at 37 degrees C. In the inducible EC cell line F9, as in fibroblasts, heat shock strongly increased the level of HSE-binding activity. In the noninducible EC cells, however, HSE-binding activity markedly decreased upon heat shock. During in vitro differentiation of the noninducible cell line PCC7-S-1009, the constitutive HSE-binding activity found at 37 degrees C disappeared and heat induction of the HSE-binding activity appeared. Therefore, a good correlation exists between the high spontaneous expression of some members of the HSP family and the constitutive level of HSE-binding activity in EC cells at 37 degrees C. Heat induction of HSP gene transcription correlates with a strong increase in HSE-binding activity, whereas a deficiency in heat induction of HSP gene transcription is associated with a loss of HSE-binding activity upon heat shock.

Animals↗
PubMed1989

Phenylephrine, vasopressin and angiotensin II as determinants of proto-oncogene and heat-shock protein gene expression in adult rat heart and aorta.

The expression of two oncogenes (conc) c-myc and c-fos, coding for nuclear proteins which play a regulatory role in growth and differentiation, and of two genes coding for two heat shock proteins (HSP) 68 (molecular weight 68,000) and 70 (molecular weight 70,000), which have a protective function during stress, have been investigated by Northern blot analysis of the total RNA, extracted from adult rat ventricle and aorta. (1) The two onc transcripts are absent from these tissues but their expression can be enhanced by a pretreatment with cycloheximide. (2) The HSP70 is, in part, constitutive, while HSP68 is not; both are thermo-inducible in an isolated coronary perfused rat heart. (3) The four messenger RNA (mRNA) are expressed in both ventricles and aorta, 1 or 2 hours after i.p. injection of 6 mg/kg phenylephrine or 12 IU/kg of vasopressin. (4) They are also induced by a continuous or discontinuous injection of angiotensin II (7.5 micrograms/kg per min) for 1-2 h, but only in the aorta. The lack of ventricular response to angiotensin II in rat ventricles has been attributed to the lack of angiotensin II receptors in this tissue. This indicates that, in addition to mechanical factors, circulating hormones which have in common the use of the phosphoinositol pathway, may activate the expression of genes coding for regulatory proteins. This may play a role in the genesis of both ventricular and aortic hypertrophy.

Angiotensin II↗
PubMed1988

Regulation of the heat-shock response by interferon in mouse L cells.

Interferon (IFN) is not able to induce heat-shock protein (HSP) synthesis. However IFN pretreatment of mouse L cells has been shown to enhance the decrease of overall protein synthesis which follows a heat shock, and to stimulate the accumulation of HSPs. We show here that the synthesis of a protein (the hepatitis B virus surface antigen) under the control of a Drosophila HSP 70 promoter is also stimulated in IFN-pretreated cells. The regulation by IFN takes place at two levels: first, the rate of HSP gene transcription is increased in nuclei isolated from IFN-treated cells; second, the synthesis of HSPs is prolonged after pretreatment with IFN. Experiments performed in the presence of actinomycin D show that this effect is due to a stabilization by IFN of mRNAs coding for HSPs.

Animals↗
PubMed1987

Deficient activation of heat shock gene transcription in embryonal carcinoma cells.

Heat shock protein (HSP) synthesis cannot be induced by stress in the cleavage stage embryos of many different species. For instance, no HSP synthesis can be induced in the mouse embryo before the formation of the blastocyst. Similarly, HSP synthesis is not stress inducible in some embryonal carcinoma (EC) cell lines such as PCC4 and PCC7 S-1009 (1009). We show that RNAs coding for the major stress inducible murine heat shock protein, HSP68, do not accumulate in PCC4 or 1009 EC cells in response to a stress. Using an in vitro nuclear transcription assay, we demonstrate that the transcription of the corresponding genes is not activated after a stress. A specific gene switch-off due to DNA methylation or chromatin conformation is unlikely to account for this result. Indeed, stress does not promote the activation of the heterologous Drosophila HSP70 heat shock promoter in transfection assays of these cells. In contrast, the same promoter, like endogenous HSP synthesis, becomes stress inducible in 1009 cells after in vitro differentiation. This suggests that, in contrast to differentiated cells, these EC cells, and maybe the very early mouse embryonic cells, could lack a transacting activating transcription factor or contain a repressor.

Actins↗