c-fos gene transcription in murine macrophages is modulated by a calcium-dependent block to elongation in intron 1.

Cultured mouse thioglycolate-elicited peritoneal macrophages exhibit a strong block to transcriptional elongation beyond the end of the c-fos gene first exon. This block is absent in freshly isolated peritoneal cells, appears slowly during culture, and does not require adherence of the cells. The extent of this block is largely responsible for the levels of c-fos mRNA in cultured macrophages, even after modulation by agents such as the tumor promoter phorbol myristate acetate and increased intracellular cyclic AMP, which also increase the activity of the c-fos promoter. When macrophages are cultured in the absence of mobilizable calcium, the block can no longer be relieved by any inducing agent. Conversely, upon calcium influxes, there is little alteration in the level of transcriptional initiation, but transcription proceeds efficiently through the entire c-fos locus. These results suggest the presence of an intragenic calcium-responsive element in the c-fos gene and illustrate its key role in the control of c-fos gene transcription.

In situ and in vitro evidence for intragenic binding of nuclear factors at the murine c-myc locus.

A block to transcriptional elongation within the c-myc proto-oncogene has been previously observed in a large number of different mouse and human cell types and its release is a potentially important element in the pathogenesis of some malignancies. We show here that the chromatin around the mouse c-myc exon 1-intron 1 boundary is differentially accessible to restriction enzymes in purified nuclei. Using a combination of in situ exonuclease III protection assay with in vitro footprints and gel band shifts, we have shown the existence of a stable nucleoprotein complex in this same region in mouse erythroleukemia cell nuclei. This situation is not peculiar to these cells and we have shown that the accessibility of the two BglII sites present at the beginning of intron 1 seems to depend not only upon the transcriptional state, but also upon the structural integrity of the gene.

In vivo footprints between the murine c-myc P1 and P2 promoters.

Assuming that when transcription starts at the P2 promoter of the c-myc gene sites located immediately upstream from P2 are occupied whereas in the absence of initiation they are not, the polymerase chain reaction (PCR)-based method of Mueller & Wold [(1989). Science, 246, 780-786] was used to map in vivo footprints upstream from the P2 promoter in various mouse cell lines. In cultured Friend erythroleukemic cells induced to differentiate with dimethysulfoxide (DMSO), a clear protection corresponding to ME1a2 and E2F sites was observed, consistent with in vitro band-shift and footprint data. However, in cell lines in which the gene was either silent or truncated the footprints were no longer visible. Friend c-myc transcripts decreased to a barely detectable level after 3 h of DMSO treatment. Transcription, as measured by in vitro run-on, was turned off at the level of RNA polymerase elongation rather than initiation [Mechti N., Piechaczyk, M. Blanchard, J.-M., Marty, L., Bonnieu, A., Jeanteur, Ph. & Lebler, B. (1986). Nucleic Acids Res., 24, 9653-9666]. The state of occupancy of the sites did not vary from the first hours up to 9 days of DMSO treatment, suggesting that DNA occupancy per se cannot explain premature termination, which rather would involve a more complex phenomenon.

Opposing functions of ATF2 and Fos-like transcription factors in c-Jun-mediated myogenin expression and terminal differentiation of avian myoblasts.

With the aim to identify the oncoprotein partners implicated in the c-Jun myogenic influence, we carried out stable transfection experiments of c-Jun and/or ATF2, Fra2, c-Fos overexpression in avian myoblasts. Before induction of differentiation, c-Jun repressed myoblast withdrawal from the cell cycle, as did a TPA treatment. However, after serum removal, unlike TPA, c-Jun significantly stimulated myoblast differentiation. In search for specific partners involved in this dual influence, we found that a reduction in the amounts of c-Fos and Fra2 and an increase in c-Jun proteins occurred at cell confluence, a situation likely to favor cooperation between c-Jun and ATF2 during terminal differentiation. Whereas c-Fos and Fra2 cooperated with c-Jun to abrogate myoblast withdrawal from the cell cycle and terminal differentiation, ATF2 co-expression potentiated the positive myogenic c-Jun influence. In addition, myogenin expression was a positive target of this cooperation and this regulation occurred through a stimulation of myogenin promoter activity: (1) whereas c-Fos or Fra2 co-expression abrogated c-Jun stimulatory activity on this promoter, ATF2 co-expression potentiated this influence; (2) using a dominant negative ATF2 mutant, we established that c-Jun transcriptional activity required functionality of endogenous ATF2. These data suggest that through this dual myogenic influence due to cooperations with different partners, c-Jun is involved in the control of duration of myoblast proliferation and thereafter of fusion efficiency.

A MGF/STAT5 binding site is necessary in the distal enhancer for high prolactin induction of transfected rabbit alpha s1-casein-CAT gene transcription.

The rabbit alphas1-casein gene contains a distal prolactin-dependent enhancer 3442-3285 bp 5' to the site of initiation of transcription. We have reported previously that four DNA/protein-binding sites (F1-F4) are located within this distal enhancer. We now show that one of this binding site (the F4 site) binds in vitro a MGF/STAT5-like factor. The functional importance of the F4 site was estimated by cotransfection of CHO cells with a chimeric gene containing or not the F4 sequence linked to the (-391/+1774)CAT gene and a plasmid encoding the rabbit mammary prolactin receptor. The F4 site is necessary for maximal response, of the enhancer to prolactin. However, this site has to be associated to the Fl-F3 fragment. It can be replaced by a genuine MGF/STAT5-binding site. A mutational analysis indicates that F4 and F1 sites are simultaneously involved to confer a high prolactin sensitivity.

Activation of STAT factors by prolactin, interferon-gamma, growth hormones, and a tyrosine phosphatase inhibitor in rabbit primary mammary epithelial cells.

In numerous studies on mammary epithelial cell lines multiple factors, added to the medium or contained in the serum, were required for casein gene expression. It has been shown in these systems that the mammary gland factor (MGF) is implicated in the activation of the beta-casein gene promoter. In the present study, we determined the relationship between known agents that affect casein gene expression and MGF activity using the properties of rabbit primary mammary epithelial cells to respond to PRL alone, when cultured in chemically defined medium. We demonstrate that MGF is rapidly activated by PRL alone or by human growth hormone, a natural ligand of many PRL receptors (PRL-Rs), in the cytoplasm and accumulated in the nucleus. The MGF activation by PRL occurred in the absence of endogenous extracellular matrix, a condition where casein synthesis is known to be markedly reduced. Different inhibitors of protein-tyrosine kinases, which have been shown to reduce casein mRNA synthesis, but not of protein kinase C, decrease the MGF activity. A tyrosine phosphatase inhibitor, sodium pervanadate, induced two GAS-binding complexes related to MGF and STAT1. Our data show that MGF is a latent cytoplasmic factor rapidly activated in mammary epithelial cells, by a mechanism involving a tyrosine kinase and a tyrosine phosphatase.

Demonstration in living cells of an intragenic negative regulatory element within the rodent c-fos gene.

We studied c-fos gene expression in rat fibroblasts by microinjection of regulatory DNA sequences, such as the serum response element (SRE) present in c-fos promotor, in order to compete directly with such sequences for binding of putative regulatory factors. We show that an additional fos intragenic regulatory element (FIRE) is located at the end of exon 1. When coinjected with an SRE oligonucleotide, it induced c-fos expression in quiescent cells, whereas injection of SRE sequence alone failed to do so. Moreover, injection in quiescent cells of an SRE oligonucleotide together with a p-fos-lacZ construct containing the c-fos SRE as well as an in-frame insertion of FIRE resulted in a block to beta-galactosidase expression that can be relieved by coinjection of the FIRE sequence.

A novel calcium signaling pathway targets the c-fos intragenic transcriptional pausing site.

In many cell types, increased intracellular calcium gives rise to a robust induction of c-fos gene expression. Here we show that in mouse Ltk(-) fibroblasts, calcium ionophore acts in synergy with either cAMP or PMA to strongly induce the endogenous c-fos gene. Run-on analysis shows that this corresponds to a substantial increase in active polymerases on downstream gene sequences, i.e. relief of an elongation block by calcium. Correspondingly a chimeric gene, in which the human metallothionein promoter is fused to the fos gene, is strongly induced by ionophore alone, unlike a c-fos promoter/beta-globin coding unit chimeric construct. Internal deletions in the hMT-fos reporter localize the intragenic calcium regulatory element to the 5' portion of intron 1, thereby confirming and extending previous in vitro mapping data. Ionophore induced cAMP response element-binding protein phosphorylation on Ser(133) without affecting the extracellular signal-regulated kinase cascade. Surprisingly, induction involved neither CaM-Ks nor calcineurin, while the calmodulin antagonist W7 activated c-fos transcription on its own. These data suggest that a novel calcium signaling pathway mediates intragenic regulation of c-fos expression via suppression of a transcriptional pause site.