An E-box element localized in the first intron mediates regulation of the prothymosin alpha gene by c-myc.

In RAT1A fibroblasts, expression of the prothymosin alpha gene is under the transcriptional control of the c-myc proto-oncogene. We have now cloned the rat gene encoding prothymosin alpha and show that the cloned gene is regulated by c-myc in vivo. We find that regulation by c-myc is mediated by sequences downstream of the transcriptional start site, whereas the promoter is constitutive and not regulated by c-myc. We have identified an enhancer element within the first intron that is sufficient to mediate a response to Myc and Max in transient transfection assays and to activation of estrogen receptor-Myc chimeras in vivo. We find that this element contains a consensus Myc-binding site (CACGTG). Disruption of this site abolishes the response to Myc and Max in both transient and stable assays. Mutants of either Myc or Max that are deficient for heterodimerization fail to regulate the prothymosin alpha gene, suggesting that a heterodimer between Myc and Max activates the prothymosin alpha gene. Our data define the prothymosin alpha gene as a bona fide target gene for c-myc.

The functions of Myc proteins.

The functions of Myc proteins have remained a mystery for a number of years. Recent data have now provided us with a clear working hypothesis of how myc may act. What appears to be still missing is a link between the biochemical properties of this oncoprotein and its observed biological effects. Further research will be necessary to fill this void.

Characterization of an Krox-24/Egr-1-responsive element in the human tumor necrosis factor promoter.

We have analyzed in various human leukemic cell lines a previously unrecognized region within the human TNF gene promoter that contains the sequence motif 5'-CCGCCCCCGCG-3'. This GC-rich sequence maps to bps -170 and -160 of the TNF gene. Electrophoretic mobility shift assays (EMSA) combined with methylation interference analysis revealed the binding of two distinct proteins with overlapping recognition sites. Supershift assays identified the constitutive transcription factor Sp1 and the immediate-early growth-response transcription factor Egr-1/Krox-24. Interestingly, this Egr-1-related factor was induced by PMA but not by TNF. The TNF gene GC-rich sequence conferred PMA responsiveness when linked to a heterologous minimal c-fos promoter. To examine the involvement of Egr-1/Krox-24 in TNF gene regulation, a Krox-24 expression vector was used, pSCTKr24. In Jurkat T cells pSCTKr24 stimulated pTNF-286CAT that contains sequences -286 to +34 of the human TNF gene fused to the chloramphenicol acetyltransferase (CAT) gene. Moreover, pSCTKr24 also stimulated the TNF gene GC-rich sequence linked to the minimal c-fos promoter. However, deletion of this site did not result in markedly reduced TNF promoter activity, suggesting that the Egr-1/Krox-24 response element may play an auxiliary role in TNF gene regulation.

PMA-responsive 5' flanking sequences of the human TNF gene.

In order to define functional domains involved in the control of TNF-gene transcription, 5'-flanking sequences of the TNF-gene were analysed by TNF-promoter deletion mutants linked to the CAT-gene and by gel retardation assays. To this, three TNF-promoter fragments of different length were fused to the CAT reporter gene and transiently transfected into several human cell lines. We found that a 315 bp long fragment encompassing positions -285 bp to +30 bp with respect to the TNF mRNA cap site is sufficient to function as a PMA inducible promoter/enhancer in all cell lines tested. By further deletion analysis of this clone we could narrow the PMA-inducible element within a 185 bp long sequence (-285 to -110 bp). In addition, we investigated specific interactions between nuclear proteins and TNF promoter sequences using gel-retardation assays. Besides several constitutive binding activities, we could demonstrate in several cell lines a nuclear protein that is induced by PMA and binds to the fragment of the TNF promoter containing the PMA-inducible element.

Protein kinase C-independent activation of nuclear factor kappa B by tumor necrosis factor.

Tumor necrosis factor (TNF) is one of the few physiological inducers of the pleiotropic transcription factor NF kappa B. NF kappa B may play a central role in mediating TNF's gene regulatory action; however, the molecular mechanisms of TNF-induced NF kappa B activation are poorly understood. In this study, we demonstrate with two human leukemic cell lines, K562 and Jurkat, that TNF induces rapid and transient activation and translocation of protein kinase C (PK-C) from the cytosol to the membranes, which is followed by the emergence of kappa B-binding activity. In order to investigate whether TNF-mediated PK-C activation can be linked to induction of NF kappa B, we tried to block TNF action by use of various protein kinase C inhibitors as well as down-regulation of PK-C. Preincubation of Jurkat cells with protein kinase inhibitor H7 or staurosporine blocked PK-C activation by either TNF or phorbol 12-myristate 12-acetate (PMA). This pretreatment regimen completely inhibited NF kappa B activation by PMA. In contrast, TNF's ability to induce NF kappa B remained unaffected. In addition, NF kappa B was TNF-inducible in Jurkat cells depleted for PK-C by long-term exposure to high dose phorbol ester. The data indicate that PK-C is not required for NF kappa B activation by TNF and imply a novel, PK-C-independent mechanism of physiological NF kappa B activation.

Differential modulation of cyclin gene expression by MYC.

We have investigated the effects of deregulated expression of the human c-MYC protooncogene on cyclin gene expression and on the transcription factor E2F. We found that constitutive expression of MYC or activation of conditional MycER chimeras led to higher levels of cyclin A and cyclin E mRNA. Activation of cyclin A expression by MYC led to a growth factor-independent association of cyclin A and cdk2 with the transcription factor E2F and correlated with an increase in E2F transcriptional activity. In contrast, expression of the G1 phase cyclin D1 was strongly reduced in MYC-transformed cells. In synchronized cells, repression of cyclin D1 by MYC occurred very early in the G1 phase of the cell cycle.