An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.     

Long-range fragmentation of the eukaryotic genome by exogenous and endogenous nucleases proceeds in a specific fashion via preferential DNA cleavage at matrix attachment sites

Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treated with different nucleases. The long-range distribution of DNA cleavage sites in the amplified c-myc gene locus was then analyzed by pulsed field gel electrophoretic separation of the released 50-kilobase to 1-megabase DNA fragments followed by indirect end labeling. Exogenous DNase I and nucleases specific for the single-stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleavage sites were located close to or even colocalized with matrix attachment regions, which were mapped independently using a recently developed procedure for DNA loop excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachment regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fragmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca(2+)- and Mg(2+)-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome.     

Inhibition of gene expression by steroid hormone receptors via a negative glucocorticoid response element: evidence for the involvement of DNA-binding and agonistic effects of the antiglucocorticoid/antiprogestin RU486

We have used a negative glucocorticoid response element (nGRE) from the bovine prolactin promoter linked to the gene for chloramphenicol acetyltransferase (PRL3CAT) to study the inhibition of gene expression by steroid hormone receptors. This nGRE increased basal expression from a heterologous promoter in COS-7 cells. In the presence of cotransfected glucocorticoid (GR), androgen, or progesterone receptor (PR) expression vectors and their cognate ligands, the expression of PRL3CAT could be repressed, indicating that these steroid receptor subfamily members could function through the same negative response element. No repression was observed with the estrogen receptor, showing that the repressive effect was specific for members of the GR-subfamily. Mutation of three amino acids within the GR-DNA binding domain that determine the specificity of GR-GRE interaction abolished the ability of the GR to inhibit the expression of PRL3CAT, demonstrating the requirement for DNA binding of the GR in the mechanism of repression. The antiglucocorticoid/antiprogestin RU486 when bound to PR or GR also repressed the expression of the PRL3CAT, but higher concentrations of RU486 were required to obtain an effect with the GR when compared to the PR. RU486 was unable to antagonize the effect of progestins on PRL3CAT and only partially antagonized the glucocorticoid repression. Thus, regarding the repression of PRL3CAT, RU486 acted as an agonist when bound to the PR and as a partial agonist when bound to the GR.     

Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression

The daily practice of radiation oncology is increasingly influenced by the late tolerance of normal tissues. The treatment decision must be based on detailed arguments and the physician's duty to extensively inform his patients is emphasised every day. The incidence and severity of radiation-induced sequelae and late complications can be reduced by decreasing the total dose to the normal tissues, and by decreasing the dose protraction, provided that the interval between fractions remains longer than 6 to 8 hours. This approach yields a selective protection of late responding normal tissues, since tumours are less sensitive to the effects of fractionation. Despite its own limitations, the linear- quadratic model is nowadays the standard method to compare the biological effects of different radiation treatments.     

Tissue-restricted expression of the cardiac alpha-myosin heavy chain gene is controlled by a downstream repressor element containing a palindrome of two ets-binding sites

The expression of the alpha-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in alpha-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac alpha-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the alpha-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the alpha-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the alpha-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the alpha-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the alpha-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the alpha-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the alpha-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.     

The construction of a beta-lactamase-encoding ApR gene cassette flanked by recognition sites for NotI, SacII, MluI, SplI, BssHII and NarI

An ApR gene cassette was constructed using the gene present in the PBR322 derivative pTZ18. The cassette is maintained in the plasmid pcLINK-1 and may be excised with any of the six rare cutting enzymes NotI, SacII, MluI, SplI, BssHII or NarI. By using a double-digestion procedure, the ApR gene may be excised with two different protruding ends. In the process of constructing the pcLINK-1 plasmid the multiplecloning site of pTZ18 was extended with recognition sites for the enzymes NotI, SacII, MluI, SplI, BssHII and NarI.     

Identification of a novel class of genomic DNA-binding sites suggests a mechanism for selectivity in target gene activation by the tumor suppressor protein p53

There are two response elements for p53 in the promoter of the gene for the cyclin-dependent kinase inhibitor p21. The binding of p53 to the 5' site was enhanced by incubation with monoclonal antibody 421, whereas the binding of p53 to the 3' site was inhibited. Mutational analysis showed that a single-base change caused one element to behave like the other. A response element in the human cdc25C promoter is bound by p53 with properties similar to the 3' site. These results identify two classes of p53-binding sites and suggest a mechanism for target gene selectivity by p53.