Amplified cellular genes in mammalian cells frequently manifest themselves as double minute chromosomes (DMs) and homogeneously staining regions of chromosomes (HSRs). With few exceptions both karyotypic abnormalities appear to be confined to tumour cells. All vertebrates possess a set of cellular genes homologous to the transforming genes of RNA tumour viruses, and there is circumstantial evidence that these cellular oncogenes are involved in tumorigenesis. We have recently shown that DMs and HSRs in cells of the mouse adrenocortical tumour Y1 and an HSR in the human colon carcinoma COLO320 contain amplified copies of the cellular oncogenes c-Ki-ras and c-myc, respectively. Both DMs and HSRs are found with remarkable frequency in cells of human neuroblastomas. We show here that a DNA domain detectable by partial homology to the myc oncogene is amplified up to 140-fold in cell lines derived from different human neuroblastomas and in a neuroblastoma tumour, but not in other tumour cells showing cytological evidence for gene amplification. By in situ hybridization we found that HSRs are the chromosomal sites of the amplified DNA. The frequency with which this amplification appears in cells from neuroblastomas and its apparent specificity raise the possibility that one or more of the genes contained within the amplified domain contribute to tumorigenesis.

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Transcriptional coactivators such as p300 and CREB-binding protein (CBP) function as important elements in the transcription factor network, linking individual transactivators via protein-protein interactions to the basal transcriptional machinery. We have investigated whether p300 plays a role in transactivation mediated by C/EBP␤, a conserved member of the C/EBP family. We show that C/EBP␤-dependent transactivation is strongly inhibited by adenovirus E1A but not by E1A mutants defective in p300 binding. Ectopic expression of p300 reverses the E1A-dependent inhibition and increases the transactivation potential of C/EBP␤. Furthermore, we show that C/EBP␤ and p300 interact with each other and demonstrate that the sequences responsible for interaction map to the E1A binding region of p300 and the amino terminus of C/EBP␤. Finally, we show that the minimal C/EBP␤ binding site of p300 acts as a dominant-negative inhibitor of C/EBP␤. These observations identify p300 as a bona fide coactivator for C/EBP␤. C/EBP␤ is highly expressed in the myelomonocytic lineage of the hematopoietic system and cooperates with Myb to activate mim-1, a gene specifically expressed during myelomonocytic differentiation. Recent evidence has shown that Myb recruits CBP (and presumably p300) as a coactivator and, in contrast to C/EBP␤, interacts with the CREB binding site of p300-CBP. We show that p300 not only stimulates the activity of Myb and C/EBP␤ individually but also increases the synergy between them. Thus, our results reveal a novel function of p300: in addition to linking specific transcription factors to the basal transcriptional machinery, p300 also mediates the cooperation between transactivators interacting with different domains of p300.Initiation of transcription by RNA polymerase II involves the cooperation of transcription factors, binding to specific regulatory sequences, with the basal transcriptional machinery. The transcriptional coactivators p300 and the CREB-binding protein (CBP) have been recognized as key molecules involved in the communication between transcription factors and the transcriptional machinery and thus appear to be important elements of gene regulation networks (for a review, see reference 22). p300 was originally identified as a cellular interaction partner of the adenovirus E1A oncoprotein (17), and inactivation of p300 by binding to E1A appears to be one of the mechanisms by which the E1A protein suppresses transcriptional activation of certain promoters (for a review, see reference 35). Since p300 has strong sequence similarity to CBP and exhibits properties similar to those of CBP, p300 and CBP are considered functional homologs (2, 33). Several transcription factors, such as CREB, Jun, Myb, Sap-1a/Elk-1, Fos, p53, MyoD, and the nuclear hormone receptors, have now been shown to require p300 and CBP as coactivators (3-5, 10, 13, 14, 21, 23, 24, 28, 31, 42, 53). p300 and CBP do not by themselves interact with specific DNA sequences; rather, they display a variety of protein interaction surfaces tha...

The retroviral oncogene v‐myb encodes a transcriptional activator which is responsible for the activation of the mim‐1 gene in myelomonocytic cells transformed by v‐myb. The mim‐1 promoter contains several myb consensus binding sites and has previously been shown to be regulated directly by v‐myb. Here we report that the mim‐1 gene is activated synergistically by v‐myb and different C/EBP transcription factors. We have cloned a chicken C/EBP‐related gene that is highly expressed in myeloid cells and identified it as the chicken homolog of C/EBP beta. A dominant‐negative variant of chicken C/EBP beta interferes with the v‐myb induced activation of the mim‐1 gene in these cells, suggesting that C/EBP beta or another C/EBP transcription factor is required for the activation of mim‐1 by v‐myb. We found that C/EBP beta and other C/EBP transcription factors confer to fibroblasts the ability to induce the mim‐1 gene in the presence of v‐myb. Finally we show that, in contrast to v‐myb, c‐myb synergizes with C/EBP transcription factors only at low concentrations of c‐myb protein. Our results suggest a role for C/EBP beta, and possibly for other C/EBP transcription factors, in v‐myb function and in myeloid‐specific gene activation.

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C‐myb encodes a transcriptional activator that is essential for the development of the hematopoietic system but appears to lack major roles in non‐hematopoietic cells. The identification of two conserved myb‐related genes, designated A‐myb and B‐myb, has raised the possibility that these genes are functional equivalents of c‐myb in non‐hematopoietic cells. Here, we report the isolation and preliminary characterization of the mouse A‐myb gene. Mouse A‐myb maps to the proximal region of chromosome 1 and encodes a transcriptional activator with properties similar to those of the c‐myb and v‐myb proteins. During embryo‐genesis A‐myb is predominantly expressed in several regions of the developing central nervous system (CNS) and the urogenital ridge. Expression in the CNS is confined to the neural tube, the hindbrain, the neural retina and the olfactory epithelium, and coincides with the presence of proliferating immature neuronal precursor cells. In the adult mouse, A‐myb is expressed during the early stages of sperm cell differentiation and in B lymphocytes located in germinal centers of the spleen. Taken together, these results suggest a role for A‐myb in the proliferation and/or differentiation of neurogenic, spermatogenic and B‐lymphoid cells.

The oncogene v‐myb and its cellular progenitor c‐myb encode nuclear, DNA binding phosphoproteins that are thought to regulate the expression of myb‐responsive genes during myeloid differentiation. To identify such myb‐regulated genes, and to explore the mechanisms by which v‐myb affects their expression, we have established a conditional expression system for v‐myb. We have converted the v‐myb protein to an estrogen‐inducible transactivator by fusing the protein to the hormone binding domain of the human estrogen receptor. Expression of the chimeric protein in a chicken macrophage cell‐line causes estrogen‐dependent, reversible changes in the differentiation state as well as alterations in the gene expression program of the cells. We have used this estrogen‐dependent v‐myb expression system to identify a novel v‐myb regulated gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which activates the myelomonocyte-specific mim-1 gene, a natural myb target gene, by cooperating with members of the C/EBP transcription factor family. The finding that v-Myb, together with C/EBP, is sufficient to activate the mim-1 gene in heterologous cell types has implicated Myb and C/EBP as a bipartite molecular switch, which regulates the expression of myelomonocyte-specific genes. To understand the relationship between v-Myb and C/EBP in more detail, we have examined the molecular basis of the activation of the mim-1 promoter by v-Myb and C/EBPbeta, a member of the C/EBP transcription factor family highly expressed in myelomonocytic cells. We have identified a composite Myb and C/EBP response element which mediates synergistic activation of the mim-1 promoter by both factors and consists of closely spaced Myb- and C/EBP-binding sites. In vitro and in vivo protein-binding studies indicate that v-Myb and C/EBPbeta interact with each other via their DNA-binding domains. We show that this interaction is essential for the synergistic activation of the mim-1 promoter by v-Myb and C/EBPbeta. Our work therefore identifies C/EBPbeta as an interaction partner of v-Myb involved in myelomonocyte gene expression.