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The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

Epstein-Barr virus (EBV) infects and transforms primary B lymphocytes in vitro. Viral infection initiates the cell cycle entry of the resting B lymphocytes. The maintenance of proliferation in the infected cells is strictly dependent on functional EBNA2. We have recently developed a conditional immortalization system for EBV by rendering the function of EBNA2, and thus proliferation of the immortalized cells, dependent on estrogen. This cellular system was used to identify early events preceding induction of proliferation. We show that LMP1 and c-myc are directly activated by EBNA2, indicating that all cellular factors essential for induction of these genes by EBNA2 are present in the resting cells. In contrast, induction of the cell cycle regulators cyclin D2 and cdk4 are secondary events, which require de novo protein synthesis.

BL67 and BL18 are Burkitt's lymphoma cell lines with t(8;14) translocations (the breakpoint is in the first exon and first intron, respectively) in which the mu‐heavy chain switch region is fused to the c‐myc gene in head to head orientation. In both cell lines only aberrant c‐myc RNAs are found. BL67 cells contain two c‐myc RNA species of 2.4 and 3.5 kb. The 2.4‐kb RNA is initiated at several cryptic promoters in the first intron. The 3.5‐kb RNA is transcribed from the immunoglobulin heavy chain anti‐sense strand across the breakpoint of the translocation into the first exon of the c‐myc gene and is then normally spliced using the physiological splice donor and acceptor sites of the c‐myc gene. BL18 contains c‐myc RNA of 2.4 kb initiated at cryptic promoters in the first intron and additional RNAs of 0.90 kb and 0.74 kb transcribed from the dual c‐myc promoters on the reciprocal fragment of the translocation. The cytoplasmic turnover of these RNAs differs significantly from that of the normal c‐myc message. The 3.5‐kb RNA of BL67 cells and the 0.90‐kb and 0.74‐kb RNAs of BL18 cells, which are both hybrid molecules consisting of c‐myc and immunoglobulin sequences, have a half‐life of several hours in contrast to the normal c‐myc message with a half‐life of 15 min. The aberrant 2.4‐kb c‐myc RNAs of BL67 and BL18 cells are also more stable than the normal c‐myc message and disappear with a half‐life of 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)

In the DNA of the adenovirus type 12 (Ad12)‐transformed hamster cell line T637 approximately 20‐22 viral DNA molecules per cell are covalently linked to cellular DNA. Spontaneously arising morphological revertants of T637 cells have lost the bulk of the viral DNA. We have been able to mimic the excision event of viral DNA, as it occurs during reversion, by autoincubation of isolated nuclei from T637 cells. The same Ad12 DNA sequences, which had been deleted in morphological revertants, proved highly sensitive to endogenous nucleases in isolated nuclei of T637 cells. Viral DNA sequences, which persisted in the revertants, are resistant to endogenous nucleases in isolated T637 nuclei. All attempts to clone the nuclease‐sensitive sites of Ad12 DNA in cell line T637 have so far failed. After denaturation and renaturation of T637 DNA followed by treatment with S1 nuclease, large fold‐back structures of DNA have been found. These snap‐back structures were derived from precisely those viral DNA restriction fragments which were uncloneable. The fragments containing palindromic sequences were both highly sensitive to endogenous nucleases in isolated T637 nuclei and were absent from the DNA of all revertant cell lines. Moreover, the palindromic sequences are susceptible to the phage T4‐specific endonuclease VII which specifically attacks cruciform structures in DNA. The peculiar structures at the termini of integrated Ad12 DNA molecules are highly sensitive to endogenous nucleases in isolated nuclei. These nucleases may be related to the reversion event.

In Burkitt's lymphoma (BL) cells the normal c‐myc allele is usually silent or expressed at very low levels. Here we demonstrate that the normal c‐myc allele can be induced in BL cells by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). TPA did activate the normal c‐myc alleles in Raji(P207), BL36, P3HR1, Jijoye and LY91 cells, but not in Raji(DE88), BL41, BL67, LY47 and KK124 cells. C‐myc RNA derived from the normal allele appeared 6 h after treatment with TPA and showed the characteristic preferential usage of the second promoter. This induction could not be inhibited by cycloheximide. Despite the differences in c‐myc induction in Raji(P207) and Raji(DE88) cells, c‐fos and the early Epstein‐Barr virus gene DR were induced to a similar extent and with similar kinetics by TPA. Nuclear run‐on experiments suggest that the normal c‐myc allele in Raji cells is activated at least in part by releasing a block to RNA elongation at the end of c‐myc exon 1. Expression of the translocated c‐myc alleles was also affected by TPA; however, only if cycloheximide was simultaneously present. TPA plus cycloheximide induced a rapid decrease of c‐myc RNA derived from the translocated allele within 6 h, whereas cycloheximide alone led to abolition of c‐myc RNA after 16‐24 h. This rapid decline of c‐myc RNA was observed in Raji and BL41 cells, but not in three cell lines with variant t(2;8) and t(8;22) translocations.

We have examined the effect of sodium butyrate, a potent inducer of differentiation in various cell systems, on the steady state RNA level and transcriptional activity of the c‐myc gene in Burkitt's lymphoma cells. Following sodium butyrate treatment a rapid decrease of c‐myc RNA was observed in all Burkitt's lymphoma cell lines studied, irrespective of the type of translocation, the location of the breakpoint relative to c‐myc or of the association with EBV. Since cellular genes induced by interferon are suspected to play a role in c‐myc regulation we have studied transcription of the 2‐5A synthetase gene in sodium butyrate‐treated Burkitt's lymphoma cells. Transcriptional activity and steady state mRNA levels of the 2‐5A synthetase gene were induced by sodium butyrate. The time course of induction excluded, however, that the decrease of c‐myc RNA is caused by induction of the 2‐5A synthetase/RNase L endonuclease system. The reduction of c‐myc RNA is caused, at least in part, by a reduced transcription rate, as shown by nuclear run‐on analysis. The fact that sodium butyrate is capable of downregulating a truncated c‐myc gene indicates that an important target site of transcriptional regulation is located outside the region encompassing the upstream regulatory sequences, the dual promoters and the leader region.

The nucleosomal structure of active and inactive c-myc genes has been analyzed in detail in undifferentiated and differentiated cells of the promyelocytic leukemia cell line HL60. The c-myc P2 promoter was never found in nucleosomal configuration, no matter whether c-myc was expressed or not. Differences in the nucleosomal structure, however, were found in the promoter upstream region proximal to a previously described DNase I-hypersensitive site I, at the P0 promoter, and at the P1 promoter and upstream thereof. In these regions nucleosomes were detected in differentiated but not undifferentiated HL60 cells. Similar patterns of nucleosomes as found for active and inactive c-myc genes in HL60 cells were found for active and inactive episomal c-myc genes in stably transfected B cell lines. In these cell lines three activation stages could be described for episomal c-myc constructs: (i) uninducible, (ii) inducible, and (iii) induced. Significant differences in the nucleosomal structure of c-myc were observed for the uninducible and inducible stages, but not for the inducible and induced stages.

The cellular proto-oncogene c-myc is involved in cell proliferation and transformation but is also implicated in the induction of programmed cell death (apoptosis). The c-Myc protein is a transcriptional activator with a carboxyl-terminal basic region/helix-loop-helix (HLH)/leucine zipper (LZ) domain. It forms heterodimers with the HLH/LZ protein Max and transactivates gene expression after binding DNA E-box elements. We have studied the phenotype of dominant-negative mutants of c-Myc and Max in microinjection experiments. Max mutants with a deleted or mutated basic region inhibited DNA synthesis in serum-stimulated 3T3-L1 mouse fibroblasts. In contrast, mutants of c-Myc expressing only the basic region/HLH/LZ or HLH/LZ domains rapidly induced apoptosis at low and high serum levels. Co-expression of the HLH/LZ domains of c-Myc and Max failed to do so. We suggest that the c-Myc HLH/LZ domain induces apoptosis by specific interaction with cellular factors different to Max.

Organization of DNA into chromatin has been shown to contribute to a repressed state of gene transcription. Disruption of nucleosomal structure is observed in response to gene induction, suggesting a model in which RNA polymerase II (pol II) is recruited to the promoter upon reorganization of nucleosomes. Here we show that induction of c-myc transcription correlates with the disruption of two nucleosomes in the upstream promoter region. This nucleosomal disruption, however, is not necessary for the binding of pol II to the promoter. Transcriptionally engaged pol II complexes can be detected when the upstream chromatin is in a more closed configuration. Thus, upstream chromatin opening is suggested to affect activation of promoter-bound pol II rather than entry of polymerases into the promoter. Interestingly, pol II complexes are detectable in both sense and antisense transcriptional directions, but only complexes in the sense direction respond to activation signals resulting in processive transcription.

Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo located with enzymatic and chemical probes. Sites hypertensive to cleavage with single-strand-specific S1 nuclease or the single-strand-selective agent potassium permanganate included the major promoters P1 and P2 as well as the far upstream sequence element (FUSE) and CT elements, which bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yielded different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggested that transcriptionally associated torsional strain might assist strand separation and facilitate factor binding. Therefore, the interaction of FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K with supercoiled DNA was studied. Remarkably, both proteins recognize their respective elements torsionally strained but not as liner duplexes. Single-strand- or supercoil-dependent gene regulatory proteins may directly link alterations in DNA conformation and topology with changes in gene expression.

Approximately 20 to 22 copies of adenovirus type 12 (Ad12) DNA per cell were integrated into the genome of the cell line T637. Only a few of these copies seemed to remain intact and colinear with virion DNA. All other persisting viral genomes exhibited deletions or inversions or both in the right-hand part of Ad12 DNA. Spontaneously arising morphological revertants of T637 cells has lost viral DNA. In most of the revertant cell lines only the intact or a part of the intact viral genome was preserved; other revertant cell lines has lost all viral DNA. In three other Ad12-transformed hamster cell lines, HA12/7, A2497-3, and CLAC3 (Stabel et al., J. Virol. 36:22-40, 1980), major rearrangements at the right end of the integrated Ad12 DNA were not found. These studies were performed to investigate the phenomena of amplification, rearrangements, and deletions of Ad12 DNA in hamster cells.