The transcriptional program of a human B cell line in response to Myc

Abstract

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.

Figure 1

Transcriptional activity of Myc target genes in nuclear run-on experiments. Nuclei were prepared from arrested P493-6 cells (–Myc) and cells 4 h after induction (+Myc). Nuclear run-on reactions were performed. Labeled RNAs were purified and hybridized to ATLAS Human Cancer 1.2 arrays (Clontech). Filters were washed, RNase treated and exposed to Kodak XAR-5 films. The arrays are divided into six sections : A, B, C (upper row) and D, E, F (lower row), from left to right. The position of a gene is given with a capital letter for the section and a small letter and number for the coordinates (for example A3c for myc). The G row represents housekeeping genes and control DNA. A complete list of all the genes on the array with a clickable array image is given at the Clontech home page at http://www.clontech.com/atlas/genelists/index.html. Reproducible candidate genes are listed in Table 2.

Figure 2

Candidate genes in nuclear run-on kinetics. Nuclei were prepared from arrested cells (0) and 2, 4, 6 and 8 h after myc induction. P indicates proliferating cells. Labeled nuclear run-on RNAs were hybridized to ATLAS Human cancer arrays 1.2. A selection of candidate genes listed in Table 2 is shown. Some candidate genes already showed an increase in transcription after 2 h (nm23-H2, RFC4, MCM4, PAC-1, FGFR3, MDC9 and LDH-A).

Figure 3

Evaluation of results by northern blot analysis. Total RNA was prepared from arrested P493-6 cells 0, 4, 8 and 24 h after myc induction and subjected to northern analysis. RNAs were hybridized with probes specific for the indicated genes. Nm23-H1, JTV-1 and p130 were identified in the cDNA screen, the other genes in the run-on screen. Genes differ in the kinetics of induction. RFC4, MCM4 and DP-1 mRNA levels do not accumulate at 8 h, although transcriptional activity is observed already after 4 h.