Myc and Mad bHLHZ domains possess identical DNA-binding specificities but only partially overlapping functions in vivo

Abstract

The opposing transcriptional activities of the basic-helix-loop-helix-leucine zipper proteins Myc and Mad, taken together with information related to their expression patterns and biological effects, have led to a model of the Myc/Max/Mad network in which Myc and Mad proteins function as antagonists. This antagonism is presumed to operate at the level of genes targeted by these complexes, where Myc:Max activates and Mad:Max represses expression of the same set of genes. However, a detailed analysis of the DNA-binding preferences for Mad proteins has not been performed. Furthermore, the model does not address the findings that Myc:Max indirectly represses transcription of several regulatory genes. To examine these issues relating to DNA-binding specificity and biological responses, we have determined the DNA-binding preferences of Mad1 by using selection and amplification of randomized oligonucleotides and demonstrated that its intrinsic specificity is identical with that of c-Myc. We have also used a chimeric Myc protein, containing a substitution of the entire Mad basic-helix-loop-helix-leucine zipper motif, and shown that it can reproduce the growth-promoting activities of Myc, but not its apoptotic function. Our results suggest that Myc and Mad, although possessing identical in vitro DNA-binding specificities, do not have an identical set of target genes in vivo, and that apoptosis is one biological outcome in which the transcriptional effects of Myc are not directly antagonized by those of Mad.

Fig 1.

Preferential binding of Mad1 protein to canonical E-boxes. (A) 20-mer sequences independently derived from the SAAB (selected and amplified binding site) selection. The upper portion contains the first 15 sequences from the negative control (untagged Max protein followed by immunoprecipitation with anti-Flag), none of which contain E-boxes. Sequence Max01 was found in two other clones, as denoted to the right of the sequence. The lower portion contains the first 23 sequences from the Flag-tagged Mad selection. Sequences without E-boxes appear on the left, whereas those with E-boxes (in bold) appear on the right. (B) Flanking sequences from Mad-selected E-boxes. Nucleotides represented in the +/−4, 5, 6 flanking positions, relative to the central G (+1) and C (−1), are shown, with the most frequently appearing nucleotide in bold. Because the E-boxes (except FMad01) are palindromic, only half-sites are shown. (C) Gel-shift assays using labeled nonmethylated E-box, or CpG-methylated E-box (mE box) oligonucleotides. Labeled probes were incubated with the indicated in vitro translated proteins.

Fig 2.

Myc and the Myc(MadbHZ) fusion protein (MM). (A) Transcription assays using a synthetic promoter with four E-boxes controlling expression of the luciferase gene. Assays were performed after transient transfection of NIH 3T3 cells with 1 μg of the M4 reporter (3T3 1 M4), 5 or 50 ng of protein expression vector (Myc5 and Myc50 for Myc; MM5 and MM50 for the Myc(MadbHZ) chimeric protein), and 0.1 μg of RSV-β-galactosidase vector. Luciferase relative luminescence units were divided by β-galactosidase units to correct for variation in transfection efficiency. (B) Schematic diagram of the c-Myc protein (N-terminal Myc box I and Myc box II in dark gray and the bHLHZ in light gray), the Mad1 protein [N-terminal Sin3 interaction domain (SID) in light gray, and the bHLHZ in black], and the Myc(MadbHZ) chimera, comprising the entire Myc protein with the exception of the bHLHZ, which has been replaced with the analogous motif from Mad1.

Fig 3.

Characterization of activity of Myc(MadbHZ) and Myc in stably transfected myc^−/− cells. (A) Equivalent expression of F-Myc and F-Myc(MadbHZ) (MM) proteins. Pools of stably transfected cells were labeled with [³⁵S]methionine, and proteins were immunoprecipitated with anti-Flag M2 antibody then resolved on an SDS/12.5% polyacrylamide gel. The figure shows the pertinent segment of the autoradiograph. (B) Proliferation of transfected Rat-1 cell. Equal numbers cells were plated on day 0 and then trypsinized and counted at days 1, 2, 3, and 4. (C) DNA content profiles of logarithmically growing cells. The graph contains the cell cycle distribution data from MULTICYCLE analysis of flow cytometry data from cells stained with propidium iodide. (D) Both Myc and Myc(MadbHZ) restore the levels of RNA in myc^−/− cells. The graph shows an overlaid set of histograms representing the G₁ RNA content of cells stained with both propidium iodide and acridine orange.

Fig 4.

Myc, but not Myc(MadbHZ), induces apoptosis. (A) Photomicrographs of Rat-1 cells following growth-factor withdrawal. Cells were grown to confluence, washed with PBS, and then refed with medium containing 10% or 0.1% serum. (B) DNA content profiles of density-arrested cells. The graph contains the cell cycle distribution data from MULTICYCLE analysis of flow cytometry data from cells stained with propidium iodide. Note the high S phase fraction of Myc-rescued cells is associated with a high sub-G₁ fraction. (C) Quantitation of DNA degradation shows apoptosis induced by Myc. The amount of intact and degraded DNA was quantitated as described (42) and used to calculate the percent degraded DNA.