A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism

Abstract

A cDNA library enriched with Myc-responsive cDNAs but depleted of myc cDNAs was used in a functional screen for growth enhancement in c-myc-null cells. A cDNA clone for mitochondrial serine hydroxymethyltransferase (mSHMT) that was capable of partial complementation of the growth defects of c-myc-null cells was identified. Expression analysis and chromatin immunoprecipitation demonstrated that mSHMT is a direct Myc target gene. Furthermore, a separate gene encoding the cytoplasmic isoform of the same enzyme is also a direct target of Myc regulation. SHMT enzymes are the major source of the one-carbon unit required for folate metabolism and for the biosynthesis of nucleotides and amino acids. Our data establish a novel functional link between Myc and the regulation of cellular metabolism.

FIG. 1.

Outline of the experimental strategy. The dotted lines indicate the CFSE profiles of initial populations before the first cell sorting. The shaded area below the peak designates cells taken for the next step of analysis. Note the gradual decrease in the intensity of CFSE staining of cells after each sorting. See details in the text and Materials and Methods.

FIG. 2.

Overexpression of mSHMT enhances proliferation of c-myc-null cells. (A) FACS profiles of DK cells (thin line), c-myc-null cells (HO15.19) infected with a c-myc-expressing vector (dotted line), and DK cells infected with a cDNA expression library (bold line) after two rounds of sorting. (B) FACS profiles of DK cells infected with either empty vector (thin line) or an mSHMT-expressing vector (bold line) after one round of sorting. c-myc-null cells (HO15.19) infected with a c-myc expression vector before the sorting are shown with a dotted line. (C) CFSE profile of c-myc-null cells (HO15.19) infected as described for panel B and selected for resistance to hygromycin (see Materials and Methods). (D) Doubling times of DK, c-myc-null, and Rat-1 cells expressing the constructs indicated below each bar.

FIG. 3.

Genes for mSHMT and cSHMT are Myc-responsive genes. (A) RNA isolated from serum-stimulated or logarithmically growing c-myc-null cells (lanes marked −), parental Rat-1 cells (lanes marked +), or c-myc-null cells expressing a c-myc cDNA (lane m) was probed in a Northern blot with an mSHMT-specific probe. Ethidium bromide-stained 28 and 18S rRNAs are shown in each lane as a loading control. The numbers above the blots indicate times after serum induction. (B) c-myc-null cells expressing c-Myc-ER were passaged several times under subconfluent conditions (<50% confluence) to obtain cells in an exponential phase of growth. 4-OH was added directly to the medium at a final concentration of 200 nM. RNA was collected following the addition of 4-OH at the time intervals shown above the blots. RNA was also collected from untreated cells at the same time intervals. RNA was probed sequentially by Northern blotting with mSHMT and GAPDH probes. A number below a blot indicates the fold induction of the mSHMT-specific message normalized to the amount of GAPDH in each lane at each time point. (C) RNA isolated from logarithmically growing c-myc-null cells (lane marked −), parental Rat-1 cells (lane marked +), or c-myc-null cells expressing a c-myc cDNA (lane m) was sequentially hybridized by Northern blotting with probes specific for mSHMT, cSHMT, and GAPDH. (D) RNA was isolated from logarithmically growing colon carcinoma cells (HCT116 and Colo320) or neuroblastoma cells (SK-NSH, SK-NAS, CHP-134, IMR32) and sequentially hybridized by Northern blotting with probes specific to the mSHMT, cSHMT, c-myc, or N-myc gene. Ethidium bromide-stained 28 and 18S rRNAs are shown in each lane as a loading control.

FIG. 4.

c-Myc binds to the promoters of mSHMT and cSHMT genes in vivo. (A) Schematic presentation of human, mouse, and rat mSHMT and cSHMT promoters. Open boxes represent exons. Myc binding sites and ATG codons are shown. Arrows indicate the positions and direction of primers used in PCR. (B and C) HO15.19 cells infected with empty vector (+vector lanes), HO15.19 cells infected with c-myc-expression vector (+myc lanes), HEK 293 cells, or Raji cells were cross-linked and lysed, and chromatin was immunoprecipitated with c-Myc-specific (M lanes) or Gal4-specific (G lanes) antibodies, followed by the reversal of the cross-linking and DNA isolation. Isolated DNA was used in PCR with radiolabeled oligonucleotides flanking Myc binding sites in the promoters of the genes indicated to the right of the blots. For negative controls, two types of PCR were performed. The first one was carried out with oligonucleotides flanking CACGTG sites that do not interact with c-Myc in the promoter of the rat glucokinase gene (GLU) (11) (B) or somewhere on human chromosome 22 (Chr. 22) (4) (C). Another PCR was performed with oligonucleotides flanking a DNA region containing no CACGTG sites in the rat PCNA gene (B) or in the third intron of a silent human β-globin gene (C). Quantitation of the PCR products was performed with the Molecular Dynamics IhoshphorImaging system. A number below the a blot indicates the fold difference in the signal intensity, determined by dividing a given PCR signal by a corresponding PCNA-specific (B) or β-globin-specific (C) signal and by the ratio of these signals in the corresponding Gal4 lane.