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c-Myc regulates mammalian body size by controlling cell number but not cell size

Abstract

Overexpression of the proto-oncogene c-myc has been implicated in the genesis of diverse human tumours. c-Myc seems to regulate diverse biological processes, but its role in tumorigenesis and normal physiology remains enigmatic1. Here we report the generation of an allelic series of mice in which c-myc expression is incrementally reduced to zero. Fibroblasts from these mice show reduced proliferation and after complete loss of c-Myc function they exit the cell cycle. We show that Myc activity is not needed for cellular growth but does determine the percentage of activated T cells that re-enter the cell cycle. In vivo, reduction of c-Myc levels results in reduced body mass owing to multiorgan hypoplasia, in contrast to Drosophila dmyc mutants, which are smaller as a result of hypotrophy2. We find that dmyc substitutes for c-myc in fibroblasts, indicating they have similar biological activities. This suggests there may be fundamental differences in the mechanisms by which mammals and insects control body size. We propose that in mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size.

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Figure 1: Generation of mutant c-myc alleles and analysis of mutant embryos.
Figure 2: Effect of altering c-myc expression on body and organ size.
Figure 3: Effect of altering c-myc expression on cell proliferation and morphology in primary and immortalized fibroblasts.
Figure 4: Activation, growth and proliferation potential of c-myc mutant naive CD4+ T cells.
Figure 5: Proliferation potential of c-mycΔORF/+ T cells in the absence of p27.

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Acknowledgements

We thank J. Roberts and M. Fero for providing the p27KIP1 mice, S. Dymecki for the β-actin-FLP mice, D. Melton for HM1 ES cells, P. Gallant for dmyc cDNA, and L. VanParijs for pMIG-Cre. We thank G. Yee for performing the RNase protection analysis, S. Kogan for help with the methylcellulose cultures, D. Ginzinger for help with the initial Taqman analysis and C. McArthur, P. Zaech and A. Wilson for FACS sorting. We thank A. C. Pasche and D. Trail for technical assistance. We also thank M. Nabholz, S. Martin and our colleagues in the Trumpp, Bishop and Martin laboratories for discussions and critical reading of the manuscript. A.T. was the recipient of postdoctoral fellowships from the Deutsche Forschungsgemeinschaft, the Human Frontiers in Science Program, and the California Division of the American Cancer Society. A.T. is now supported by grants from the Swiss National Science Foundation and the Swiss Cancer League. Y.R. is a Merck fellow of the Life Sciences Research Foundation. This work was supported by the NIH (G.R.M. and J.M.B.), and the G.W. Hooper Foundation.

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Correspondence to Andreas Trumpp.

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Supplementary information

Detailed methods:

Generation and analysis of mutant mice.

To generate the c-myc∆ORF allele we constructed a targeting vector in which 4.1 kb of c-myc DNA (from the Not1 site in exon 1 to the Xho1 site in exon 3) was replaced by a Pgk-Hprt cassette. The deleted region contains the entire c-myc open reading frame (ORF). The homology region of the targeting vector extended 1.8 kb 5' from the Not1 and 7.4 kb 3' from the Xho1 site. Homologous recombinants in HM-1 ES-cells were identified by Southern blot analysis using probes indicated in Fig. 1a and were obtained at a frequency of 1:80. Mutant ES cells were used to produce chimeras that transmitted the c-myc∆ORF allele through the germline using the aggregation technique . Mice carrying the c-myc∆ORF allele were genotyped by PCR. Primers used: AT6 (5'TTTCTGACTCGCTGTAGTAATTCC3') with 703T (5'GCTAAAGCGCATGCTCCAGACTGCC3') to detect the c-myc∆ORF allele and AT6 with AT7 (5'CAGCCTTCAAACAGCTCGAGGA3') to detect the c-myc wild-type allele.

To generate the c-mycfloxN allele we inserted a flrted (flanked by FRT sites) Pgk-neo cassette with a 3' loxP site into the XhoI site in exon 3. A second loxP site was inserted into the NheI site in intron 1 (Fig. 1a). The regions of homology, screening method and frequency of homologous recombination were similar to those described above for c-myc∆ORF. Mice carrying c-mycfloxN were routinely genotyped by PCR using standard neo primers or alternatively using the primers 5'flox (5'CACCGCCTACATCCTGTCCATTC3') and 3'flox (5'TACAGTCCCAAAGCCCCAGCCAAG3'). The c-mycflox allele was generated by crossing mice carrying the c-mycfloxN allele to b-actin-FLP mice .

The genetic background of the c-myc∆ORF/+ mice used to assess effects on body and organ weight was mixed, but predominantly FVB/N (4 to 6 backcrosses). The genetic background of all other mice was mixed. For the growth curves of the allelic series three litters were followed with one typical experiment shown. In addition more than 5 other litters in which one of the genotypes was absent were analyzed and gave consistent results. The c-myc∆ORF allele was bred onto several other mutant backgrounds (such as e.g. Rb, p21, and p107 , A. Trumpp unpublished observation). In all cases we observed a body size reduction in mice carrying c-myc∆ORF compared to the parental mutant strain with wild-type c-myc.

To induce Cre expression by the MxCre transgene, MxCre; c-myc∆ORF/flox and MxCre; c-mycflox/flox mice received five i.p. injections (10mg/g body weight) of polyI-polyC (Sigma) at two days intervals . After 5 weeks, the mice were sacrificed, peripheral T-cells and livers were isolated, and genomic DNA was prepared and used to determine the deletion frequency by Taqman PCR (see below). Recombination occurred in 91% ± 4% of liver cells and in 62% ±10% of CD4/8 cells, similar to what has been previously reported .

RNA was prepared from E9.5 embryos using the RNAeasy kit (Qiagen). RNase protection analysis was performed essentially as described . using probes obtained from Pharmingen.

Hematopoietic progenitor colony assays.

E9.5 yolk sacs were isolated, incubated in 0.1% collagenase (Sigma) (in PBS supplemented with 20% Fetal bovine serum (FBS) for 30 min at 37°C, and disaggregated by passage through a 22-gauge needle. Cells from individual yolk sacs were cultured as previously described . Colonies were scored after 8 days of incubation.

Isolation and culture of primary mouse embryonic fibroblasts (MEFs).

Primary MEFs from E13.5 wild-type and mutant mouse embryos were isolated and cultured using an established protocol . After three passages at 1:4, control and c-mycfloxN/floxN MEFs (which behave like c-myc∆ORF/+ cells ) were infected using pMIG or pMIG-Cre virus-containing supernatant produced in BOSC 23 cells as previously described . Cells were sorted 36 hrs after infection into GFP+/Cre+ and GFP-/Cre- populations using a high speed cell sorter (Moflo). Sorted populations were plated at the same density and cell numbers were determined at regular intervals.

3T9 cells were obtained from primary wild-type and c-mycflox/flox MEFs (both have similar growth behavior in culture) using a standard passage protocol . To obtain dmyc expression in wild-type and c-mycflox/flox 3T9 cells, we produced a viral expression vector by cloning a dmyc cDNA (kindly provided by Peter Gallant) into pBABE-puro, and infected the cells with pBABE-puro-dmyc virus produced in BOSC 23 cells as previously described . To delete the c-myc gene from dmyc-expressing cells, Puromycin-resistant 3T3 cells were infected with pMIG-Cre and 48h later sorted into GFP+ (Cre-expressing) and GFP- populations. The two populations were mixed in approximately equal numbers and the ratio of GFP+ to GFP- cells was monitored every two days by flow cytometry. All experiments were done at least three times with similar results.

Real Time PCR assay.

For Taqman PCR analysis of the wild-type c-myc gene a 5’VIC/3’ TAMRA labeled probe (5’CAGACAGCCACGACGATGCCCC3’) was used in conjunction with the following primer pair: Tmfloxunrec5 (5’TCTAGACTTGCTTCCCTTGCTGT3’) and Tmfloxunrec3 (5’TTCCTGTTGGTGAAGTTCACGT3’). For Taqman PCR analysis of the c-myc∆ORFrec allele a 5’6-FAM/3’ TAMRA labeled probe (CCCGCGGCACATGGACTTGA) was used in conjunction with the following primer pair: TMdel1(AAATAGTGATCGTAGTAAAATTTAGCCTG) and TMdel2 (ACCGTTCTCCTTAGCTCTCACG). Expression of c-myc and N-myc was analyzed using Real Time Taqman RT-PCR using the following primers and 5’ 6-FAM/3’ TAMRA labelled probes: RTcmyc1 (CTGGATTTCCTTTGGGCGTT), RTcmyc2: (TGGTGAAGTTCACGTTGAGGG), RTcmyc probe (AAACCCCGCAGACAGCCACGAC), RTNmyc1 (AAGCCCCTCAGCACCTCC), RTNmyc2: (TGACCACATCGATTTCCTCCT), RTNmyc probe (AGAGGATACCTTGAGCGACTCAGGATGATGA). N-myc expression in normal and c-myc mutant T- cells, fibroblasts and livers was in all cases below the detection limit of our assay and at least 5000-fold lower than N-myc expression in E9.5 mouse embryos. Expression of dmyc and L-myc was assayed using SYBR Green real time RT-PCR. dmyc1 (TCGCAGATCTGGACTACACG), dmyc2: (CGATTTGCGGATAATGTCCT), L-myc1: (GGACGTGACCAAGAGGAAGA), L-myc2: (TCTAACGCCTTGCTGAGGAT). All cDNAs were normalized by b2microglobulin expression. The following primers were used: b2m1: (GTGTATGCTATCCAGAAAACCC), b2m2 (TCACATGTCTCGATCCCAGTAG)

Expression of L-myc was > 100fold lower in naïve and activated T-cells or fibroblasts than in E9.5 embryos. Furthermore, expression did not change after TCR activation by ConA stimulation, nor was it dependent on c-myc mutant allelic constitution. We therefore considered the L-myc expression as not significant.

T-cell purification and in vitro proliferation assays.

Naïve CD4+ T-cells were purified from pooled spleen and lymph nodes harvested from c-myc mutant mice, as described previously . T cell preparations were typically 96% CD4+, as determined by staining and flow cytometry. CFSE labelling was performed by washing the purified, naïve CD4+ T-cells twice in PBS and then incubating them with 10mM CFSE in PBS, for 7 minutes, in the dark. The labelling reaction was quenched by adding an equal volume of FBS, and washing the cells twice in complete medium. Proliferative responses to antigen were determined by intracellular fluorescent dye staining the cells before they were incubated in RPMI 1640 supplemented with 1mM L-glutamine, penicillin/streptomycin, non-essential amino acids, sodium pyruvate and Hepes (Gibco/BRL, Grand Island, NY) and 10% FCS. TCR-induced proliferation was assayed by incubating 106 CFSE-stained CD4+ T-cells with 1mg/ml soluble anti-CD3 (clone 2C11, Pharmingen) and 10mg/ml soluble anti-CD28 (clone 37N1, Pharmingen), or alternatively with Concavalin A (ConA) for three days, and determining cell division number by flow cytometry. For Western analysis, anti-c-Myc antibody sc-764 (Santa. Cruz) was used. All experiments have been performed at least three times with similar outcomes.

Supplementary Figures

Figure 1. Analysis of mutant

c-myc alleles. (JPG 27.7 KB)

a, Southern analysis of EcoRI digested DNA isolated from E9.5 embryos using probe1 (see Fig. 1a main text). b, Genotyping by PCR of mice carrying different allelic combinations. The locations of the primers are indicated in Fig. 1a (main text).

Figure 2. Effect of altering

c-myc expression on body size. (GIF 7.53 KB)

Growth curves of individual male wild-type and c-myc∆ORF/+ littermates.

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Trumpp, A., Refaeli, Y., Oskarsson, T. et al. c-Myc regulates mammalian body size by controlling cell number but not cell size. Nature 414, 768–773 (2001). https://doi.org/10.1038/414768a

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