Correspondence to Peter J. Hurlin: pjh{at}shcc.org
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Consistent with a key role for Myc proteins in stimulating cell cycle entry, primary mouse embryo fibroblasts (MEFs) lacking c-Myc accumulate in G0 of the cell cycle and are rendered incompetent to proliferate in response to mitogenic stimulation (de Alboran et al., 2001; Trumpp et al., 2001). In contrast to primary cells, deletion of c-Myc in the "immortal" Rat1A fibroblast cell line significantly slows, but does not abrogate cell proliferation (Mateyak et al., 1997). The slowed proliferation in the latter cells appears to be the result of both a decreased rate at which cells traverse the cell cycle and a failure of some cells to enter a productive cell cycle (Holzel et al., 2001; Schorl and Sedivy, 2003). From these and many other results, it can be concluded that deregulated and elevated Myc production, so common in tumors, has the consequence of both stimulating cells to enter the cell cycle and preventing cells from properly exiting the cell cycle.
The ability of Myc family proteins to promote cell proliferation and contribute to tumor formation is dependent on its bHLHZip domain, which mediates heterodimerization with Max and DNA binding (Blackwood and Eisenman, 1991). The MycMax heterodimer, but not Myc alone, is able to bind DNA at the E-box consensus sequence CANNTG and activate transcription (Amati et al., 1992; Kretzner et al., 1992). In logarithmically proliferating cells it appears that most, if not all newly synthesized c-Myc enters into a complex with Max and that the rapid turnover of c-Myc protein (half-life of approximately 15 min) is not affected by heterodimerization with Max (Blackwood et al., 1992). Together with results showing that Max is a highly stable protein with a half-life in excess of 24 h (Blackwood et al., 1992), these results support the idea that a pool of Max is always available for interaction with newly synthesized c-Myc. However, in addition to Myc family proteins, Max interacts with a number of additional bHLHZip proteins that have the potential to limit the supply of Max for Myc heterodimerization. Moreover, these additional Max-interacting proteins, which include Mad family proteins (Mad1, Mxi1, Mad3, and Mad4), Mnt, and Mga (Zhou and Hurlin, 2001), are transcriptional repressors that have been demonstrated to antagonize Myc-dependent cell transformation in cell culture experiments.
Mnt is unique among these putative Myc antagonists in that it is expressed ubiquitously and like Max, Mnt levels do not fluctuate during the G0 to S-phase transition (Hurlin et al., 1997a, 2003). Importantly, Mnt appears to play a role in cell cycle entry, as cells lacking Mnt were found to exhibit an accelerated G0 to S-phase transition (Hurlin et al., 2003). These results, together with the finding that deletion of Mnt can predispose cells in vivo to apoptosis and tumorigenesis (Hurlin et al., 2003, Nilsson et al., 2004), suggest that MntMax regulation of Myc activity during cell cycle entry may endow it with a unique role as a cellular Myc antagonist. In this study, we investigate the role Mnt plays in cell cycle entry and provide evidence that a transient switch in the ratio of MntMax to c-MycMax underlies the basic decision of cells to enter the cell cycle and proliferate.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Switching between MntMax and c-MycMax binding at shared target genes during cell cycle entry
To directly examine the consequences of complex switching between MntMax and c-MycMax during cell cycle entry, chromatin immunoprecipitation (ChIP) experiments were performed. Early passage Mnt null and wild-type MEFs were made quiescent and stimulated to enter the cell cycle as described above. c-Myc expression was monitored by Western blot (Fig. 2 a) and tritiated thymidine incorporation assays performed in parallel to confirm cells were arrested and entered into the cell cycle (not depicted). Binding to specific sites in a number of known or suspected Myc target genes, including Cdk4, Cyclin D2 (ccnd2), ODC, E2F2, Nucleolin, Tert, and CAD (for review see Cole and McMahon, 1999) was determined in quiescent cells and at 4 and 24 h after serum stimulation. One of several E-boxcontaining regions within the Cyclin E1 (ccne1) gene where no c-Myc or Mnt binding was observed was used as a negative control.
|
Previous resulted showed that Mnt interacts with Sin3 in vitro and that deletion of Mnt's Sin3 interaction domain abolished its ability to repress transcription (Hurlin et al., 1997a; Meroni et al., 1997). To examine endogenous Mnt-Sin3 interactions as a function of cell cycle entry, low stringency anti-Mnt immunoprecipitations were performed, followed by immunoblotting with anti-Sin3A and Max antibodies. These results showed that Mnt interacts with Sin3 in vivo and that Mnt-Sin3 interaction is not regulated during the cell cycle (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200411013/DC1). Furthermore, ChIP assays performed by first immunoprecipitating with Mnt antibody, then reimmunoprecipitating (Metivier et al., 2003) with either anti-Sin3A or anti-HDAC1 (an Sin3A associated protein; Ayer, 1999) demonstrated association of Sin3A and HDAC1 with Mnt at Mnt-Myc binding regions within the Cyclin D2, Nucleolin, and TERT genes (Fig. 2 c). These results suggest that MntSin3HDAC complexes are present at Mnt binding sites in chromatin.
Mnt/c-Myc target gene regulation in the absence of Mnt
To determine how loss of Mnt affects transcription of c-Myc/Mnt target genes, real-time RT-PCR was used to analyze transcript abundance for genes identified above or previously (Hurlin et al., 2003; Nilsson et al., 2004) in the context of cell cycle entry. RNA was extracted from serum starved, quiescent Mnt null and wild-type cells and at 4, 12, 16, 20, and 24 h after serum stimulation. Levels of ARBP P0 RNA, encoded by a gene whose transcription is not regulated during cell cycle entry (Fig. 3; Humbert et al., 2000), was used to normalize all RT-PCR values obtained. As expected, c-Myc RNA levels were strongly induced in MEFs after serum stimulation (Fig. 3). However, the dynamics of induction were different between wild-type and Mnt null MEFs, with the latter cells showing higher levels at 12 h, but lower levels at other time points. Mnt and Max RNA levels, like their respective encoded proteins (Hurlin et al., 2003), were maintained at a near constant level. In contrast, expression levels of Nucleolin, E2F2, and CAD RNA were consistently altered, with each showing markedly increased levels in Mnt null MEFs, especially 12 h after serum stimulation (Fig. 3). The E1F4E gene, a putative Myc target gene (Jones et al., 1996) encoding elongation factor 4E, was also up-regulated at the 12-h time point (Fig. 3), although we have yet to identify Myc-Mnt binding sites in the mouse version of this gene. In contrast, ODC levels were not significantly affected by loss of Mnt. Interestingly, although the amplitude of Cyclin D2 and CDK4 expression did not appear to be significantly affected by Mnt loss, the expression patterns of Cyclin D2 and CDK4 in Mnt null MEFs appeared to reflect the accelerated cell cycle entry profile of these cells compared with wild-type MEFs (Fig. 5; Hurlin et al., 2003).
|
|
Mnt deficiency rescues proliferation arrest caused by c-Myc deletion in primary MEFs
To better define the relationship between Mnt and c-Myc in cell proliferation and cell cycle entry, we examined the consequences of acute, simultaneous deletion of both Mnt and c-Myc in primary MEFs. Crosses between Mntflox/flox (Hurlin et al., 2003) and c-MycfloxN/floxN mice (Trumpp et al., 2001) were performed to generate mice containing homozygous floxed alleles for both Mnt and c-Myc (Mnt/c-MycdCKO). MEFs were then isolated from the latter mice and from Mntflox/flox mice and c-Mycflox/flox mice. MEFs at passage 1 were infected with adenovirus expressing Cre recombinase and GFP. GFP expression indicated that >95% of cells were infected and PCR genotyping and Western blot analysis confirmed that a very high percentage of floxed alleles were successfully targeted (Fig. 5, a and b, not depicted). Proliferation assays performed 48 h after infection cells confirmed previous results (de Alboran et al., 2001; Trumpp et al., 2001) showing that deletion of c-Myc leads to rapid cessation of primary MEF proliferation (Fig. 5 c). In contrast, primary MEFs in which c-Myc and Mnt were simultaneously ablated continued to proliferate (Fig. 5 c). Very similar results were achieved after AdCre infection of immortal (i) Mntflox/flox, ic-Mycflox/flox, and iMnt-c-MycdCKO MEF cell lines (Fig. 5 d). The latter cell lines were developed by continuously passaging primary cell populations until they escaped senescence (Todaro and Green, 1963).
|
To further characterize how Mnt deletion rescued proliferation arrest caused by loss of c-Myc, a series of Western blots were performed examining the expression of proteins that perform critical functions in cell cycle control (Fig. 6). From this analysis, it was observed that several proteins up-regulated by loss of Mnt, including Cyclin D2, Cyclin A, Cyclin B, Cyclin E, and p107, were also up-regulated in Mnt/c-MycdCKO MEFs, relative to their expression in c-Mycdeficient MEFs (Fig. 6). In addition, pRB was hyperphosphorylated by Mnt deletion, both in the presence and absence of c-Myc. These results suggest that Mnt deletion rescues the proliferation arrest caused by c-Myc deletion at least partly through up-regulation of critical cell cycle regulatory proteins.
|
Finally, the relationship between Mnt and c-Myc specifically in the context of cell cycle entry was examined after AdCre-mediated deletion of Mnt, c-Myc, and Mnt plus c-Myc in primary MEF cell lines. Whereas ablation of c-Myc completely blocked the ability of MEFs to enter S-phase, MEFs in which c-Myc and Mnt were simultaneously ablated incorporated tritiated thymidine with kinetics similar to control AdCre-infected MEFs (Fig. 7 a). Consistent with the observed rescue of cell cycle entry kinetics, deletion of Mnt rescued the depressed RNA levels of the Cyclin D2, E2F2, ODC, and Cyclin E caused by c-Myc deletion (Fig. 7 b). Interestingly, Gadd45a, a target of c-Myc repression was up-regulated during cell cycle entry in the absence of c-Myc, but its regulation MEFs deficient for both Mnt and c-Myc was similar to that observed in wild-type cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200411013/DC1). However, other putative targets of Myc repression were not similarly affected (Fig. S2).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The importance of maintaining a proper ratio of Myc to Mnt during cell cycle entry is suggested by results showing that overexpression of Mnt retards cell cycle entry and cell proliferation (Fig. 4) and loss of Mnt accelerates cell cycle entry (Figs. 4 and 5; Hurlin et al., 2003). Moreover, the notion that Mnt and c-Myc function as a binary system to regulate cell cycle entry and proliferation is firmly supported by the ability of Mnt deletion to rescue the proliferative arrest caused by c-Myc ablation in primary and immortal MEFs (Fig. 5). It is important to stress however, that Mnt ablation in primary and immortal MEFs does not completely rescue the proliferative block caused by loss of c-Myc (Fig. 5). Although increased apoptosis after Mnt deletion (Fig. 5; Hurlin et al., 2003; Nilsson et al., 2004) may be responsible for the partial, instead of full rescue of primary MEFs, it is also possible that antagonism of Myc by Mad family proteins or other unknown mechanisms may contribute as well.
While this work was in progress, it was reported that siRNA-mediated knockdown of Mnt in Myc-deficient immortal Rat1A fibroblasts (Mateyak et al., 1997) rescued the slow proliferation of these cells (Nilsson et al., 2004). However, whereas deletion of c-Myc in the Rat1A cell line causes slowed proliferation, deletion of c-Myc in primary and in immortal c-Mycflox/flox MEFs caused a complete block in proliferation (MEFs; Fig. 5, c and d; de Alboran et al., 2001; Trumpp et al., 2001). These results suggest that Rat1A cells acquired defects over the course of their extensive growth in culture that partially substitute for loss of c-Myc. The possibility and perhaps likelihood that such acquired defects affect cell cycle control, argue that primary or "freshly" immortalized Mnt/c-MycdCKO MEFs are better suited to rigorously establish the relationship between c-Myc and Mnt in governing cell cycle entry and cell proliferation.
It should also be noted that there are significant differences observed between siRNA-mediated knockdown of Mnt (Nilsson et al., 2004) and Cre-mediated Mnt gene deletion in the regulation of gene targets and downstream effector protein levels. Whereas siRNA-mediated knockdown of Mnt is associated with a dramatic down-regulation of antiapoptotic BclXL and strong up-regulation of pro-apoptotic p19ARF and p53 protein levels and ODC RNA and protein levels (Nilsson et al., 2004), we find that chronic Mnt deletion or acute Mnt deletion in primary MEFs has only a modest or no effect on their expression levels. (Figs. 3, 6, and 7; Hurlin et al., 2003). Moreover, siRNA-mediated Mnt knockdown was found to dramatically down-regulate p27Kip protein levels in Myc/ Rat1A fibroblasts, NIH3T3s, and MEFs (Nilsson et al., 2004), but we surprisingly find that p27Kip1 levels are up-regulated as a result of both chronic and acute deletion of Mnt, as well as in tumors caused by Mnt deletion in vivo (Fig. 6; Fig. S2; and not depicted). It is not clear why these differences are seen, but they are likely due to nonspecific effects associated with the different methods used to remove Mnt.
The displacement of MntMax complexes in favor of c-MycMax complexes at shared target genes during cell cycle entry (Fig. 2) is consistent with the proposal that MycMax complexes operate, at least in part, by derepressing MntMax bound target genes (Hurlin et al., 1997a, 2003; Nilsson et al., 2004). Although it is unlikely that MntMax and c-MycMax complexes bind to and regulate exactly the same set of target genes, we find that they do bind and regulate at least a subset of overlapping target genes (Figs. 2, 3, and 7). What remains unclear is the precise relationship between c-Myc and Mnt binding and transcriptional status. For example, we have been unable to establish a clear link between the presence of Mnt at different target genes and local histone acetylation status (not depicted), despite being able to detect MntSin3 and presumably MntSin3HDAC interactions at target gene binding sites (Fig. 2). However, chromatin deacetylation at the Cyclin D2 gene does appear to correspond to Mnt binding (unpublished data). Whatever the mechanism of Mnt repression, the finding that acute Mnt deletion rescues, at least partially, the severe down-regulation of Myc targets such as Cyclin D2 and E2F2 caused by acute c-Myc deletion in primary MEFs (Fig. 7 b) is consistent with Mnt and c-Myc functioning as a repression-activation system to regulate the expression of critical target genes involved in cell cycle progression. Although we observe strong down-regulation of some Myc target genes after c-Myc deletion, others such as ODC were only modestly down-regulated (Fig. 7 b). The slight affect on ODC levels are consistent with studies showing that most Myc target genes are only modestly affected by loss of c-Myc in immortal Rat1A cells (Bush et al., 1998; Cole and McMahon, 1999). Furthermore, transcript levels of most of the various Myc target genes examined in this and previous studies were only modestly affected by loss of Mnt (Fig. 3; Hurlin et al., 2003; Nilsson et al., 2004). One potential explanation for these findings is that the transcriptional activity of Mnt and Myc is inherently weak (Eisenman, 2001) and that their biological impact derives from eliciting small changes in the expression levels of perhaps thousands of transcriptional targets (for reviews see Fernandez et al., 2003; Orian et al., 2003; Cawley et al., 2004; see http//www.myc-cancer-gene.org). Alternatively, transcriptional regulation of Myc and Mnt target genes may be constrained to specific physiological settings that have yet to be fully recognized. For example, during cell cycle entry in Mnt null MEFs, up-regulation of target genes was most apparent 12 h after serum stimulation (Fig. 3). Thus, because c-Myc binding to target gene promoters was observed by 4 h after serum stimulation (Fig. 2 b), secondary events, perhaps related to cell cycleregulated binding of other transcription factors to DNA or to Myc, may dictate the time and amplitude of Myc-dependent transcription activation (Cole and McMahon, 1999). The different responses in gene expression observed in Mnt-deficient cells may also reflect the existence of different classes of Myc target genes (Haggerty et al., 2003). The development of MEF cell lines containing conditional, floxed alleles of Mnt, c-Myc, and both Mnt and c-Myc (Fig. 5) should prove useful for more precisely defining the mechanisms that underlie the regulation of specific target genes by Myc and Mnt.
Although up-regulation of c-Myc expression plays an important role in stimulating cell cycle entry, it is of paramount importance that it is then rapidly down-regulated. Sustained high level Myc expression is not well tolerated and sensitizes cells to apoptosis and tumorigenesis (Pelengaris et al., 2002; Nilsson and Cleveland, 2003). Because both MycMax and MntMax complexes are present in cells during the normal proliferative cell cycle (Hurlin et al., 1997a), we speculate that Myc-driven apoptosis and tumorigenesis results when the proper balance between MycMax and MntMax in cells is breached. This idea is strongly supported by results showing that loss of Mnt triggers a "Myc-like" response of accelerated proliferation and apoptosis in fibroblasts and leads to tumor formation when deleted in breast epithelium (Hurlin et al., 2003) and T cells (unpublished data). Though it is not yet clear whether tumor formation caused by loss of Mnt is mechanistically equivalent to that caused by Myc, our results establish that Myc-Mnt antagonism underlies the basic decision of cells to enter the cell cycle and proliferate.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of MEFs
MEFs were obtained from Mnt null and Mntflox/flox (Hurlin et al., 2003; Toyo-oka et al., 2004), c-Mycflox/flox (Trumpp et al., 2001), and Mntflox/flox c-MycFloxN/FloxN (or Mnt/CKO c-Myc/CKO) embryos at embryonic day 13.5 as previously described (Hurlin et al., 2003). Immortal MEF cell lines were established by continuous passaging of cultures using a 3T9 protocol (Todaro and Green, 1963). Genotyping of cells was performed by PCR. Primer sets for the c-Myc locus were previously described (Trumpp et al., 2001).
Cell cycle entry and proliferation assays
For cell cycle entry experiments, MEFs or HFFs were grown to confluence and switched from medium containing 10% FCS to medium containing 0.1% FCS. Cells were maintained in 0.1% FCS for 23 d before stimulating with medium containing 10% FCS. Tritiated thymidine incorporation assays were performed in triplicate in 6-well dishes. The cells were labeled by incubating in 2 ml DME containing 1 µCi/ml methyl [3H]thymidine (ICN Biomedicals) for 2 h at 37°C, 5% CO2. Cells were washed twice with ice-cold PBS and incubated for 20 min at 4°C in 5% TCA to remove acid soluble radioactivity. The cells were washed twice with 70% EtOH and solubilized for 30 min at 37°C in 1 ml 0.1 M NaOH, 2% Na2CO3, and 1% SDS. 5 ml of Ecolume scintillation cocktail (ICN Biomedicals) was added to lysates and counts per minute determined using a scintillation counter (model LS5000TD; Beckman Coulter). Proliferation assays were performed as previously described (Hurlin et al., 2003).
ChIP analysis
For cell cycle entry assays, cells from four 150-mm tissue culture dishes (containing cells to grown to confluence, serum starved and serum stimulated) were washed once with PBS and fixed with 1.5% formaldehyde (in PBS) for 5 min at 37°C. Fixed cells were collected, lysed, sonicated to attain an average length of 7501,200 bp, and immunoprecipitations were performed using equal amounts of sonicated lysates. Immunoprecipitations, washes, and elution of immunoprecipitated material were performed as previously described (Metivier et al., 2003). Preimmune serum was used as a negative control. Re-immunoprecipitations of primary immunoprecipitations were performed as described by Metivier et al. (2003) and PCR (3035 cycles) was performed on equal amounts of eluate. Antibodies used for ChIP experiments included affinity-purified anti-Mnt (H6823), c-Myc (N-262), Max (C-17), Sin3A (AK-11) were obtained from Santa Cruz Biotechnology, Inc., and HDAC1 (2E10) was obtained from Upstate Biotechnology.
Real-time PCR
RNA was harvested using TRIzol reagent (Invitrogen), purified for mRNA using oligotex particles (QIAGEN), and quantified by spectrophotometer. 0.5 µg mRNA per sample was random primed using Superscript II reverse transcriptase (Invitrogen). Samples in triplicate were amplified using SYBR green I dye in an Applied Biosystems 7900HT sequence detection system. Analysis of data was performed using the 2Ct method (Livak and Schmittgen, 2001) and quantitated relative to the ARBO PO gene. Gene expression was normalized to unstimulated (0 time point) wild-type samples, which provided an arbitrary constant for comparative fold expression.
Online supplemental material
Fig. S1 shows co-immunoprecipitation of Mnt and Sin3 in cells. Low stringency (LS) Mnt immunoprecipitates were separated by SDS-PAGE and Western blots performed using the antibodies against the indicated proteins. Max was used as a positive control for Mnt interaction and high stringency (HS) Mnt immunoprecipitates were used as a negative control. Fig. S2 shows expression of Myc repression targets during cell cycle entry of MEFs deficient in either Mnt, c-Myc, or both Mnt and c_Myc. RNA was harvested at the indicated times after serum stimulation and real-time RT-PCR performed for the indicated genes. Experiments were performed in triplicate and SDs are shown. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200411013/DC1.
![]() |
Acknowledgments |
---|
Submitted: 2 November 2004
Accepted: 8 March 2005
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amati, B., S. Dalton, M.W. Brooks, T.D. Littlewood, G.I. Evan, and H. Land. 1992. Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max. Nature. 359:423426.[CrossRef][Medline]
Askew, D.S., R.A. Ashmun, B.C. Simmons, and J.L. Cleveland. 1991. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene. 6:19151922.[Medline]
Ayer, D.E. 1999. Histone deacetylases: transcriptional repression with SINers and NuRDs. Trends Cell Biol. 9:193198.[CrossRef][Medline]
Blackwood, E.M., and R.N. Eisenman. 1991. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 251:12111217.[Medline]
Blackwood, E.M., B. Luscher, and R.N. Eisenman. 1992. Myc and Max associate in vivo. Genes Dev. 6:7180.[Abstract]
Bush, A., M. Mateyak, K. Dugan, A. Obaya, S. Adachi, J. Sedivy, and M. Cole. 1998. c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets. Genes Dev. 12:37973802.
Cawley, S., S. Bekiranov, H.H. Ng, P. Kapranov, E.A. Sekinger, D. Kampa, A. Piccolboni, V. Sementchenko, J. Cheng, A.J. Williams, et al., 2004. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell. 116:499509.[CrossRef][Medline]
Cole, M.D., and S.B. McMahon. 1999. The Myc oncoprotein: a critical evaluation of transactivation and target gene regulation. Oncogene. 18:29162924.[CrossRef][Medline]
de Alboran, I.M., R.C. O'Hagan, F. Gartner, B. Malynn, L. Davidson, R. Rickert, K. Rajewsky, R.A. DePinho, and F.W. Alt. 2001. Analysis of C-MYC function in normal cells via conditional gene-targeted mutation. Immunity. 14:4555.[CrossRef][Medline]
Eilers, M., S. Schirm, and J.M. Bishop. 1991. The MYC protein activates transcription of the alpha-prothymosin gene. EMBO J. 10:133141.[Abstract]
Eisenman, R.N. 2001. Deconstructing myc. Genes Dev. 15:20232030.
Evan, G.I., A.H. Wyllie, C.S. Gilbert, T.D. Littlewood, H. Land, M. Brooks, C.M. Waters, L.Z. Penn, and D.C. Hancock. 1992. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 69:119128.[CrossRef][Medline]
Fernandez, P.C., S.R. Frank, L. Wang, M. Schroeder, S. Liu, J. Greene, A. Cocito, and B. Amati. 2003. Genomic targets of the human c-Myc protein. Genes Dev. 17:11151129.
Haggerty, T.J., K.I. Zeller, R.C. Osthus, D.R. Wonsey, and C.V. Dang. 2003. A strategy for identifying transcription factor binding sites reveals two classes of genomic c-Myc target sites. Proc. Natl. Acad. Sci. USA. 100:53135318.
Hann, S.R., C.B. Thompson, and R.N. Eisenman. 1985. c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cells. Nature. 314:366369.[CrossRef][Medline]
Holzel, M., F. Kohlhuber, I. Schlosser, D. Holzel, B. Luscher, and D. Eick. 2001. Myc/Max/Mad regulate the frequency but not the duration of productive cell cycles. EMBO Rep. 2:11251132.
Humbert, P.O., R. Verona, J.M. Trimarchi, C. Rogers, S. Dandapani, and J.A. Lees. 2000. E2f3 is critical for normal cellular proliferation. Genes Dev. 14:690703.
Hurlin, P.J., C. Queva, and R.N. Eisenman. 1997a. Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites. Genes Dev. 11:4458.[Abstract]
Hurlin, P.J., C. Queva, and R.N. Eisenman. 1997b. Mnt: a novel Max-interacting protein and Myc antagonist. Curr. Top. Microbiol. Immunol. 224:115121.[Medline]
Hurlin, P.J., Z.Q. Zhou, K. Toyo-Oka, S. Ota, W.L. Walker, S. Hirotsune, and A. Wynshaw-Boris. 2003. Deletion of Mnt leads to disrupted cell cycle control and tumorigenesis. EMBO J. 22:45844596.
Jones, R.M., J. Branda, K.A. Johnston, M. Polymenis, M. Gadd, A. Rustgi, L. Callanan, and E.V. Schmidt. 1996. An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc. Mol. Cell. Biol. 16:47544764.[Abstract]
Kretzner, L., E.M. Blackwood, and R.N. Eisenman. 1992. Myc and Max proteins possess distinct transcriptional activities. Nature. 359:426429.[CrossRef][Medline]
Livak, K.J., and T.D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods. 25:402408.[CrossRef][Medline]
Mateyak, M.K., A.J. Obaya, S. Adachi, and J.M. Sedivy. 1997. Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination. Cell Growth Differ. 8:10391048.[Abstract]
Meroni, G., A. Reymond, M. Alcalay, G. Borsani, A. Tanigami, R. Tonlorenzi, C.L. Nigro, S. Messali, M. Zollo, D.H. Ledbetter, et al., 1997. Rox, a novel bHLHZip protein expressed in quiescent cells that heterodimerizes with Max, binds a non-canonical E box and acts as a transcriptional repressor. EMBO J. 16:28922906.
Meroni, G., S. Cairo, G. Merla, S. Messali, R. Brent, A. Ballabio, and A. Reymond. 2000. Mlx, a new Max-like bHLHZip family member: the center stage of a novel transcription factors regulatory pathway? Oncogene. 19:32663277.[CrossRef][Medline]
Metivier, R., G. Penot, M.R. Hubner, G. Reid, H. Brand, M. Kos, and F. Gannon. 2003. Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell. 115:751763.[CrossRef][Medline]
Nilsson, J.A., and J.L. Cleveland. 2003. Myc pathways provoking cell suicide and cancer. Oncogene. 22:90079021.[CrossRef][Medline]
Nilsson, J.A., K.H. Maclean, U.B. Keller, H. Pendeville, T.A. Baudino, and J.L. Cleveland. 2004. Mnt loss triggers Myc transcription targets, proliferation, apoptosis, and transformation. Mol. Cell. Biol. 24:15601569.
Orian, A., B. van Steensel, J. Delrow, H.J. Bussemaker, L. Li, T. Sawado, E. Williams, L.W. Loo, S.M. Cowley, C. Yost, et al., 2003. Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev. 17:11011114.
Pelengaris, S., M. Khan, and G. Evan. 2002. c-MYC: more than just a matter of life and death. Nat. Rev. Cancer. 2:764776.[CrossRef][Medline]
Persson, H., L. Hennighausen, R. Taub, W. DeGrado, and P. Leder. 1984. Antibodies to human c-myc oncogene product: evidence of an evolutionarily conserved protein induced during cell proliferation. Science. 225:687693.[Medline]
Schorl, C., and J.M. Sedivy. 2003. Loss of protooncogene c-Myc function impedes G1 phase progression both before and after the restriction point. Mol. Biol. Cell. 14:823835.
Todaro, G.J., and H. Green. 1963. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17:299313.
Toyo-oka, K., S. Hirotsune, M.J. Gambello, Z.Q. Zhou, L. Olson, M.G. Rosenfeld, R.N. Eisenman, P.J. Hurlin, and A. Wynshaw-Boris. 2004. Loss of the Max-interacting protein Mnt contained within the murine Miller-Dieker syndrome region results in defective embryonic growth and craniofacial defects. Hum. Mol. Genet. 13:10571067.
Trumpp, A., Y. Refaeli, T. Oskarsson, S. Gasser, M. Murphym, G.R. Martin, and J.M. Bishop. 2001. c-Myc regulates mammalian body size by controlling cell number but not cell size. Nature. 414:768773.[CrossRef][Medline]
Zhou, Z.Q., and P.J. Hurlin. 2001. The interplay between Mad and Myc in proliferation and differentiation. Trends Cell Biol. 11:S10S14.[CrossRef][Medline]