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Mnt-Max to Myc-Max complex switching regulates cell cycle entry.

Walker W, Zhou ZQ, Ota S, Wynshaw-Boris A, Hurlin PJ - J. Cell Biol. (2005)

Bottom Line: Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes.Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry.These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Shriners Hospitals for Children, Portland, OR 97201, USA.

ABSTRACT
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

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Regulation of c-Myc/Mnt target genes during cell cycle entry in the absence of Mnt. RNA from wild-type and Mnt  primary MEFs was harvested at the indicated times after serum stimulation and quantitative real-time RT-PCR assays performed in triplicate for the indicated genes. The data are representative of two or more independent experiments. RNA levels of the ARBP P0 gene, which do not change during cell cycle entry (not depicted), were used to standardize samples. Fold expression values were calculated relative to the 0-h time point in wild-type cells for each gene as previously described (Livak and Schmittgen, 2001). SDs are shown.
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fig3: Regulation of c-Myc/Mnt target genes during cell cycle entry in the absence of Mnt. RNA from wild-type and Mnt primary MEFs was harvested at the indicated times after serum stimulation and quantitative real-time RT-PCR assays performed in triplicate for the indicated genes. The data are representative of two or more independent experiments. RNA levels of the ARBP P0 gene, which do not change during cell cycle entry (not depicted), were used to standardize samples. Fold expression values were calculated relative to the 0-h time point in wild-type cells for each gene as previously described (Livak and Schmittgen, 2001). SDs are shown.

Mentions: 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 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 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 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 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-Max to Myc-Max complex switching regulates cell cycle entry.

Walker W, Zhou ZQ, Ota S, Wynshaw-Boris A, Hurlin PJ - J. Cell Biol. (2005)

Regulation of c-Myc/Mnt target genes during cell cycle entry in the absence of Mnt. RNA from wild-type and Mnt  primary MEFs was harvested at the indicated times after serum stimulation and quantitative real-time RT-PCR assays performed in triplicate for the indicated genes. The data are representative of two or more independent experiments. RNA levels of the ARBP P0 gene, which do not change during cell cycle entry (not depicted), were used to standardize samples. Fold expression values were calculated relative to the 0-h time point in wild-type cells for each gene as previously described (Livak and Schmittgen, 2001). SDs are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171929&req=5

fig3: Regulation of c-Myc/Mnt target genes during cell cycle entry in the absence of Mnt. RNA from wild-type and Mnt primary MEFs was harvested at the indicated times after serum stimulation and quantitative real-time RT-PCR assays performed in triplicate for the indicated genes. The data are representative of two or more independent experiments. RNA levels of the ARBP P0 gene, which do not change during cell cycle entry (not depicted), were used to standardize samples. Fold expression values were calculated relative to the 0-h time point in wild-type cells for each gene as previously described (Livak and Schmittgen, 2001). SDs are shown.
Mentions: 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 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 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 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 MEFs appeared to reflect the accelerated cell cycle entry profile of these cells compared with wild-type MEFs (Fig. 5; Hurlin et al., 2003).

Bottom Line: Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes.Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry.These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Shriners Hospitals for Children, Portland, OR 97201, USA.

ABSTRACT
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

Show MeSH
Related in: MedlinePlus