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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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Mnt–Max and Myc–Max complex switching on shared target genes. (a) Western blot showing c-Myc expression in wild-type (Mnt+/+) and Mnt  (Mnt−/−) MEFs at 0, 4, and 24 h after serum stimulation of quiescent cells. (b) ChIP analysis, performed in parallel with the Western blot shown in panel a, examining Mnt, c-Myc, and Max binding to specific E-box–containing regions in the Cdk4, Cyclin D2, ODC, Nucleolin, Telomerase (Tert) E2F2, CAD, and Cyclin E1 genes at 0, 4, and 24 h after serum stimulation. (c) Chromatin reimmunoprecipitation (ReIP) assays showing the presence of Sin3A and HDAC1 at several Mnt-Myc target genes. Pre, preimmune serum; Total, total input.
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fig2: Mnt–Max and Myc–Max complex switching on shared target genes. (a) Western blot showing c-Myc expression in wild-type (Mnt+/+) and Mnt (Mnt−/−) MEFs at 0, 4, and 24 h after serum stimulation of quiescent cells. (b) ChIP analysis, performed in parallel with the Western blot shown in panel a, examining Mnt, c-Myc, and Max binding to specific E-box–containing regions in the Cdk4, Cyclin D2, ODC, Nucleolin, Telomerase (Tert) E2F2, CAD, and Cyclin E1 genes at 0, 4, and 24 h after serum stimulation. (c) Chromatin reimmunoprecipitation (ReIP) assays showing the presence of Sin3A and HDAC1 at several Mnt-Myc target genes. Pre, preimmune serum; Total, total input.

Mentions: To directly examine the consequences of complex switching between Mnt–Max and c-Myc–Max during cell cycle entry, chromatin immunoprecipitation (ChIP) experiments were performed. Early passage Mnt 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-box–containing regions within the Cyclin E1 (ccne1) gene where no c-Myc or Mnt binding was observed was used as a negative control.


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)

Mnt–Max and Myc–Max complex switching on shared target genes. (a) Western blot showing c-Myc expression in wild-type (Mnt+/+) and Mnt  (Mnt−/−) MEFs at 0, 4, and 24 h after serum stimulation of quiescent cells. (b) ChIP analysis, performed in parallel with the Western blot shown in panel a, examining Mnt, c-Myc, and Max binding to specific E-box–containing regions in the Cdk4, Cyclin D2, ODC, Nucleolin, Telomerase (Tert) E2F2, CAD, and Cyclin E1 genes at 0, 4, and 24 h after serum stimulation. (c) Chromatin reimmunoprecipitation (ReIP) assays showing the presence of Sin3A and HDAC1 at several Mnt-Myc target genes. Pre, preimmune serum; Total, total input.
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Related In: Results  -  Collection

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fig2: Mnt–Max and Myc–Max complex switching on shared target genes. (a) Western blot showing c-Myc expression in wild-type (Mnt+/+) and Mnt (Mnt−/−) MEFs at 0, 4, and 24 h after serum stimulation of quiescent cells. (b) ChIP analysis, performed in parallel with the Western blot shown in panel a, examining Mnt, c-Myc, and Max binding to specific E-box–containing regions in the Cdk4, Cyclin D2, ODC, Nucleolin, Telomerase (Tert) E2F2, CAD, and Cyclin E1 genes at 0, 4, and 24 h after serum stimulation. (c) Chromatin reimmunoprecipitation (ReIP) assays showing the presence of Sin3A and HDAC1 at several Mnt-Myc target genes. Pre, preimmune serum; Total, total input.
Mentions: To directly examine the consequences of complex switching between Mnt–Max and c-Myc–Max during cell cycle entry, chromatin immunoprecipitation (ChIP) experiments were performed. Early passage Mnt 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-box–containing regions within the Cyclin E1 (ccne1) gene where no c-Myc or Mnt binding was observed was used as a negative control.

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