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Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells.

Zhang K, Sha J, Harter ML - J. Cell Biol. (2010)

Bottom Line: MyoD and Cdc6 are both expressed after quiescent C2C12 myoblasts or satellite cells in association with myofibers are stimulated for growth, but MyoD appears at least 2-3 h earlier than Cdc6.Finally, knockdown of MyoD impairs the ability of C2C12 cells to express Cdc6 after leaving quiescence, and as a result, they cannot fully progress into S phase.Our results define a mechanism by which MyoD helps myogenic satellite cells to enter into the first round of DNA replication after transitioning out of quiescence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
MyoD is a transcriptional factor that is required for the differentiation of muscle stem cells (satellite cells). In this study, we describe a previously unknown function for MyoD in regulating a gene (Cdc6) that is vital to endowing chromatin with the capability of replicating DNA. In C2C12 and primary mouse myoblasts, we show that MyoD can occupy an E-box within the promoter of Cdc6 and that this association, along with E2F3a, is required for its activity. MyoD and Cdc6 are both expressed after quiescent C2C12 myoblasts or satellite cells in association with myofibers are stimulated for growth, but MyoD appears at least 2-3 h earlier than Cdc6. Finally, knockdown of MyoD impairs the ability of C2C12 cells to express Cdc6 after leaving quiescence, and as a result, they cannot fully progress into S phase. Our results define a mechanism by which MyoD helps myogenic satellite cells to enter into the first round of DNA replication after transitioning out of quiescence.

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E2F3a occupies the Cdc6 promoter in myoblasts, but it is replaced by E2F4 after differentiation. (A) ChIP of C2C12 myoblasts cultured in GM with antibodies (Ab) detecting E2F1, E2F3a, and E2F4. As a control, ChIP was also performed in parallel with normal rabbit IgG (NR IgG). Purified DNA from enriched chromatin fragments was amplified by PCR with the E1 primers. (B and C) ChIP of C2C12 myoblasts cultured in DM at the indicated times using normal rabbit IgG and either anti-E2F3a or anti-E2F4, respectively. Precipitated DNA was analyzed for the presence of the Cdc6 promoter by PCR. (D) The occupation of the Cdc6 promoter by E2F4 correlates with its silencing in differentiated myoblasts. Immunofluorescent staining of C2C12 myoblasts cultured in GM or DM for 96 h was performed by using a Cdc6-specific antibody. Antibody staining is visualized as red. RT-PCR detection of Cdc6 transcripts in C2C12 myoblasts cultured in GM or in DM for 72 or 96 h. GAPDH expression is used as a loading control. White lines indicate that intervening lanes have been spliced out. Bars, 20 µm.
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fig2: E2F3a occupies the Cdc6 promoter in myoblasts, but it is replaced by E2F4 after differentiation. (A) ChIP of C2C12 myoblasts cultured in GM with antibodies (Ab) detecting E2F1, E2F3a, and E2F4. As a control, ChIP was also performed in parallel with normal rabbit IgG (NR IgG). Purified DNA from enriched chromatin fragments was amplified by PCR with the E1 primers. (B and C) ChIP of C2C12 myoblasts cultured in DM at the indicated times using normal rabbit IgG and either anti-E2F3a or anti-E2F4, respectively. Precipitated DNA was analyzed for the presence of the Cdc6 promoter by PCR. (D) The occupation of the Cdc6 promoter by E2F4 correlates with its silencing in differentiated myoblasts. Immunofluorescent staining of C2C12 myoblasts cultured in GM or DM for 96 h was performed by using a Cdc6-specific antibody. Antibody staining is visualized as red. RT-PCR detection of Cdc6 transcripts in C2C12 myoblasts cultured in GM or in DM for 72 or 96 h. GAPDH expression is used as a loading control. White lines indicate that intervening lanes have been spliced out. Bars, 20 µm.

Mentions: ChIP experiments were also performed to determine whether any member of the E2F family of transcription factors (E2F1–7) might be interacting with the Cdc6 promoter in proliferating myoblasts. As shown in Fig. 2 A, E2F3a, an isoform mainly expressed in growing cells (Leone et al., 2000), was found to be the most predominant factor in association with this promoter, although a weak binding by E2F4 could also be observed. Note that the near absence of E2F4 from the Cdc6 promoter was not the result of an antibody that had lost its specificity when used in a ChIP assay (Fig. 2 C). Although the site on the promoter to which E2F3a is binding has yet to be identified, it is possible that it may be the one closest to the transcriptional start site (Fig. 1 A), as has been previously shown in the human Cdc6 promoter (Schlisio et al., 2002). We also observed in this analysis that E2F1 was noticeably absent from the Cdc6 promoter (Fig. 2 A), and again, this result is consistent with what has been previous reported for the human Cdc6 promoter (Takahashi et al., 2000; Ren et al., 2002; Schlisio et al., 2002). E2F3a has been reported to play an important role in controlling genes that are critical to the replication of DNA. In fact, the ability of cells to enter S phase after being released from a G1/S block becomes markedly diminished if E2F3a is purposely made inactive (Leone et al., 1998). Because of this and the fact that the cessation of DNA synthesis is concurrent with the differentiation of muscle cells, we examined by ChIP assays the relationship between E2F3a and the Cdc6 promoter after myoblasts were induced to differentiate. We found by temporal analysis that E2F3a could no longer be detected on the Cdc6 promoter after 24 h of differentiation (Fig. 2 B). Perhaps equally important is that this loss also corresponded to a linear increase in the association of E2F4 with this promoter (Fig. 2 C). This observation and the fact that E2F4 functions primarily as a repressor of transcription (Blais et al., 2005) may be one reason why the expression of Cdc6 is no longer seen in differentiated muscle cells, either by immunofluorescence or by RT-PCR analysis (Fig. 2 D). We note that Cdc6 is primarily located in the nucleus of proliferating myoblasts, with some evidence of it in the cytoplasm (Fig. 2 D). This phenomenon has also been observed by others (Petersen et al., 1999; Delmolino et al., 2001) presumably because Cdc6 remains chromatin bound after S phase is initiated so that it can then participate in other cellular functions such as mitotic entry (Clay-Farrace et al., 2003).


Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells.

Zhang K, Sha J, Harter ML - J. Cell Biol. (2010)

E2F3a occupies the Cdc6 promoter in myoblasts, but it is replaced by E2F4 after differentiation. (A) ChIP of C2C12 myoblasts cultured in GM with antibodies (Ab) detecting E2F1, E2F3a, and E2F4. As a control, ChIP was also performed in parallel with normal rabbit IgG (NR IgG). Purified DNA from enriched chromatin fragments was amplified by PCR with the E1 primers. (B and C) ChIP of C2C12 myoblasts cultured in DM at the indicated times using normal rabbit IgG and either anti-E2F3a or anti-E2F4, respectively. Precipitated DNA was analyzed for the presence of the Cdc6 promoter by PCR. (D) The occupation of the Cdc6 promoter by E2F4 correlates with its silencing in differentiated myoblasts. Immunofluorescent staining of C2C12 myoblasts cultured in GM or DM for 96 h was performed by using a Cdc6-specific antibody. Antibody staining is visualized as red. RT-PCR detection of Cdc6 transcripts in C2C12 myoblasts cultured in GM or in DM for 72 or 96 h. GAPDH expression is used as a loading control. White lines indicate that intervening lanes have been spliced out. Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig2: E2F3a occupies the Cdc6 promoter in myoblasts, but it is replaced by E2F4 after differentiation. (A) ChIP of C2C12 myoblasts cultured in GM with antibodies (Ab) detecting E2F1, E2F3a, and E2F4. As a control, ChIP was also performed in parallel with normal rabbit IgG (NR IgG). Purified DNA from enriched chromatin fragments was amplified by PCR with the E1 primers. (B and C) ChIP of C2C12 myoblasts cultured in DM at the indicated times using normal rabbit IgG and either anti-E2F3a or anti-E2F4, respectively. Precipitated DNA was analyzed for the presence of the Cdc6 promoter by PCR. (D) The occupation of the Cdc6 promoter by E2F4 correlates with its silencing in differentiated myoblasts. Immunofluorescent staining of C2C12 myoblasts cultured in GM or DM for 96 h was performed by using a Cdc6-specific antibody. Antibody staining is visualized as red. RT-PCR detection of Cdc6 transcripts in C2C12 myoblasts cultured in GM or in DM for 72 or 96 h. GAPDH expression is used as a loading control. White lines indicate that intervening lanes have been spliced out. Bars, 20 µm.
Mentions: ChIP experiments were also performed to determine whether any member of the E2F family of transcription factors (E2F1–7) might be interacting with the Cdc6 promoter in proliferating myoblasts. As shown in Fig. 2 A, E2F3a, an isoform mainly expressed in growing cells (Leone et al., 2000), was found to be the most predominant factor in association with this promoter, although a weak binding by E2F4 could also be observed. Note that the near absence of E2F4 from the Cdc6 promoter was not the result of an antibody that had lost its specificity when used in a ChIP assay (Fig. 2 C). Although the site on the promoter to which E2F3a is binding has yet to be identified, it is possible that it may be the one closest to the transcriptional start site (Fig. 1 A), as has been previously shown in the human Cdc6 promoter (Schlisio et al., 2002). We also observed in this analysis that E2F1 was noticeably absent from the Cdc6 promoter (Fig. 2 A), and again, this result is consistent with what has been previous reported for the human Cdc6 promoter (Takahashi et al., 2000; Ren et al., 2002; Schlisio et al., 2002). E2F3a has been reported to play an important role in controlling genes that are critical to the replication of DNA. In fact, the ability of cells to enter S phase after being released from a G1/S block becomes markedly diminished if E2F3a is purposely made inactive (Leone et al., 1998). Because of this and the fact that the cessation of DNA synthesis is concurrent with the differentiation of muscle cells, we examined by ChIP assays the relationship between E2F3a and the Cdc6 promoter after myoblasts were induced to differentiate. We found by temporal analysis that E2F3a could no longer be detected on the Cdc6 promoter after 24 h of differentiation (Fig. 2 B). Perhaps equally important is that this loss also corresponded to a linear increase in the association of E2F4 with this promoter (Fig. 2 C). This observation and the fact that E2F4 functions primarily as a repressor of transcription (Blais et al., 2005) may be one reason why the expression of Cdc6 is no longer seen in differentiated muscle cells, either by immunofluorescence or by RT-PCR analysis (Fig. 2 D). We note that Cdc6 is primarily located in the nucleus of proliferating myoblasts, with some evidence of it in the cytoplasm (Fig. 2 D). This phenomenon has also been observed by others (Petersen et al., 1999; Delmolino et al., 2001) presumably because Cdc6 remains chromatin bound after S phase is initiated so that it can then participate in other cellular functions such as mitotic entry (Clay-Farrace et al., 2003).

Bottom Line: MyoD and Cdc6 are both expressed after quiescent C2C12 myoblasts or satellite cells in association with myofibers are stimulated for growth, but MyoD appears at least 2-3 h earlier than Cdc6.Finally, knockdown of MyoD impairs the ability of C2C12 cells to express Cdc6 after leaving quiescence, and as a result, they cannot fully progress into S phase.Our results define a mechanism by which MyoD helps myogenic satellite cells to enter into the first round of DNA replication after transitioning out of quiescence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
MyoD is a transcriptional factor that is required for the differentiation of muscle stem cells (satellite cells). In this study, we describe a previously unknown function for MyoD in regulating a gene (Cdc6) that is vital to endowing chromatin with the capability of replicating DNA. In C2C12 and primary mouse myoblasts, we show that MyoD can occupy an E-box within the promoter of Cdc6 and that this association, along with E2F3a, is required for its activity. MyoD and Cdc6 are both expressed after quiescent C2C12 myoblasts or satellite cells in association with myofibers are stimulated for growth, but MyoD appears at least 2-3 h earlier than Cdc6. Finally, knockdown of MyoD impairs the ability of C2C12 cells to express Cdc6 after leaving quiescence, and as a result, they cannot fully progress into S phase. Our results define a mechanism by which MyoD helps myogenic satellite cells to enter into the first round of DNA replication after transitioning out of quiescence.

Show MeSH