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MyoD induces myogenic differentiation through cooperation of its NH2- and COOH-terminal regions.

Ishibashi J, Perry RL, Asakura A, Rudnicki MA - J. Cell Biol. (2005)

Bottom Line: MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes.This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions.Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.

ABSTRACT
MyoD and Myf5 are basic helix-loop-helix transcription factors that play key but redundant roles in specifying myogenic progenitors during embryogenesis. However, there are functional differences between the two transcription factors that impact myoblast proliferation and differentiation. Target gene activation could be one such difference. We have used microarray and polymerase chain reaction approaches to measure the induction of muscle gene expression by MyoD and Myf5 in an in vitro model. In proliferating cells, MyoD and Myf5 function very similarly to activate the expression of likely growth phase target genes such as L-myc, m-cadherin, Mcpt8, Runx1, Spp1, Six1, IGFBP5, and Chrnbeta1. MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes. This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions. Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation.

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MyoD NH2 and COOH termini cooperatively promote differentiation. The expression of MyoD/Myf5 chimeric MRFs that included the MyoD NH2- or COOH-terminal regions in MyoD−/−;Myf5−/− fibroblasts produced more efficient differentiation in low serum conditions than those with the corresponding Myf5 region. (A) Percentage of infected cells in each pool based on GFP expression immediately before differentiation. (B) Percentage of total nuclei (n > 1,000) found within a differentiated myosin heavy chain+ cell, normalized to A. (C) Myosin heavy chain immunostaining of differentiated pools (MF20, red; DAPI, blue). Bar, 100 μm.
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fig5: MyoD NH2 and COOH termini cooperatively promote differentiation. The expression of MyoD/Myf5 chimeric MRFs that included the MyoD NH2- or COOH-terminal regions in MyoD−/−;Myf5−/− fibroblasts produced more efficient differentiation in low serum conditions than those with the corresponding Myf5 region. (A) Percentage of infected cells in each pool based on GFP expression immediately before differentiation. (B) Percentage of total nuclei (n > 1,000) found within a differentiated myosin heavy chain+ cell, normalized to A. (C) Myosin heavy chain immunostaining of differentiated pools (MF20, red; DAPI, blue). Bar, 100 μm.

Mentions: A significant number of potential MyoD target genes that were identified by GeneChip analysis were differentiation markers. Wild-type MyoD and Myf5 and chimeras were expressed in dblKO cells using retrovirus and were maintained for 3 d in growth conditions before harvest. The proportion of infected cells was very similar between pools (see Fig. 5 A). Differentiation marker expression was examined by real-time RT-PCR (Fig. 4 A). This set of vectors included a COOH-terminal FLAG epitope tag that allowed for the normalization of gene expression against MRF protein levels (Fig. 4 B). Two growth phase markers (Chrnβ1 and Runx1, identified in the aforementioned GeneChip experiment) showed little relative difference between MyoD, Myf5, and the chimeras. In concordance with the GeneChip results, however, the expression of MyoD produced a considerable activation of genes such as the cholinergic receptor α and γ subunits, myogenin, α-actin, myosin, and troponin (Fig. 4; Table II shows unnormalized changes vs. empty vector controls). In comparison, the level of Myf5 induction of these genes was moderate relative to the empty vector control.


MyoD induces myogenic differentiation through cooperation of its NH2- and COOH-terminal regions.

Ishibashi J, Perry RL, Asakura A, Rudnicki MA - J. Cell Biol. (2005)

MyoD NH2 and COOH termini cooperatively promote differentiation. The expression of MyoD/Myf5 chimeric MRFs that included the MyoD NH2- or COOH-terminal regions in MyoD−/−;Myf5−/− fibroblasts produced more efficient differentiation in low serum conditions than those with the corresponding Myf5 region. (A) Percentage of infected cells in each pool based on GFP expression immediately before differentiation. (B) Percentage of total nuclei (n > 1,000) found within a differentiated myosin heavy chain+ cell, normalized to A. (C) Myosin heavy chain immunostaining of differentiated pools (MF20, red; DAPI, blue). Bar, 100 μm.
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Related In: Results  -  Collection

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fig5: MyoD NH2 and COOH termini cooperatively promote differentiation. The expression of MyoD/Myf5 chimeric MRFs that included the MyoD NH2- or COOH-terminal regions in MyoD−/−;Myf5−/− fibroblasts produced more efficient differentiation in low serum conditions than those with the corresponding Myf5 region. (A) Percentage of infected cells in each pool based on GFP expression immediately before differentiation. (B) Percentage of total nuclei (n > 1,000) found within a differentiated myosin heavy chain+ cell, normalized to A. (C) Myosin heavy chain immunostaining of differentiated pools (MF20, red; DAPI, blue). Bar, 100 μm.
Mentions: A significant number of potential MyoD target genes that were identified by GeneChip analysis were differentiation markers. Wild-type MyoD and Myf5 and chimeras were expressed in dblKO cells using retrovirus and were maintained for 3 d in growth conditions before harvest. The proportion of infected cells was very similar between pools (see Fig. 5 A). Differentiation marker expression was examined by real-time RT-PCR (Fig. 4 A). This set of vectors included a COOH-terminal FLAG epitope tag that allowed for the normalization of gene expression against MRF protein levels (Fig. 4 B). Two growth phase markers (Chrnβ1 and Runx1, identified in the aforementioned GeneChip experiment) showed little relative difference between MyoD, Myf5, and the chimeras. In concordance with the GeneChip results, however, the expression of MyoD produced a considerable activation of genes such as the cholinergic receptor α and γ subunits, myogenin, α-actin, myosin, and troponin (Fig. 4; Table II shows unnormalized changes vs. empty vector controls). In comparison, the level of Myf5 induction of these genes was moderate relative to the empty vector control.

Bottom Line: MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes.This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions.Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.

ABSTRACT
MyoD and Myf5 are basic helix-loop-helix transcription factors that play key but redundant roles in specifying myogenic progenitors during embryogenesis. However, there are functional differences between the two transcription factors that impact myoblast proliferation and differentiation. Target gene activation could be one such difference. We have used microarray and polymerase chain reaction approaches to measure the induction of muscle gene expression by MyoD and Myf5 in an in vitro model. In proliferating cells, MyoD and Myf5 function very similarly to activate the expression of likely growth phase target genes such as L-myc, m-cadherin, Mcpt8, Runx1, Spp1, Six1, IGFBP5, and Chrnbeta1. MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes. This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions. Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation.

Show MeSH