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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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Gene expression induced by chimeras in growing dblKO cells. (A) Schematic of MyoD/Myf5 chimeras. Chimeric MRFs were constructed by interchanging the corresponding NH2-terminal, bHLH-, and COOH-terminal regions of MyoD and Myf5. (B) Levels of MRF and tubulin protein expression in each pool. The combination of three different epitopes that were recognized by MyoD and Myf5 antibodies was used to normalize the expression results in C for relative MRF expression. Puro, puromycin-resistant empty vector negative control. (C) Induction of transcripts for potential growth phase targets by each of MyoD, Myf5, and the chimeric MRFs (normalized to MRF protein and GAPDH transcript levels). Numbers on y axis indicates fold changes.
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fig3: Gene expression induced by chimeras in growing dblKO cells. (A) Schematic of MyoD/Myf5 chimeras. Chimeric MRFs were constructed by interchanging the corresponding NH2-terminal, bHLH-, and COOH-terminal regions of MyoD and Myf5. (B) Levels of MRF and tubulin protein expression in each pool. The combination of three different epitopes that were recognized by MyoD and Myf5 antibodies was used to normalize the expression results in C for relative MRF expression. Puro, puromycin-resistant empty vector negative control. (C) Induction of transcripts for potential growth phase targets by each of MyoD, Myf5, and the chimeric MRFs (normalized to MRF protein and GAPDH transcript levels). Numbers on y axis indicates fold changes.

Mentions: Within their bHLH regions, Myf5 and MyoD exhibit 88% identity and >95% homology at the amino acid level. In contrast, the regions that are NH2 and COOH terminal to this highly conserved DNA-binding region exhibit considerably more differences in sequence and function (Gerber et al., 1997). Therefore, we hypothesized that functional differences between MyoD and Myf5 would be a consequence of their divergent NH2- and COOH-terminal domains rather than a result of the bHLH recognition of discrete DNA sequences. To test this, chimeric MRFs were built, interchanging the MyoD and Myf5 NH2- and COOH-terminal regions around their bHLH domains. This yielded six chimeric MRF genes (termed d5d, d55, 55d, 5d5, 5dd, and dd5, where d denotes the portion derived from MyoD and 5 denotes the portion derived from Myf5) in addition to the full-length wild-type MyoD and Myf5 (Fig. 3 A).


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)

Gene expression induced by chimeras in growing dblKO cells. (A) Schematic of MyoD/Myf5 chimeras. Chimeric MRFs were constructed by interchanging the corresponding NH2-terminal, bHLH-, and COOH-terminal regions of MyoD and Myf5. (B) Levels of MRF and tubulin protein expression in each pool. The combination of three different epitopes that were recognized by MyoD and Myf5 antibodies was used to normalize the expression results in C for relative MRF expression. Puro, puromycin-resistant empty vector negative control. (C) Induction of transcripts for potential growth phase targets by each of MyoD, Myf5, and the chimeric MRFs (normalized to MRF protein and GAPDH transcript levels). Numbers on y axis indicates fold changes.
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Related In: Results  -  Collection

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fig3: Gene expression induced by chimeras in growing dblKO cells. (A) Schematic of MyoD/Myf5 chimeras. Chimeric MRFs were constructed by interchanging the corresponding NH2-terminal, bHLH-, and COOH-terminal regions of MyoD and Myf5. (B) Levels of MRF and tubulin protein expression in each pool. The combination of three different epitopes that were recognized by MyoD and Myf5 antibodies was used to normalize the expression results in C for relative MRF expression. Puro, puromycin-resistant empty vector negative control. (C) Induction of transcripts for potential growth phase targets by each of MyoD, Myf5, and the chimeric MRFs (normalized to MRF protein and GAPDH transcript levels). Numbers on y axis indicates fold changes.
Mentions: Within their bHLH regions, Myf5 and MyoD exhibit 88% identity and >95% homology at the amino acid level. In contrast, the regions that are NH2 and COOH terminal to this highly conserved DNA-binding region exhibit considerably more differences in sequence and function (Gerber et al., 1997). Therefore, we hypothesized that functional differences between MyoD and Myf5 would be a consequence of their divergent NH2- and COOH-terminal domains rather than a result of the bHLH recognition of discrete DNA sequences. To test this, chimeric MRFs were built, interchanging the MyoD and Myf5 NH2- and COOH-terminal regions around their bHLH domains. This yielded six chimeric MRF genes (termed d5d, d55, 55d, 5d5, 5dd, and dd5, where d denotes the portion derived from MyoD and 5 denotes the portion derived from Myf5) in addition to the full-length wild-type MyoD and Myf5 (Fig. 3 A).

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
Related in: MedlinePlus