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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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Preparation of RNA for GeneChip analysis. (A) Representative FACS plots of MyoD−/−;Myf5−/− fibroblasts infected with retrovirus expressing MyoD, Myf5, or no gene as well as GFP from an internal ribosomal entry site (IRES) within the same transcript. GFP expression amongst sorted cells after 24 h of culture was verified by fluorescence microscopy immediately before harvesting for total RNA. SSC, side scatter; FL1, fluorescence channel 1; G, gate; R, region. The circled regions denote the sorted populations. Bar, 100 μm. (B) Northern blot demonstrating equivalent levels of retroviral transcript expression amongst samples. (C) Western blots demonstrating robust MyoD or Myf5 expression in corresponding samples.
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fig1: Preparation of RNA for GeneChip analysis. (A) Representative FACS plots of MyoD−/−;Myf5−/− fibroblasts infected with retrovirus expressing MyoD, Myf5, or no gene as well as GFP from an internal ribosomal entry site (IRES) within the same transcript. GFP expression amongst sorted cells after 24 h of culture was verified by fluorescence microscopy immediately before harvesting for total RNA. SSC, side scatter; FL1, fluorescence channel 1; G, gate; R, region. The circled regions denote the sorted populations. Bar, 100 μm. (B) Northern blot demonstrating equivalent levels of retroviral transcript expression amongst samples. (C) Western blots demonstrating robust MyoD or Myf5 expression in corresponding samples.

Mentions: Shortly after infection of the dblKO target cells with retrovirus, positive-expressing cells were purified by FACS based on GFP fluorescence expressed from the bicistronic retroviral transcript (Fig. 1, A and B). Pools of >106 cells were then harvested for total RNA after a further 24 h of culture in high serum growth conditions. Fluorescently labeled probes generated from biological–triplicate RNA samples were hybridized to MG-U74Av2 GeneChips, each containing probesets directed at ∼6,000 genes and an additional ∼6,000 ESTs. Comparison of MyoD or Myf5 arrays with control arrays produced candidate lists that were considered to contain genes potentially regulated by MyoD or Myf5 during growth phase (Table I and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200502101/DC1).


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)

Preparation of RNA for GeneChip analysis. (A) Representative FACS plots of MyoD−/−;Myf5−/− fibroblasts infected with retrovirus expressing MyoD, Myf5, or no gene as well as GFP from an internal ribosomal entry site (IRES) within the same transcript. GFP expression amongst sorted cells after 24 h of culture was verified by fluorescence microscopy immediately before harvesting for total RNA. SSC, side scatter; FL1, fluorescence channel 1; G, gate; R, region. The circled regions denote the sorted populations. Bar, 100 μm. (B) Northern blot demonstrating equivalent levels of retroviral transcript expression amongst samples. (C) Western blots demonstrating robust MyoD or Myf5 expression in corresponding samples.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171269&req=5

fig1: Preparation of RNA for GeneChip analysis. (A) Representative FACS plots of MyoD−/−;Myf5−/− fibroblasts infected with retrovirus expressing MyoD, Myf5, or no gene as well as GFP from an internal ribosomal entry site (IRES) within the same transcript. GFP expression amongst sorted cells after 24 h of culture was verified by fluorescence microscopy immediately before harvesting for total RNA. SSC, side scatter; FL1, fluorescence channel 1; G, gate; R, region. The circled regions denote the sorted populations. Bar, 100 μm. (B) Northern blot demonstrating equivalent levels of retroviral transcript expression amongst samples. (C) Western blots demonstrating robust MyoD or Myf5 expression in corresponding samples.
Mentions: Shortly after infection of the dblKO target cells with retrovirus, positive-expressing cells were purified by FACS based on GFP fluorescence expressed from the bicistronic retroviral transcript (Fig. 1, A and B). Pools of >106 cells were then harvested for total RNA after a further 24 h of culture in high serum growth conditions. Fluorescently labeled probes generated from biological–triplicate RNA samples were hybridized to MG-U74Av2 GeneChips, each containing probesets directed at ∼6,000 genes and an additional ∼6,000 ESTs. Comparison of MyoD or Myf5 arrays with control arrays produced candidate lists that were considered to contain genes potentially regulated by MyoD or Myf5 during growth phase (Table I and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200502101/DC1).

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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