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Setdb1 is required for myogenic differentiation of C2C12 myoblast cells via maintenance of MyoD expression.

Song YJ, Choi JH, Lee H - Mol. Cells (2015)

Bottom Line: Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification.We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on.Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, College of Natural Science, Inha University, Incheon 402-751, Korea.

ABSTRACT
Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

No MeSH data available.


Setdb1 maintains endogenous MyoD expression without interfering transactivation by exogenous MyoD. (A, B) Setdb1 shRNA inhibits MyoD-luciferase reporter in C2C12 cells but not in C3H 10T1/2 cells. C2C12 myoblast cells (A) or C3H 10T1/2 mesenchymal cells (B) were transiently transfected for 24 h with plasmid expressing myc-MyoD and/or Setdb1 shRNA together with a MyoD-luciferase reporter. (C–D) Exogenous expression of Flag-Setdb1 had little effect on MyoD-luciferase reporter in both C2C12 and C3H 10T1/2 cells. C2C12 cells (C) or C3H 10T1/2 cells (D) were transfected with plasmid expressing myc-MyoD and/or Flag-Setdb1. For all reporter analysis, empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector); Shown are representative data of three independent experiments performed in triplicate, and error bars indicate standard deviation. (E) Setdb1 did not bind to MyoD or myogenin promoter. Chromatin Immunoprecipitation (ChIP) was performed in C2C12 cells infected with retroviruses expressing Flag-Setdb1. The empty vector (pLZRS- IRES-GFP) was used as a control. Immunoprecipitated DNA was analyzed by PCR with specific primer sets described in Materials and methods followed by real-time qRT-PCR and agarose gel electrophoresis. Shown are representative data of three independent experiments. Nnat is a known Setdb1 target and was used as a positive control for Setdb1 binding, whereas Actin was used as a negative control.
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f5-molce-38-4-362: Setdb1 maintains endogenous MyoD expression without interfering transactivation by exogenous MyoD. (A, B) Setdb1 shRNA inhibits MyoD-luciferase reporter in C2C12 cells but not in C3H 10T1/2 cells. C2C12 myoblast cells (A) or C3H 10T1/2 mesenchymal cells (B) were transiently transfected for 24 h with plasmid expressing myc-MyoD and/or Setdb1 shRNA together with a MyoD-luciferase reporter. (C–D) Exogenous expression of Flag-Setdb1 had little effect on MyoD-luciferase reporter in both C2C12 and C3H 10T1/2 cells. C2C12 cells (C) or C3H 10T1/2 cells (D) were transfected with plasmid expressing myc-MyoD and/or Flag-Setdb1. For all reporter analysis, empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector); Shown are representative data of three independent experiments performed in triplicate, and error bars indicate standard deviation. (E) Setdb1 did not bind to MyoD or myogenin promoter. Chromatin Immunoprecipitation (ChIP) was performed in C2C12 cells infected with retroviruses expressing Flag-Setdb1. The empty vector (pLZRS- IRES-GFP) was used as a control. Immunoprecipitated DNA was analyzed by PCR with specific primer sets described in Materials and methods followed by real-time qRT-PCR and agarose gel electrophoresis. Shown are representative data of three independent experiments. Nnat is a known Setdb1 target and was used as a positive control for Setdb1 binding, whereas Actin was used as a negative control.

Mentions: C2C12 cells were grown to 50% confluency in 6-well plates in DMEM with 10% fetal bovine serum and transfected with 0.2 μg of pMyoD-Luciferase containing F3/-2.5 fragment and MyoD promoter, 0.1 μg of pSV-β-Gal and 0.3 μg of Setdb1 shRNA expression vector and/or 0.4 μg of myc-MyoD expression vector. For Figs. 5C and 5D, 0.3 μg of Flag-Setdb1 expression vector was used for transfection. The total amount of plasmid DNA was adjusted to 1.0 μg by adding empty vector (pcDNA3). Cells were harvested at 24 h after transfection and analyzed for luciferase activity following the manufacturer’s protocol (Promega).


Setdb1 is required for myogenic differentiation of C2C12 myoblast cells via maintenance of MyoD expression.

Song YJ, Choi JH, Lee H - Mol. Cells (2015)

Setdb1 maintains endogenous MyoD expression without interfering transactivation by exogenous MyoD. (A, B) Setdb1 shRNA inhibits MyoD-luciferase reporter in C2C12 cells but not in C3H 10T1/2 cells. C2C12 myoblast cells (A) or C3H 10T1/2 mesenchymal cells (B) were transiently transfected for 24 h with plasmid expressing myc-MyoD and/or Setdb1 shRNA together with a MyoD-luciferase reporter. (C–D) Exogenous expression of Flag-Setdb1 had little effect on MyoD-luciferase reporter in both C2C12 and C3H 10T1/2 cells. C2C12 cells (C) or C3H 10T1/2 cells (D) were transfected with plasmid expressing myc-MyoD and/or Flag-Setdb1. For all reporter analysis, empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector); Shown are representative data of three independent experiments performed in triplicate, and error bars indicate standard deviation. (E) Setdb1 did not bind to MyoD or myogenin promoter. Chromatin Immunoprecipitation (ChIP) was performed in C2C12 cells infected with retroviruses expressing Flag-Setdb1. The empty vector (pLZRS- IRES-GFP) was used as a control. Immunoprecipitated DNA was analyzed by PCR with specific primer sets described in Materials and methods followed by real-time qRT-PCR and agarose gel electrophoresis. Shown are representative data of three independent experiments. Nnat is a known Setdb1 target and was used as a positive control for Setdb1 binding, whereas Actin was used as a negative control.
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f5-molce-38-4-362: Setdb1 maintains endogenous MyoD expression without interfering transactivation by exogenous MyoD. (A, B) Setdb1 shRNA inhibits MyoD-luciferase reporter in C2C12 cells but not in C3H 10T1/2 cells. C2C12 myoblast cells (A) or C3H 10T1/2 mesenchymal cells (B) were transiently transfected for 24 h with plasmid expressing myc-MyoD and/or Setdb1 shRNA together with a MyoD-luciferase reporter. (C–D) Exogenous expression of Flag-Setdb1 had little effect on MyoD-luciferase reporter in both C2C12 and C3H 10T1/2 cells. C2C12 cells (C) or C3H 10T1/2 cells (D) were transfected with plasmid expressing myc-MyoD and/or Flag-Setdb1. For all reporter analysis, empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector); Shown are representative data of three independent experiments performed in triplicate, and error bars indicate standard deviation. (E) Setdb1 did not bind to MyoD or myogenin promoter. Chromatin Immunoprecipitation (ChIP) was performed in C2C12 cells infected with retroviruses expressing Flag-Setdb1. The empty vector (pLZRS- IRES-GFP) was used as a control. Immunoprecipitated DNA was analyzed by PCR with specific primer sets described in Materials and methods followed by real-time qRT-PCR and agarose gel electrophoresis. Shown are representative data of three independent experiments. Nnat is a known Setdb1 target and was used as a positive control for Setdb1 binding, whereas Actin was used as a negative control.
Mentions: C2C12 cells were grown to 50% confluency in 6-well plates in DMEM with 10% fetal bovine serum and transfected with 0.2 μg of pMyoD-Luciferase containing F3/-2.5 fragment and MyoD promoter, 0.1 μg of pSV-β-Gal and 0.3 μg of Setdb1 shRNA expression vector and/or 0.4 μg of myc-MyoD expression vector. For Figs. 5C and 5D, 0.3 μg of Flag-Setdb1 expression vector was used for transfection. The total amount of plasmid DNA was adjusted to 1.0 μg by adding empty vector (pcDNA3). Cells were harvested at 24 h after transfection and analyzed for luciferase activity following the manufacturer’s protocol (Promega).

Bottom Line: Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification.We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on.Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, College of Natural Science, Inha University, Incheon 402-751, Korea.

ABSTRACT
Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

No MeSH data available.