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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.


Related in: MedlinePlus

Knockdown of Setdb1 in C2C12 cells delay cell cycle progression. (A) The 2 × 103 cells stably expressing Setdb1 shRNAs or control vector (pLKO.1) were grown in 24-well plates, harvested at indicated time points, and stained with NBB staining solution following fixation with 10% formalin. Stained cells were extracted with 50 mM NaOH and absorbance for each sample was measured at 595 nm. Experiments were performed at least three times with triplicate for each time point. Error bars indicate standard deviation. (B) To measure relative proportion of cells at each stage of the cell cycle, 5 × 105 proliferating myoblast cells with indicated shRNA were labeled with propidium iodide in the presence of RNase A and analyzed using an FACS-Caliber flowcytometer. Data shown are representative of at least three independent experiments performed in triplicate. FlowJo V 7software was used for quantitation and graphic presentation. (C–E) Overexpression of Setdb1 had little or no effect on myogenic differentiation. (C) Undifferentiated C2C12 myoblast cells were infected with retroviruses expressing Flag-Setdb1. Empty vector (pLZRS-IRES-GFP) was used as a control. After 2 consecutive days of infection, cells were grown for 72 h under differentiation condition. Differentiation was assessed by Western blot analysis using antibody against MHC and exogenous expression of Flag-Setdb1 was shown to confirm retroviral infection. (DE) Levels of endogenous MyoD (D) and myogenin (E) were quantified by real-time RT-PCR as described in “Materials and Methods”. Shown are representative data of at least three independent experiments in triplicate, error bars indicate standard deviation.
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f3-molce-38-4-362: Knockdown of Setdb1 in C2C12 cells delay cell cycle progression. (A) The 2 × 103 cells stably expressing Setdb1 shRNAs or control vector (pLKO.1) were grown in 24-well plates, harvested at indicated time points, and stained with NBB staining solution following fixation with 10% formalin. Stained cells were extracted with 50 mM NaOH and absorbance for each sample was measured at 595 nm. Experiments were performed at least three times with triplicate for each time point. Error bars indicate standard deviation. (B) To measure relative proportion of cells at each stage of the cell cycle, 5 × 105 proliferating myoblast cells with indicated shRNA were labeled with propidium iodide in the presence of RNase A and analyzed using an FACS-Caliber flowcytometer. Data shown are representative of at least three independent experiments performed in triplicate. FlowJo V 7software was used for quantitation and graphic presentation. (C–E) Overexpression of Setdb1 had little or no effect on myogenic differentiation. (C) Undifferentiated C2C12 myoblast cells were infected with retroviruses expressing Flag-Setdb1. Empty vector (pLZRS-IRES-GFP) was used as a control. After 2 consecutive days of infection, cells were grown for 72 h under differentiation condition. Differentiation was assessed by Western blot analysis using antibody against MHC and exogenous expression of Flag-Setdb1 was shown to confirm retroviral infection. (DE) Levels of endogenous MyoD (D) and myogenin (E) were quantified by real-time RT-PCR as described in “Materials and Methods”. Shown are representative data of at least three independent experiments in triplicate, error bars indicate standard deviation.

Mentions: It is often the case that factors that regulate cell cycle progression inhibit myogenic differentiation by preventing exit from the cell cycle (Rao and Kohtz, 1995; Wang et al., 1995). So, we tested whether depletion of Setdb1 affects on cell proliferation by measuring cell growth and relative proportions of cells at different stages of cell cycle using flowcytometry (Figs. 3A and 3B). C2C12 myoblast cells harboring Setdb1 shRNA showed slower growth at low confluence (2 × 103 cells/24-well plate) and higher proportion of cells at G2/M stage of cell cycle compared to control C2C12 myoblast cells (Figs. 3A and 3B). Although it is unclear whether delayed cell growth is directly linked to the inhibition of differentiation, these data demonstrate that Setdb1 is associated with both cell proliferation and differentiation of C2C12 myoblast cells.


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

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

Knockdown of Setdb1 in C2C12 cells delay cell cycle progression. (A) The 2 × 103 cells stably expressing Setdb1 shRNAs or control vector (pLKO.1) were grown in 24-well plates, harvested at indicated time points, and stained with NBB staining solution following fixation with 10% formalin. Stained cells were extracted with 50 mM NaOH and absorbance for each sample was measured at 595 nm. Experiments were performed at least three times with triplicate for each time point. Error bars indicate standard deviation. (B) To measure relative proportion of cells at each stage of the cell cycle, 5 × 105 proliferating myoblast cells with indicated shRNA were labeled with propidium iodide in the presence of RNase A and analyzed using an FACS-Caliber flowcytometer. Data shown are representative of at least three independent experiments performed in triplicate. FlowJo V 7software was used for quantitation and graphic presentation. (C–E) Overexpression of Setdb1 had little or no effect on myogenic differentiation. (C) Undifferentiated C2C12 myoblast cells were infected with retroviruses expressing Flag-Setdb1. Empty vector (pLZRS-IRES-GFP) was used as a control. After 2 consecutive days of infection, cells were grown for 72 h under differentiation condition. Differentiation was assessed by Western blot analysis using antibody against MHC and exogenous expression of Flag-Setdb1 was shown to confirm retroviral infection. (DE) Levels of endogenous MyoD (D) and myogenin (E) were quantified by real-time RT-PCR as described in “Materials and Methods”. Shown are representative data of at least three independent experiments in triplicate, error bars indicate standard deviation.
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f3-molce-38-4-362: Knockdown of Setdb1 in C2C12 cells delay cell cycle progression. (A) The 2 × 103 cells stably expressing Setdb1 shRNAs or control vector (pLKO.1) were grown in 24-well plates, harvested at indicated time points, and stained with NBB staining solution following fixation with 10% formalin. Stained cells were extracted with 50 mM NaOH and absorbance for each sample was measured at 595 nm. Experiments were performed at least three times with triplicate for each time point. Error bars indicate standard deviation. (B) To measure relative proportion of cells at each stage of the cell cycle, 5 × 105 proliferating myoblast cells with indicated shRNA were labeled with propidium iodide in the presence of RNase A and analyzed using an FACS-Caliber flowcytometer. Data shown are representative of at least three independent experiments performed in triplicate. FlowJo V 7software was used for quantitation and graphic presentation. (C–E) Overexpression of Setdb1 had little or no effect on myogenic differentiation. (C) Undifferentiated C2C12 myoblast cells were infected with retroviruses expressing Flag-Setdb1. Empty vector (pLZRS-IRES-GFP) was used as a control. After 2 consecutive days of infection, cells were grown for 72 h under differentiation condition. Differentiation was assessed by Western blot analysis using antibody against MHC and exogenous expression of Flag-Setdb1 was shown to confirm retroviral infection. (DE) Levels of endogenous MyoD (D) and myogenin (E) were quantified by real-time RT-PCR as described in “Materials and Methods”. Shown are representative data of at least three independent experiments in triplicate, error bars indicate standard deviation.
Mentions: It is often the case that factors that regulate cell cycle progression inhibit myogenic differentiation by preventing exit from the cell cycle (Rao and Kohtz, 1995; Wang et al., 1995). So, we tested whether depletion of Setdb1 affects on cell proliferation by measuring cell growth and relative proportions of cells at different stages of cell cycle using flowcytometry (Figs. 3A and 3B). C2C12 myoblast cells harboring Setdb1 shRNA showed slower growth at low confluence (2 × 103 cells/24-well plate) and higher proportion of cells at G2/M stage of cell cycle compared to control C2C12 myoblast cells (Figs. 3A and 3B). Although it is unclear whether delayed cell growth is directly linked to the inhibition of differentiation, these data demonstrate that Setdb1 is associated with both cell proliferation and differentiation of C2C12 myoblast cells.

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.


Related in: MedlinePlus