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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 depletion leads to downregulation of myogenic regulatory factors in proliferating C2C12 cells. (A) Exogenous Setdb1 not targeted by Setdb1 shRNA rescues myogenic differentiation in Setdb1-depleted myoblast cells. C2C12 cells depleted of endogenous Setdb1 were infected with retrovirus expressing Flag-Setdb1 which is not targeted by Setdb1 shRNA used for cell line establishment. Empty vector was used as a control. Differentiation was assessed by the appearance of myotubes after 72 h in serum-deprived growth medium (left) and expressions of MHC and MyoD (right). (B, C) Total RNAs were prepared from both control and Setdb1 shRNA-expressing C2C12 cells prior to or 72 h after initiation of differentiation. RNA was analyzed by real-time PCR using primers specific for MyoD (B) and myogenin (C). Results were quantified using the standard curve method and normalized to GAPDH. Data were represented as relative expression. Shown are representative data of at least three independent experiments performed in triplicate, and error bars indicate standard deviation. *p-value < 0.05 and **p-value < 0.01
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f2-molce-38-4-362: Setdb1 depletion leads to downregulation of myogenic regulatory factors in proliferating C2C12 cells. (A) Exogenous Setdb1 not targeted by Setdb1 shRNA rescues myogenic differentiation in Setdb1-depleted myoblast cells. C2C12 cells depleted of endogenous Setdb1 were infected with retrovirus expressing Flag-Setdb1 which is not targeted by Setdb1 shRNA used for cell line establishment. Empty vector was used as a control. Differentiation was assessed by the appearance of myotubes after 72 h in serum-deprived growth medium (left) and expressions of MHC and MyoD (right). (B, C) Total RNAs were prepared from both control and Setdb1 shRNA-expressing C2C12 cells prior to or 72 h after initiation of differentiation. RNA was analyzed by real-time PCR using primers specific for MyoD (B) and myogenin (C). Results were quantified using the standard curve method and normalized to GAPDH. Data were represented as relative expression. Shown are representative data of at least three independent experiments performed in triplicate, and error bars indicate standard deviation. *p-value < 0.05 and **p-value < 0.01

Mentions: We next investigated whether or not the defects in myogenic differentiation observed in Setdb1-depleted C2C12 cells can be rescued by exogenous expression of Setdb1 that is not targeted by Setdb1 shRNA (Fig. 2). Retroviral infection of human Setdb1 followed by in vitro differentiation appeared to restore myogenic potential in C2C12 cells depleted of Setdb1, which is evident by the formation of myotubes as well as the expressions of both myosin heavy chain (MHC) and MyoD (Fig. 2A). These data suggest that inhibition of differentiation in our established C2C12 cells was due to decreased Setdb1 level and not off-site effects caused by shRNAs. Interestingly, steady state mRNAs levels of both MyoD and myogenin, which are two key regulators of skeletal muscle differentiation, were significantly lower in cells depleted of Setdb1 compared to cells with control myoblast cells in undifferentiated state and these lower levels of expressions are not fully restored in a condition that promotes differentiation (Figs. 2B and 2C). This result implies that the role of Setdb1 in C2C12 cells might be associated with maintaining steady state levels of key myogenic regulatory factors (MRFs) such as MyoD and myogenin.


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 depletion leads to downregulation of myogenic regulatory factors in proliferating C2C12 cells. (A) Exogenous Setdb1 not targeted by Setdb1 shRNA rescues myogenic differentiation in Setdb1-depleted myoblast cells. C2C12 cells depleted of endogenous Setdb1 were infected with retrovirus expressing Flag-Setdb1 which is not targeted by Setdb1 shRNA used for cell line establishment. Empty vector was used as a control. Differentiation was assessed by the appearance of myotubes after 72 h in serum-deprived growth medium (left) and expressions of MHC and MyoD (right). (B, C) Total RNAs were prepared from both control and Setdb1 shRNA-expressing C2C12 cells prior to or 72 h after initiation of differentiation. RNA was analyzed by real-time PCR using primers specific for MyoD (B) and myogenin (C). Results were quantified using the standard curve method and normalized to GAPDH. Data were represented as relative expression. Shown are representative data of at least three independent experiments performed in triplicate, and error bars indicate standard deviation. *p-value < 0.05 and **p-value < 0.01
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Related In: Results  -  Collection

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f2-molce-38-4-362: Setdb1 depletion leads to downregulation of myogenic regulatory factors in proliferating C2C12 cells. (A) Exogenous Setdb1 not targeted by Setdb1 shRNA rescues myogenic differentiation in Setdb1-depleted myoblast cells. C2C12 cells depleted of endogenous Setdb1 were infected with retrovirus expressing Flag-Setdb1 which is not targeted by Setdb1 shRNA used for cell line establishment. Empty vector was used as a control. Differentiation was assessed by the appearance of myotubes after 72 h in serum-deprived growth medium (left) and expressions of MHC and MyoD (right). (B, C) Total RNAs were prepared from both control and Setdb1 shRNA-expressing C2C12 cells prior to or 72 h after initiation of differentiation. RNA was analyzed by real-time PCR using primers specific for MyoD (B) and myogenin (C). Results were quantified using the standard curve method and normalized to GAPDH. Data were represented as relative expression. Shown are representative data of at least three independent experiments performed in triplicate, and error bars indicate standard deviation. *p-value < 0.05 and **p-value < 0.01
Mentions: We next investigated whether or not the defects in myogenic differentiation observed in Setdb1-depleted C2C12 cells can be rescued by exogenous expression of Setdb1 that is not targeted by Setdb1 shRNA (Fig. 2). Retroviral infection of human Setdb1 followed by in vitro differentiation appeared to restore myogenic potential in C2C12 cells depleted of Setdb1, which is evident by the formation of myotubes as well as the expressions of both myosin heavy chain (MHC) and MyoD (Fig. 2A). These data suggest that inhibition of differentiation in our established C2C12 cells was due to decreased Setdb1 level and not off-site effects caused by shRNAs. Interestingly, steady state mRNAs levels of both MyoD and myogenin, which are two key regulators of skeletal muscle differentiation, were significantly lower in cells depleted of Setdb1 compared to cells with control myoblast cells in undifferentiated state and these lower levels of expressions are not fully restored in a condition that promotes differentiation (Figs. 2B and 2C). This result implies that the role of Setdb1 in C2C12 cells might be associated with maintaining steady state levels of key myogenic regulatory factors (MRFs) such as MyoD and myogenin.

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.