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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

Inhibition of myogenic differentiation by Setdb1 depletion. (A) levels of Setdb1 decrease during C2C12 myoblast differentiation. C2C12 myoblast cells were grown to confluency in DMEM supplemented with 10% fetal bovine serum and differentiation was induced by serum withdrawal. Cells were harvested at the indicated time points and total RNAs or total protein extracts were prepared as described in “Materials and Methods”. RNA was analyzed by quantitative real-time RT-PCR using primers specific for Setdb1 and GAPDH. Relative expression of Setdb1 was determined using the standard curve method and then normalized to GAPDH. Error bars indicate standard deviation (left). Proteins were resolved on 7.5% (for Setdb1 and MHC) or 12% (for MyoD, myogenin, and Actin) SDS-PAGE and detected with antibodies against indicated proteins (right). (B-E) C2C12 myoblast cells with Setdb1 shRNA displayed severely delayed differentiation under serum-deprived conditions. C2C12 myoblast cells stably expressing control vector (pLKO.1) or Setdb1 shRNA were maintained in DMEM containing 10% fetal bovine serum and differentiation was initiated as described in Materials and methods. After 72 h, differentiation was assessed by the appearance of myotubes using photomicrograph (B), expression of MHC as well as endogenous MyoD using Western blot analysis (C), and number of MHC-positive nuclei per 103 cells using immunofluorescence (D, E).
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f1-molce-38-4-362: Inhibition of myogenic differentiation by Setdb1 depletion. (A) levels of Setdb1 decrease during C2C12 myoblast differentiation. C2C12 myoblast cells were grown to confluency in DMEM supplemented with 10% fetal bovine serum and differentiation was induced by serum withdrawal. Cells were harvested at the indicated time points and total RNAs or total protein extracts were prepared as described in “Materials and Methods”. RNA was analyzed by quantitative real-time RT-PCR using primers specific for Setdb1 and GAPDH. Relative expression of Setdb1 was determined using the standard curve method and then normalized to GAPDH. Error bars indicate standard deviation (left). Proteins were resolved on 7.5% (for Setdb1 and MHC) or 12% (for MyoD, myogenin, and Actin) SDS-PAGE and detected with antibodies against indicated proteins (right). (B-E) C2C12 myoblast cells with Setdb1 shRNA displayed severely delayed differentiation under serum-deprived conditions. C2C12 myoblast cells stably expressing control vector (pLKO.1) or Setdb1 shRNA were maintained in DMEM containing 10% fetal bovine serum and differentiation was initiated as described in Materials and methods. After 72 h, differentiation was assessed by the appearance of myotubes using photomicrograph (B), expression of MHC as well as endogenous MyoD using Western blot analysis (C), and number of MHC-positive nuclei per 103 cells using immunofluorescence (D, E).

Mentions: To investigate the roles of histone modifying enzymes that target lysine 9 of histone H3 in myogenic differentiation, we depleted C2C12 myoblast cells of five H3-K9 specific histone methyltransferases, namely Suv39h1, Suv39h2, GLP, G9a, and Setdb1, by using relevant shRNAs and then tested for myogenic differentiation. Among those tested, we found that transfection of Setdb1 shRNA severely impaired the ability of myoblast cells to differentiate in a serum-deprived growth condition (Fig. 1 and data not shown). Of particular interest, decreased expression of endogenous Setdb1 inhibited differentiation rather than promoted it, whereas depletion of Suv39h1 or G9a that also targets H3-K9 has been shown to increase myogenic potential in C2C12 myoblast cells (Ling et al., 2012; Mal, 2006). To confirm that Setdb1 is indeed required for myogenic differentiation, we first examined the changes in Setdb1 expression during differentiation of C2C12 myoblast cells (Fig. 1A). Quantitative RT-PCR and Western blot analysis showed that levels of both Setdb1 mRNA and protein decrease during differentiation, which is inversely correlated with that of myosin heavy chain (MHC), a marker of terminal differentiation (Fig. 1A). Next, we established stable myoblast cell lines in which endogenous Setdb1 expression is depleted by shRNAs that target different regions of mouse Setdb1 gene and then tested for their myogenic differentiation potential (Fig. 1B and Supplementary Fig. S1). In vitro differentiation assay revealed that C2C12 cells with decreased level of Setdb1 protein displayed severely delayed differentiation even after 72 h in differentiation medium (Fig. 1B). In addition, western blot analysis and immunofluorescence using anti-MHC antibody showed that knockdown of Setdb1 resulted in significantly lower level of myosin heavy chain expression (MHC) as well as myogenic index during an extended differentiation period (96 h), suggesting that Setdb1 is required for the differentiation of C2C12 myoblast cells (Figs. 1C–1E).


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

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

Inhibition of myogenic differentiation by Setdb1 depletion. (A) levels of Setdb1 decrease during C2C12 myoblast differentiation. C2C12 myoblast cells were grown to confluency in DMEM supplemented with 10% fetal bovine serum and differentiation was induced by serum withdrawal. Cells were harvested at the indicated time points and total RNAs or total protein extracts were prepared as described in “Materials and Methods”. RNA was analyzed by quantitative real-time RT-PCR using primers specific for Setdb1 and GAPDH. Relative expression of Setdb1 was determined using the standard curve method and then normalized to GAPDH. Error bars indicate standard deviation (left). Proteins were resolved on 7.5% (for Setdb1 and MHC) or 12% (for MyoD, myogenin, and Actin) SDS-PAGE and detected with antibodies against indicated proteins (right). (B-E) C2C12 myoblast cells with Setdb1 shRNA displayed severely delayed differentiation under serum-deprived conditions. C2C12 myoblast cells stably expressing control vector (pLKO.1) or Setdb1 shRNA were maintained in DMEM containing 10% fetal bovine serum and differentiation was initiated as described in Materials and methods. After 72 h, differentiation was assessed by the appearance of myotubes using photomicrograph (B), expression of MHC as well as endogenous MyoD using Western blot analysis (C), and number of MHC-positive nuclei per 103 cells using immunofluorescence (D, E).
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Related In: Results  -  Collection

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f1-molce-38-4-362: Inhibition of myogenic differentiation by Setdb1 depletion. (A) levels of Setdb1 decrease during C2C12 myoblast differentiation. C2C12 myoblast cells were grown to confluency in DMEM supplemented with 10% fetal bovine serum and differentiation was induced by serum withdrawal. Cells were harvested at the indicated time points and total RNAs or total protein extracts were prepared as described in “Materials and Methods”. RNA was analyzed by quantitative real-time RT-PCR using primers specific for Setdb1 and GAPDH. Relative expression of Setdb1 was determined using the standard curve method and then normalized to GAPDH. Error bars indicate standard deviation (left). Proteins were resolved on 7.5% (for Setdb1 and MHC) or 12% (for MyoD, myogenin, and Actin) SDS-PAGE and detected with antibodies against indicated proteins (right). (B-E) C2C12 myoblast cells with Setdb1 shRNA displayed severely delayed differentiation under serum-deprived conditions. C2C12 myoblast cells stably expressing control vector (pLKO.1) or Setdb1 shRNA were maintained in DMEM containing 10% fetal bovine serum and differentiation was initiated as described in Materials and methods. After 72 h, differentiation was assessed by the appearance of myotubes using photomicrograph (B), expression of MHC as well as endogenous MyoD using Western blot analysis (C), and number of MHC-positive nuclei per 103 cells using immunofluorescence (D, E).
Mentions: To investigate the roles of histone modifying enzymes that target lysine 9 of histone H3 in myogenic differentiation, we depleted C2C12 myoblast cells of five H3-K9 specific histone methyltransferases, namely Suv39h1, Suv39h2, GLP, G9a, and Setdb1, by using relevant shRNAs and then tested for myogenic differentiation. Among those tested, we found that transfection of Setdb1 shRNA severely impaired the ability of myoblast cells to differentiate in a serum-deprived growth condition (Fig. 1 and data not shown). Of particular interest, decreased expression of endogenous Setdb1 inhibited differentiation rather than promoted it, whereas depletion of Suv39h1 or G9a that also targets H3-K9 has been shown to increase myogenic potential in C2C12 myoblast cells (Ling et al., 2012; Mal, 2006). To confirm that Setdb1 is indeed required for myogenic differentiation, we first examined the changes in Setdb1 expression during differentiation of C2C12 myoblast cells (Fig. 1A). Quantitative RT-PCR and Western blot analysis showed that levels of both Setdb1 mRNA and protein decrease during differentiation, which is inversely correlated with that of myosin heavy chain (MHC), a marker of terminal differentiation (Fig. 1A). Next, we established stable myoblast cell lines in which endogenous Setdb1 expression is depleted by shRNAs that target different regions of mouse Setdb1 gene and then tested for their myogenic differentiation potential (Fig. 1B and Supplementary Fig. S1). In vitro differentiation assay revealed that C2C12 cells with decreased level of Setdb1 protein displayed severely delayed differentiation even after 72 h in differentiation medium (Fig. 1B). In addition, western blot analysis and immunofluorescence using anti-MHC antibody showed that knockdown of Setdb1 resulted in significantly lower level of myosin heavy chain expression (MHC) as well as myogenic index during an extended differentiation period (96 h), suggesting that Setdb1 is required for the differentiation of C2C12 myoblast cells (Figs. 1C–1E).

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