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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

Depletion of Setdb1 leads to downregulation of muscle-specific genes in proliferating myoblast cells. (A) Venn diagrams show overlap of genes altered by serum withdrawal, which initiates myogenic differentiation of control C2C12 cells. Depletion of Setdb1 leads to deregulation of a comparable number of genes without induction of myogenic differentiation. Only a subset of upregulated genes (51/352) in C2C12 cells with Setdb1 depletion increase during normal differentiation (left panel), while 50% of downregulated genes (207/414) overlaps with those reduced by serum withdrawal (right panel). (B) Venn diagrams show upregulated (left) and downregulated (right) genes by Setdb1 depletion that are identified by combination of oligonucleotide microarray (light gray) and RNA-seq analsyis (dark gray). (C) Setdb1 is required to maintain levels of differentiation-dependent genes. Venn diagrams show that 32 of 75 genes downregulated by Setdb1 knockdown are induced during normal myogenic differentiation (left panel), whereas only two upregulated genes overlap with genes reduced during differentiation (right panel). P-values were calculated by hypergeometric test.
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f4-molce-38-4-362: Depletion of Setdb1 leads to downregulation of muscle-specific genes in proliferating myoblast cells. (A) Venn diagrams show overlap of genes altered by serum withdrawal, which initiates myogenic differentiation of control C2C12 cells. Depletion of Setdb1 leads to deregulation of a comparable number of genes without induction of myogenic differentiation. Only a subset of upregulated genes (51/352) in C2C12 cells with Setdb1 depletion increase during normal differentiation (left panel), while 50% of downregulated genes (207/414) overlaps with those reduced by serum withdrawal (right panel). (B) Venn diagrams show upregulated (left) and downregulated (right) genes by Setdb1 depletion that are identified by combination of oligonucleotide microarray (light gray) and RNA-seq analsyis (dark gray). (C) Setdb1 is required to maintain levels of differentiation-dependent genes. Venn diagrams show that 32 of 75 genes downregulated by Setdb1 knockdown are induced during normal myogenic differentiation (left panel), whereas only two upregulated genes overlap with genes reduced during differentiation (right panel). P-values were calculated by hypergeometric test.

Mentions: Setdb1 depletion by applying high-density oligonucleotide microarray (Illumina MouseWG-6 v2 expression BeadChip) and direct sequencing of total RNAs (Fig. 4). Gene expression profiles were obtained from three stable cell lines derived from C2C12 myoblast cells, one harboring control vector (pLKO.1) that maintain myogenic potential and two independently established cells with decreased Setdb1 respectively. By using criteria of a two-fold threshold and a false discovery rate (FDR) < 0.05, we first identified 757 genes (347 genes up and 410 genes down) that are differentially expressed between proliferating and differentiating C2C12 myoblast cells (72 h after induction of differentiation) (Fig. 4A), most of which were also presented in previously reported data (Cao et al., 2006; Delgado et al., 2003; Moran et al., 2002; Tomczak et al., 2004). Interestingly, 766 genes (352 genes up and 414 genes down) were found to be differentially expressed in myoblast cells with decreased Setdb1 under same experimental conditions even though they could not differentiate into myotube (Fig. 4A). Comparison of these expression profiles revealed that more than 85% of genes (296/347) induced in differentiated myotubes, which includes myogenin and muscle-specific genes, were not found in the list of genes upregulated in Setdb1 shRNA expressing cells. In addition, 50% of genes (207/414) that are downregulated by serum withdrawal in Setdb1-depleted myoblast cells overlapped with genes decreased during myogenic differentiation, demonstrating that knockdown of Setdb1 in C2C12 cells leads to the deregulation of genes involved in muscle differentiation.


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

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

Depletion of Setdb1 leads to downregulation of muscle-specific genes in proliferating myoblast cells. (A) Venn diagrams show overlap of genes altered by serum withdrawal, which initiates myogenic differentiation of control C2C12 cells. Depletion of Setdb1 leads to deregulation of a comparable number of genes without induction of myogenic differentiation. Only a subset of upregulated genes (51/352) in C2C12 cells with Setdb1 depletion increase during normal differentiation (left panel), while 50% of downregulated genes (207/414) overlaps with those reduced by serum withdrawal (right panel). (B) Venn diagrams show upregulated (left) and downregulated (right) genes by Setdb1 depletion that are identified by combination of oligonucleotide microarray (light gray) and RNA-seq analsyis (dark gray). (C) Setdb1 is required to maintain levels of differentiation-dependent genes. Venn diagrams show that 32 of 75 genes downregulated by Setdb1 knockdown are induced during normal myogenic differentiation (left panel), whereas only two upregulated genes overlap with genes reduced during differentiation (right panel). P-values were calculated by hypergeometric test.
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Related In: Results  -  Collection

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f4-molce-38-4-362: Depletion of Setdb1 leads to downregulation of muscle-specific genes in proliferating myoblast cells. (A) Venn diagrams show overlap of genes altered by serum withdrawal, which initiates myogenic differentiation of control C2C12 cells. Depletion of Setdb1 leads to deregulation of a comparable number of genes without induction of myogenic differentiation. Only a subset of upregulated genes (51/352) in C2C12 cells with Setdb1 depletion increase during normal differentiation (left panel), while 50% of downregulated genes (207/414) overlaps with those reduced by serum withdrawal (right panel). (B) Venn diagrams show upregulated (left) and downregulated (right) genes by Setdb1 depletion that are identified by combination of oligonucleotide microarray (light gray) and RNA-seq analsyis (dark gray). (C) Setdb1 is required to maintain levels of differentiation-dependent genes. Venn diagrams show that 32 of 75 genes downregulated by Setdb1 knockdown are induced during normal myogenic differentiation (left panel), whereas only two upregulated genes overlap with genes reduced during differentiation (right panel). P-values were calculated by hypergeometric test.
Mentions: Setdb1 depletion by applying high-density oligonucleotide microarray (Illumina MouseWG-6 v2 expression BeadChip) and direct sequencing of total RNAs (Fig. 4). Gene expression profiles were obtained from three stable cell lines derived from C2C12 myoblast cells, one harboring control vector (pLKO.1) that maintain myogenic potential and two independently established cells with decreased Setdb1 respectively. By using criteria of a two-fold threshold and a false discovery rate (FDR) < 0.05, we first identified 757 genes (347 genes up and 410 genes down) that are differentially expressed between proliferating and differentiating C2C12 myoblast cells (72 h after induction of differentiation) (Fig. 4A), most of which were also presented in previously reported data (Cao et al., 2006; Delgado et al., 2003; Moran et al., 2002; Tomczak et al., 2004). Interestingly, 766 genes (352 genes up and 414 genes down) were found to be differentially expressed in myoblast cells with decreased Setdb1 under same experimental conditions even though they could not differentiate into myotube (Fig. 4A). Comparison of these expression profiles revealed that more than 85% of genes (296/347) induced in differentiated myotubes, which includes myogenin and muscle-specific genes, were not found in the list of genes upregulated in Setdb1 shRNA expressing cells. In addition, 50% of genes (207/414) that are downregulated by serum withdrawal in Setdb1-depleted myoblast cells overlapped with genes decreased during myogenic differentiation, demonstrating that knockdown of Setdb1 in C2C12 cells leads to the deregulation of genes involved in muscle differentiation.

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