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Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD-related gene 1 (FRG1) expression during human myogenic differentiation.

Bodega B, Ramirez GD, Grasser F, Cheli S, Brunelli S, Mora M, Meneveri R, Marozzi A, Mueller S, Battaglioli E, Ginelli E - BMC Biol. (2009)

Bottom Line: Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes.The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy. beatrice.bodega@unimi.it

ABSTRACT

Background: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects.

Results: In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.

Conclusion: We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.

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Related in: MedlinePlus

Facioscapulohumeral muscular dystrophy-related gene 1 (FRG1) upregulation during myogenic differentiation is marked by a switch between H3K27me3 and Polycomb factors with H3K4me3 on its promoter. FRG1 mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) (a) and western blotting (b) in myoblasts and myotubes (8 days of differentiation); 18S rRNA and b tubulin were used as controls. (c) Total RNA from adult tissues was tested for FRG1 expression by means of RT-PCR; 18S rRNA was used as a control. Histograms in A and C represent FRG1 expression over 18S rRNA. (d) Chromatin immunoprecipitation (ChIP) assays of (i) myoblasts and myotubes, and (ii) HeLa and lymphoblasts, using antibodies against H3K4me3 (K4), H3K9me3 (K9) and H3K27me3 (K27). Input DNA (+) represents total chromatin, and IgG the immunoprecipitation by normal rabbit IgG. The amplified FRG1 promoter subregion corresponds to FRG1 A in (f)(i). The G6PD promoter was amplified as a negative H3K27me3 control. (e) ChIP analyses of control (CN) and facioscapulohumeral muscular dystrophy (FSHD) myoblasts and myotubes, indicating the standard error of the mean. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05. CN-K27me3/CN-K4me3 in myoblasts: P = 0.0167, n = 3; FSHD-K27me3/FSHD-K4me3 in myoblasts: P = 0.0157, n = 4; CN-K27me3/CN-K4me3 in myotubes: P = 0.0006, n = 3; FSHD-K27me3/FSHD-K4me3 in myotubes: P < 0.0001, n = 4. The RT-PCR primer pairs were 4q specific [11], and are shown in Additional file 3; the anti-FRG1P antibody is specific for a 4q FRG1 peptide [21]. (f)(i) A schema of the FRG1 promoter showing the position of one CarG box responsive element (in red) and two E-boxes (in green) in relation to the ATG and transcription start site (+1), and the PvuII site. The arrowheads indicate the primer positions for the FRG1 A and FRG1 B PCRs. (ii) ChIP and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using the anti-H3K27me3 (K27me3), anti-Ezh2, anti-YY1, and anti-5-methyl cytidine (5meCy) antibodies. All PCR experiments were performed in a linear range of amplification, and band intensities were measured using a Typhoon 9200 phosphoscanner and Image Quant analysis software; after subtracting the signals derived from IgG immunoprecipitation, the results were expressed as percentages of input DNA.
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Figure 1: Facioscapulohumeral muscular dystrophy-related gene 1 (FRG1) upregulation during myogenic differentiation is marked by a switch between H3K27me3 and Polycomb factors with H3K4me3 on its promoter. FRG1 mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) (a) and western blotting (b) in myoblasts and myotubes (8 days of differentiation); 18S rRNA and b tubulin were used as controls. (c) Total RNA from adult tissues was tested for FRG1 expression by means of RT-PCR; 18S rRNA was used as a control. Histograms in A and C represent FRG1 expression over 18S rRNA. (d) Chromatin immunoprecipitation (ChIP) assays of (i) myoblasts and myotubes, and (ii) HeLa and lymphoblasts, using antibodies against H3K4me3 (K4), H3K9me3 (K9) and H3K27me3 (K27). Input DNA (+) represents total chromatin, and IgG the immunoprecipitation by normal rabbit IgG. The amplified FRG1 promoter subregion corresponds to FRG1 A in (f)(i). The G6PD promoter was amplified as a negative H3K27me3 control. (e) ChIP analyses of control (CN) and facioscapulohumeral muscular dystrophy (FSHD) myoblasts and myotubes, indicating the standard error of the mean. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05. CN-K27me3/CN-K4me3 in myoblasts: P = 0.0167, n = 3; FSHD-K27me3/FSHD-K4me3 in myoblasts: P = 0.0157, n = 4; CN-K27me3/CN-K4me3 in myotubes: P = 0.0006, n = 3; FSHD-K27me3/FSHD-K4me3 in myotubes: P < 0.0001, n = 4. The RT-PCR primer pairs were 4q specific [11], and are shown in Additional file 3; the anti-FRG1P antibody is specific for a 4q FRG1 peptide [21]. (f)(i) A schema of the FRG1 promoter showing the position of one CarG box responsive element (in red) and two E-boxes (in green) in relation to the ATG and transcription start site (+1), and the PvuII site. The arrowheads indicate the primer positions for the FRG1 A and FRG1 B PCRs. (ii) ChIP and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using the anti-H3K27me3 (K27me3), anti-Ezh2, anti-YY1, and anti-5-methyl cytidine (5meCy) antibodies. All PCR experiments were performed in a linear range of amplification, and band intensities were measured using a Typhoon 9200 phosphoscanner and Image Quant analysis software; after subtracting the signals derived from IgG immunoprecipitation, the results were expressed as percentages of input DNA.

Mentions: These experimental conditions were used to analyze FRG1 expression at transcriptional and protein levels by RT-PCR and western blotting. FRG1 mRNA and protein expression was higher in the myotubes than in the myoblasts (Figure 1a, b); these data were confirmed in all the available cell lines (data not shown). RT-PCR analysis of a panel of adult human tissue RNA showed that FRG1 expression was not restricted to muscle cells (Figure 1c). FRG1 therefore seems to be subject to muscle-specific regulation, but is not a classic muscle gene.


Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD-related gene 1 (FRG1) expression during human myogenic differentiation.

Bodega B, Ramirez GD, Grasser F, Cheli S, Brunelli S, Mora M, Meneveri R, Marozzi A, Mueller S, Battaglioli E, Ginelli E - BMC Biol. (2009)

Facioscapulohumeral muscular dystrophy-related gene 1 (FRG1) upregulation during myogenic differentiation is marked by a switch between H3K27me3 and Polycomb factors with H3K4me3 on its promoter. FRG1 mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) (a) and western blotting (b) in myoblasts and myotubes (8 days of differentiation); 18S rRNA and b tubulin were used as controls. (c) Total RNA from adult tissues was tested for FRG1 expression by means of RT-PCR; 18S rRNA was used as a control. Histograms in A and C represent FRG1 expression over 18S rRNA. (d) Chromatin immunoprecipitation (ChIP) assays of (i) myoblasts and myotubes, and (ii) HeLa and lymphoblasts, using antibodies against H3K4me3 (K4), H3K9me3 (K9) and H3K27me3 (K27). Input DNA (+) represents total chromatin, and IgG the immunoprecipitation by normal rabbit IgG. The amplified FRG1 promoter subregion corresponds to FRG1 A in (f)(i). The G6PD promoter was amplified as a negative H3K27me3 control. (e) ChIP analyses of control (CN) and facioscapulohumeral muscular dystrophy (FSHD) myoblasts and myotubes, indicating the standard error of the mean. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05. CN-K27me3/CN-K4me3 in myoblasts: P = 0.0167, n = 3; FSHD-K27me3/FSHD-K4me3 in myoblasts: P = 0.0157, n = 4; CN-K27me3/CN-K4me3 in myotubes: P = 0.0006, n = 3; FSHD-K27me3/FSHD-K4me3 in myotubes: P < 0.0001, n = 4. The RT-PCR primer pairs were 4q specific [11], and are shown in Additional file 3; the anti-FRG1P antibody is specific for a 4q FRG1 peptide [21]. (f)(i) A schema of the FRG1 promoter showing the position of one CarG box responsive element (in red) and two E-boxes (in green) in relation to the ATG and transcription start site (+1), and the PvuII site. The arrowheads indicate the primer positions for the FRG1 A and FRG1 B PCRs. (ii) ChIP and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using the anti-H3K27me3 (K27me3), anti-Ezh2, anti-YY1, and anti-5-methyl cytidine (5meCy) antibodies. All PCR experiments were performed in a linear range of amplification, and band intensities were measured using a Typhoon 9200 phosphoscanner and Image Quant analysis software; after subtracting the signals derived from IgG immunoprecipitation, the results were expressed as percentages of input DNA.
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Related In: Results  -  Collection

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Figure 1: Facioscapulohumeral muscular dystrophy-related gene 1 (FRG1) upregulation during myogenic differentiation is marked by a switch between H3K27me3 and Polycomb factors with H3K4me3 on its promoter. FRG1 mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) (a) and western blotting (b) in myoblasts and myotubes (8 days of differentiation); 18S rRNA and b tubulin were used as controls. (c) Total RNA from adult tissues was tested for FRG1 expression by means of RT-PCR; 18S rRNA was used as a control. Histograms in A and C represent FRG1 expression over 18S rRNA. (d) Chromatin immunoprecipitation (ChIP) assays of (i) myoblasts and myotubes, and (ii) HeLa and lymphoblasts, using antibodies against H3K4me3 (K4), H3K9me3 (K9) and H3K27me3 (K27). Input DNA (+) represents total chromatin, and IgG the immunoprecipitation by normal rabbit IgG. The amplified FRG1 promoter subregion corresponds to FRG1 A in (f)(i). The G6PD promoter was amplified as a negative H3K27me3 control. (e) ChIP analyses of control (CN) and facioscapulohumeral muscular dystrophy (FSHD) myoblasts and myotubes, indicating the standard error of the mean. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05. CN-K27me3/CN-K4me3 in myoblasts: P = 0.0167, n = 3; FSHD-K27me3/FSHD-K4me3 in myoblasts: P = 0.0157, n = 4; CN-K27me3/CN-K4me3 in myotubes: P = 0.0006, n = 3; FSHD-K27me3/FSHD-K4me3 in myotubes: P < 0.0001, n = 4. The RT-PCR primer pairs were 4q specific [11], and are shown in Additional file 3; the anti-FRG1P antibody is specific for a 4q FRG1 peptide [21]. (f)(i) A schema of the FRG1 promoter showing the position of one CarG box responsive element (in red) and two E-boxes (in green) in relation to the ATG and transcription start site (+1), and the PvuII site. The arrowheads indicate the primer positions for the FRG1 A and FRG1 B PCRs. (ii) ChIP and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using the anti-H3K27me3 (K27me3), anti-Ezh2, anti-YY1, and anti-5-methyl cytidine (5meCy) antibodies. All PCR experiments were performed in a linear range of amplification, and band intensities were measured using a Typhoon 9200 phosphoscanner and Image Quant analysis software; after subtracting the signals derived from IgG immunoprecipitation, the results were expressed as percentages of input DNA.
Mentions: These experimental conditions were used to analyze FRG1 expression at transcriptional and protein levels by RT-PCR and western blotting. FRG1 mRNA and protein expression was higher in the myotubes than in the myoblasts (Figure 1a, b); these data were confirmed in all the available cell lines (data not shown). RT-PCR analysis of a panel of adult human tissue RNA showed that FRG1 expression was not restricted to muscle cells (Figure 1c). FRG1 therefore seems to be subject to muscle-specific regulation, but is not a classic muscle gene.

Bottom Line: Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes.The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy. beatrice.bodega@unimi.it

ABSTRACT

Background: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects.

Results: In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.

Conclusion: We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.

Show MeSH
Related in: MedlinePlus