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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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Chromatin structure of D4Z4 units in human myoblasts. (a)(i) A simplified schema of the D4Z4 unit showing the position of two CarG box responsive elements (sequences in red). The arrowheads indicate the primer positions for the Lsau, D4Z4 binding element (DBE)1 and DBE2 subregions; the PvuII restriction site positions are indicated. (ii) Chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using anti-H3K27me3 (K27me3), Ezh2, YY1, and 5-methyl cytidine (5meCy) antibodies (iii) Examples of ChIP experiments on the Lsau and DBE1 subregions in myoblasts and myotubes. (b) H3K27 trimethylation of D4Z4 sequences before (day 0) and after (day 8) myogenic differentiation in healthy control (CN) and facioscapulohumeral muscular dystrophy (FSHD) cell lines, as revealed by ChIP experiments on the DBE1 subregion using anti-H3K27me3 antibody (red), 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-day 0/CN-day 8: P = 0.0172, n = 3; FSHD-day 0/FSHD-day 8: P = 0.0003, n = 4. All of the polymerase chain reaction (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 immunoprecipitation with IgG antibody, the results were expressed as percentages of input DNA. The primer pairs are shown in Additional file 3.
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Figure 3: Chromatin structure of D4Z4 units in human myoblasts. (a)(i) A simplified schema of the D4Z4 unit showing the position of two CarG box responsive elements (sequences in red). The arrowheads indicate the primer positions for the Lsau, D4Z4 binding element (DBE)1 and DBE2 subregions; the PvuII restriction site positions are indicated. (ii) Chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using anti-H3K27me3 (K27me3), Ezh2, YY1, and 5-methyl cytidine (5meCy) antibodies (iii) Examples of ChIP experiments on the Lsau and DBE1 subregions in myoblasts and myotubes. (b) H3K27 trimethylation of D4Z4 sequences before (day 0) and after (day 8) myogenic differentiation in healthy control (CN) and facioscapulohumeral muscular dystrophy (FSHD) cell lines, as revealed by ChIP experiments on the DBE1 subregion using anti-H3K27me3 antibody (red), 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-day 0/CN-day 8: P = 0.0172, n = 3; FSHD-day 0/FSHD-day 8: P = 0.0003, n = 4. All of the polymerase chain reaction (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 immunoprecipitation with IgG antibody, the results were expressed as percentages of input DNA. The primer pairs are shown in Additional file 3.

Mentions: The same chromatin analyses as those described above were used to examine the D4Z4 sequences. The MatInspector software revealed two CarG boxes in specific subregions of the repeat, which are indicated as D4Z4 binding element (DBE)1 and DBE2 in the schema of Figure 3a part i. A YY1 binding site has been previously described in the D4Z4 repeat [18] and, in our schema, it coincides with the CarG box within the DBE1 region (Figure 3a part i).


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)

Chromatin structure of D4Z4 units in human myoblasts. (a)(i) A simplified schema of the D4Z4 unit showing the position of two CarG box responsive elements (sequences in red). The arrowheads indicate the primer positions for the Lsau, D4Z4 binding element (DBE)1 and DBE2 subregions; the PvuII restriction site positions are indicated. (ii) Chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using anti-H3K27me3 (K27me3), Ezh2, YY1, and 5-methyl cytidine (5meCy) antibodies (iii) Examples of ChIP experiments on the Lsau and DBE1 subregions in myoblasts and myotubes. (b) H3K27 trimethylation of D4Z4 sequences before (day 0) and after (day 8) myogenic differentiation in healthy control (CN) and facioscapulohumeral muscular dystrophy (FSHD) cell lines, as revealed by ChIP experiments on the DBE1 subregion using anti-H3K27me3 antibody (red), 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-day 0/CN-day 8: P = 0.0172, n = 3; FSHD-day 0/FSHD-day 8: P = 0.0003, n = 4. All of the polymerase chain reaction (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 immunoprecipitation with IgG antibody, the results were expressed as percentages of input DNA. The primer pairs are shown in Additional file 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719609&req=5

Figure 3: Chromatin structure of D4Z4 units in human myoblasts. (a)(i) A simplified schema of the D4Z4 unit showing the position of two CarG box responsive elements (sequences in red). The arrowheads indicate the primer positions for the Lsau, D4Z4 binding element (DBE)1 and DBE2 subregions; the PvuII restriction site positions are indicated. (ii) Chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using anti-H3K27me3 (K27me3), Ezh2, YY1, and 5-methyl cytidine (5meCy) antibodies (iii) Examples of ChIP experiments on the Lsau and DBE1 subregions in myoblasts and myotubes. (b) H3K27 trimethylation of D4Z4 sequences before (day 0) and after (day 8) myogenic differentiation in healthy control (CN) and facioscapulohumeral muscular dystrophy (FSHD) cell lines, as revealed by ChIP experiments on the DBE1 subregion using anti-H3K27me3 antibody (red), 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-day 0/CN-day 8: P = 0.0172, n = 3; FSHD-day 0/FSHD-day 8: P = 0.0003, n = 4. All of the polymerase chain reaction (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 immunoprecipitation with IgG antibody, the results were expressed as percentages of input DNA. The primer pairs are shown in Additional file 3.
Mentions: The same chromatin analyses as those described above were used to examine the D4Z4 sequences. The MatInspector software revealed two CarG boxes in specific subregions of the repeat, which are indicated as D4Z4 binding element (DBE)1 and DBE2 in the schema of Figure 3a part i. A YY1 binding site has been previously described in the D4Z4 repeat [18] and, in our schema, it coincides with the CarG box within the DBE1 region (Figure 3a part i).

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