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 - PubMed (original) (raw)
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
Beatrice Bodega et al. BMC Biol. 2009.
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.
Figures
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 _Pvu_II 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.
Figure 2
Anticipated facioscapulohumeral muscular dystrophy-related gene 1 (FRG1) expression during facioscapulohumeral muscular dystrophy (FSHD) myoblast differentiation. (a) FRG1 expression in three healthy (CN) and three FSHD muscle biopsies (FSHD) as revealed by means of a quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. (see Additional file 3 for primers), indicating the standard deviation of the mean. A two-tailed t test was used for statistical analysis; the asterisk indicates the statistical significant difference between FSHD1 and CN3 (the control with the highest level of FRG1 expression): a = 0.05, P = 0.023. (b) FRG1 expression during myogenic differentiation in healthy (CN) and FSHD (FSHD) samples, expressed as fold of GAPDH expression, indicating the standard deviation of the mean. A two-tailed t test was used for statistical analysis; asterisks indicate the statistical significant differences between: FSHD1 day 1/FSHD1 day 0, α = 0.05, P = 0.0031; FSHD2 day 1/FSHD2 day 0, α = 0.05, P = 0.0035; FSHD3 day 1/FSHD3 day 0, α = 0.05, P = 0.0036; FSHD4 day 1/FSHD4 day 0, α = 0.05, P = 0.0033. (c) Kinetics of FRG1 expression during myogenic differentiation in control and FSHD cell lines; the values were determined as median of FRG1 expression at each step of differentiation (days 0, 1, 4 and 8) after subtracting the median value at day 0. The standard error of the mean was indicated. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05; controls, n = 3; FSHD, n = 4; CN day 1/FSHD day 1, P = 0.0001. These results were derived from at least three independent RNA extractions for each human cell line.
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 _Pvu_II 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.
Figure 4
Nuclear topology of D4Z4 units in control and FSHD myoblasts. 4q subtelomere architecture in 1.4 μm midprojections of 3D-preserved interphase nuclei from healthy (i, iii, iv and v) and FSHD myoblasts (ii, vi and vii) immunofluorescence in situ hybridization (immuno-FISH) using anti-H3K27me3 antibody (scale bar = 5 μm). The chromosome 4q territories are shown in blue, the bacterial artificial chromosome (BAC) upstream of the FRG1 gene in red, and the BAC containing a D4Z4 array in green. The arrows highlight the 4q subtelomeres identified by the cohybridization of both BACs and the chromosome 4 painting. The arrowhead in (ii) identifies a 4q subtelomere showing reduced hybridization with the green BAC and probably corresponding to the contracted D4Z4 allele. (iii) and (vi) The same nuclei as those respectively shown in (i) and (ii) were immunostained with anti-H3K27me3 antibody (red); the green spots correspond to hybridization with the D4Z4-containing BAC; the arrows and arrowhead identify the 4q alleles. (iv) and (v) Representative confocal sections of a nucleus from healthy myoblasts consisting of 4q D4Z4 alleles (green) that were negative ((iv), only green) or positive ((v), green and yellow) for colocalization with anti-H3K27me3 immunofluorescence (red). (vii) A 3 × enlargement of the confocal section in (vi) showing a nucleus from FSHD myoblast consisting of 4q D4Z4 alleles (green) that were negative (arrowhead, only green) or positive (arrow, green and yellow) for colocalization with anti-H3K27me3 immunofluorescence (red).
Figure 5
Higher order structure of the facioscapulohumeral muscular dystrophy (FSHD) locus. (a) Diagram of the genomic region analyzed in the chromosome conformation capture (3C) experiments, indicating the _Pvu_II restriction sites (thin vertical lines); the arrowheads indicate the primer positions, the F series in FSHD-related gene 1(FRG1), the C series in DUX4c, the D series in the D4Z4 repeats; the numbers from 1 to 5 indicate the restriction sites near the D1 bait used as positive controls. The red rectangles show the locations of CarG boxes. (b) The Y axis represents the crosslinking frequency expressed as the ratio of polymerase chain reaction (PCR) performed on 3C samples relative to bacterial artificial chromosome (BAC) controls between the fixed _Pvu_II fragment D1 (D4Z4 repeats) and the rest of the FSHD locus after the correction for digestion and ligation. The calculation of the relative crosslinking frequency between two given fragments, performed as described previously [43], allows a direct comparison between the different cell types used in the 3C assay by correcting for possible variants. All data points were generated from an average of three independent experiments. The standard error of the mean is indicated. One-way analysis of variance (ANOVA) was applied for statistical analysis; control myoblasts: α = 0.05, P = 1.81*10-12; control myotubes: α = 0.05, P = 3.14*10-6. To compare myoblasts and myotubes, two-way ANOVA was applied for statistical analysis: α = 0.05, P = 0.0073. (c) Frequency of interaction between the FRG1 promoter and D4Z4 sequences in FSHD and control myoblasts, indicating the standard error of the mean. Quantitative (q)PCR was performed on 3C templates using a TaqMan probe complementary to the FRG1 promoter portion of the PCR product obtained using F7 and D1 primers. The results are normalized to digestion, ligation and crosslinking efficiency, as described in Additional file 5. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05. Controls, n = 3; FSHD, n = 4; P = 0.021. The primers are shown in the detailed 3C protocol section of Additional file 5.
References
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