Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD)

University of Cambridge, United Kingdom
PLoS Genetics (Impact Factor: 7.53). 08/2009; 5(7):e1000559. DOI: 10.1371/journal.pgen.1000559
Source: PubMed


Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed "phenotypic" FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4-specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)-treated cells. We found that SUV39H1-mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1gamma and cohesin are co-recruited to D4Z4 in an H3K9me3-dependent and cell type-specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type-specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1gamma/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.

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Available from: Kyoko Yokomori
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    • "Most large tandem repeats are arranged into heterochromatin (22,35,36,37). Notable exceptions include the X-linked macrosatellite DXZ4 (6), that adopts both euchromatin and heterochromatin arrangements in response to X chromosome inactivation (22,23), and the chromosome 4 macrosatellite D4Z4, that adopts a more euchromatic organization in response to a reduction in the tandem repeat copy number (37,38), or due to haploinsufficiency of the heterochromatin protein SMCHD1 (39). "
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    • "DNA hypomethylation of the D4Z4 region is common to both FSHD1 and 2 and causes transcriptional de-repression, which allows the DUX4 gene to be transcribed. The FSHD permissive alleles further present a poly-adenylation signal in the pLAM region distal to the repeat array [6], which allows stabilization of the DUX4 transcripts derived from the last D4Z4 unit and their translation [6]–[14]. "
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    ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35. Recent studies suggested that aberrant expression of double homeobox 4 (DUX4) from the last D4Z4 repeat causes FSHD. The aim of this study is to determine transcriptomic responses to ectopically expressed DUX4 in human and mouse cells of muscle lineage. We expression profiled human rhabdomyosarcoma (RD) cells and mouse C2C12 cells transfected with expression vectors of DUX4 using the Affymetrix Human Genome U133 Plus 2.0 Arrays and Mouse Genome 430 2.0 Arrays, respectively. A total of 2267 and 150 transcripts were identified to be differentially expressed in the RD and C2C12 cells, respectively. Amongst the transcripts differentially expressed in the RD cells, MYOD and MYOG (2 fold, p<0.05), and six MYOD downstream targets were up-regulated in RD but not C2C12 cells. Furthermore, 13 transcripts involved in germline function were dramatically induced only in the RD cells expressing DUX4. The top 3 IPA canonical pathways affected by DUX4 were different between the RD (inflammation, BMP signaling and NRF-2 mediated oxidative stress) and the C2C12 cells (p53 signaling, cell cycle regulation and cellular energy metabolism). Amongst the 40 transcripts shared by the RD and C2C12 cells, UTS2 was significantly induced by 76 fold and 224 fold in the RD and C2C12 cells, respectively. The differential expression of MYOD, MYOG and UTS2 were validated using real-time quantitative RT-PCR. We further validated the differentially expressed genes in immortalized FSHD myoblasts and showed up-regulation of MYOD, MYOG, ZSCAN4 and UTS2. The results suggest that DUX4 regulates overlapped and distinct groups of genes and pathways in human and mouse cells as evident by the selective up-regulation of genes involved in myogenesis and gametogenesis in human RD and immortalized cells as well as the different molecular pathways identified in the cells.
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    • "For D4Z4, the epigenetic make-up is copy number-dependent. The non-contracted array, which retains more than 11 D4Z4 units, displays heterochromatic features like the repressive histone marks H3K9me3 (Zeng et al., 2009) and H3K27me3 (Bodega et al., 2009; Cabianca et al., 2012), histone hypoacetylation (Jiang et al., 2003), as well as a high level of DNA methylation (Van Overveld et al., 2003). Reduction of D4Z4 copy number below 11 units is associated with reduced levels of repressive histone marks (Bodega et al., 2009; Zeng et al., 2009; Cabianca et al., 2012), acquisition of the activating histone marks H3K4me3 and H3K36me2 (Cabianca et al., 2012), DNA hypomethylation (Van Overveld et al., 2003), binding of CTCF (Ottaviani et al., 2009) and loss of Polycomb silencing (Cabianca et al., 2012). "
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