Additional file 1: of Diversification of the muscle proteome through alternative splicing

List of genes that undergo alternative splicing in muscle tissue [218–226]. (DOCX 36 kb

DNA Methylation Analysis of the Macrosatellite Repeat Associated with FSHD Muscular Dystrophy at Single Nucleotide Level

<div><p>Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common inherited diseases of the skeletal muscle. It is characterized by asymmetric muscle weakness and variable penetrance. FSHD is linked to a reduction in copy number of the D4Z4 3.3 kb macrosatellite repeat, located in 4q35. This causes the epigenetic de-repression of FSHD candidate genes leading to disease. Nevertheless, the molecular mechanism responsible for silencing of FSHD candidate genes in healthy subjects is not fully understood. While a role for DNA methylation has been suggested, so far there is limited information regarding the methylation status of the 325 CpGs contained in each D4Z4 unit. Using a human/rodent monochromosomal hybrid cell line containing a single human chromosome 4, we performed an in depth analysis of DNA methylation for the majority of the CpGs inside D4Z4 at single nucleotide level. We found that D4Z4 is not uniformly methylated and that the level of DNA methylation does not correlate with the density of CpG dinucleotides. Moreover, in several D4Z4 regions characterized by near complete methylation, we found specific unmethylated CpGs. These elements are enriched in transcription factor binding sites that could be involved in muscle-specific D4Z4 activity. Our approach also detected differential methylation among different D4Z4 units, suggesting that the D4Z4 array is a mosaic of euchromatic and heterochromatic domains. Finally, we found that DNA methylation and histone de-acetylation are required to maintain FSHD candidate genes repressed. Taken together, our data underscore new players involved in the epigenetic regulation of the FSHD locus that could be targeted for therapeutic purposes.</p></div

Chr4/CHO cells recapitulate the epigenetic status present at the FSHD locus in healthy subjects.

<p>ChIP survey for repressive and actives marks as well as enzymes present inside D4Z4. Recruitment was analyzed by ChIP followed by qPCR with primers located throughout D4Z4. Primers located in the D4Z4 proximal region p13E-11 were used as control. Results are normalized for D4Z4 copy number. <b>a.</b> Set of primers spanning D4Z4 used in qPCR and Bisulfite Studies, including location of DR primers from Hartweck et al and Methylation sensitive sites previously reported in literature. <b>b.</b> ChIP for H3K9me3. <b>c.</b> ChIP for HP1 gamma. <b>d.</b> ChIP for Cohesin. <b>e</b> and <b>f.</b> ChIP for pan acetylated histones H3 and Histone H4 compared with the highly transcribed gene <i>beta</i>-<i>Actin</i> used as control. <b>g.</b> ChIP for HMGB2. <b>h.</b> ChIP for YY1. <b>i.</b> ChIP for Total Histone H3 showing similar enrichment for all the regions. Error bars correspond to SEM. Panel b, e and f are normalized for the signal of total histone H3. Panel c and d, signal is normalized for D4Z4 copy number as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115278#s2" target="_blank">Materials and Methods</a>.</p

Primers used in Bisulfite sequencing analysis.

<p>Primers used in Bisulfite sequencing analysis.</p

HDACs are enriched to the FSHD locus.

<p>ChIP survey for Hdac1, Hdac2 and Hdac3 in Chr4/CHO cells. Recruitment was analyzed by qPCR with primers located throughout D4Z4. Primers located in the D4Z4 proximal region p13E-11 were used as control. <b>a.</b> ChIP for Hdac1. <b>b.</b> ChIP for Hdac2. <b>c.</b> ChIP for Hdac3. Error bars correspond to SEM. Panel a through c signal was normalized for D4Z4 copy number as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115278#s2" target="_blank">Material and Methods</a>.</p

Primers used in RT-qPCR for gene expression analysis.

<p>Primers used in RT-qPCR for gene expression analysis.</p

Model for DNMTs and HDACs contribution in 4q35 gene repression.

<p>Healthy individuals carrying from 11 to 100 D4Z4 units display a high enrichment for DNMTs and HDACs, redundantly contributing to the repressive environment of the FSHD locus. Deletion of D4Z4 units is associated to reduced DNMTs and HDACs, leading to de-repression of several FSHD candidate genes in FSHD.</p

Effect of DNMTs and HDACs inhibitors on the expression of FSHD candidate genes.

<p>Expression analyses for 4q35 genes upon single treatment with AZA, TSA or a combined treatment with AZA plus TSA. Relative expression of 4q35 genes, using GAPDH as normalizator and mock as reference. Graphs represent average of three independent experiments. Error bar correspond to SEM. Statistic analysis was performed using Graphpad Software. Samples were analyzed by One way ANOVA followed by Dunnett's Multiple Comparison Test. * P≤0.05; ** p≤0.01; *** p≤0.001. <b>a.</b> MeDIP inside D4Z4 showing reduction in DNA methylation upon AZA + TSA treatment. <b>b.</b> ChIP showing reduced acH3 in AZA + TSA treated cells. <b>c.</b> ChIP showing reduced acH4 in AZA + TSA treated cells. <b>d.</b><i>FRG1</i> expression. <b>e.</b><i>FRG2</i> expression analysis. <b>f.</b><i>ANT1</i> expression analysis. <b>g.</b><i>DUX4</i> expression analysis. <b>h.</b> LncRNA <i>DBE-T</i> expression analysis. <b>i.</b><i>Beta</i>-<i>Actin</i> expression analysis, as control. <b>j.</b> Scheme showing location of genes of interest in the 4q35 region.</p

D4Z4 methylation analysis by bisulfite sequencing.

<p>At least 30 clones were analyzed by Quantification Tool for Methylation Analysis (QUMA). The figure represents cumulative results for ≥10 independents clones sequenced in both directions. <b>a.</b> Scheme representing bisulfite primer location. <b>b.</b> Region B1 contains 30 CpGs dinucleotides. <b>c.</b> Region B2 contains 41 CpGs dinucleotides. <b>d.</b> Region B3 contains 23 CpGs dinucleotides. <b>e.</b> Region B4 contains 36 CpGs dinucleotides. <b>f.</b> Region B5 contains 21 CpGs dinucleotides. <b>g.</b> Region B6 contains 43 CpGs dinucleotides. <b>h.</b> Region B7 contains 25 CpGs dinucleotides. Red boxes highlight single non-methylated CpGs. Open Circle correspond to unmethylated CpG. Close circle correspond to methylated CpG dinucleotide.</p

Primers used in ChIP experiments.

<p>Primers used in ChIP experiments.</p