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DNA methylation and differentiation: HOX genes in muscle cells.

ABSTRACT: BACKGROUND: Tight regulation of homeobox genes is essential for vertebrate development. In a study of genome-wide differential methylation, we recently found that homeobox genes, including those in the HOX gene clusters, were highly overrepresented among the genes with hypermethylation in the skeletal muscle lineage. Methylation was analyzed by reduced representation bisulfite sequencing (RRBS) of postnatal myoblasts, myotubes and adult skeletal muscle tissue and 30 types of non-muscle-cell cultures or tissues. RESULTS: In this study, we found that myogenic hypermethylation was present in specific subregions of all four HOX gene clusters and was associated with various chromatin epigenetic features. Although the 3' half of the HOXD cluster was silenced and enriched in polycomb repression-associated H3 lysine 27 trimethylation in most examined cell types, including myoblasts and myotubes, myogenic samples were unusual in also displaying much DNA methylation in this region. In contrast, both HOXA and HOXC clusters displayed myogenic hypermethylation bordering a central region containing many genes preferentially expressed in myogenic progenitor cells and consisting largely of chromatin with modifications typical of promoters and enhancers in these cells. A particularly interesting example of myogenic hypermethylation was HOTAIR, a HOXC noncoding RNA gene, which can silence HOXD genes in trans via recruitment of polycomb proteins. In myogenic progenitor cells, the preferential expression of HOTAIR was associated with hypermethylation immediately downstream of the gene. Other HOX gene regions also displayed myogenic DNA hypermethylation despite being moderately expressed in myogenic cells. Analysis of representative myogenic hypermethylated sites for 5-hydroxymethylcytosine revealed little or none of this base, except for an intragenic site in HOXB5 which was specifically enriched in this base in skeletal muscle tissue, whereas myoblasts had predominantly 5-methylcytosine at the same CpG site. CONCLUSIONS: Our results suggest that myogenic hypermethylation of HOX genes helps fine-tune HOX sense and antisense gene expression through effects on 5' promoters, intragenic and intergenic enhancers and internal promoters. Myogenic hypermethylation might also affect the relative abundance of different RNA isoforms, facilitate transcription termination, help stop the spread of activation-associated chromatin domains and stabilize repressive chromatin structures.

SUBMITTER: Tsumagari K

PROVIDER: S-EPMC3750649 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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DNA methylation and differentiation: HOX genes in muscle cells.

Tsumagari Koji K Baribault Carl C Terragni Jolyon J Chandra Sruti S Renshaw Chloe C Sun Zhiyi Z Song Lingyun L Crawford Gregory E GE Pradhan Sriharsa S Lacey Michelle M Ehrlich Melanie M

Epigenetics & chromatin 20130802 1

<h4>Background</h4>Tight regulation of homeobox genes is essential for vertebrate development. In a study of genome-wide differential methylation, we recently found that homeobox genes, including those in the HOX gene clusters, were highly overrepresented among the genes with hypermethylation in the skeletal muscle lineage. Methylation was analyzed by reduced representation bisulfite sequencing (RRBS) of postnatal myoblasts, myotubes and adult skeletal muscle tissue and 30 types of non-muscle-ce ...[more]

PMID: 23916067

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Project description:Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P. Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

Project description:The goals were to investigate in umbilical cord tissue if gestational obesity: (1) was associated with changes in DNA methylation of skeletal muscle-specific genes; (2) could modulate the co-methylation interactions among these genes. Additionally, we assessed the associations between DNA methylation levels and infant's variables at birth and at age 6. DNA methylation was measured in sixteen pregnant women [8-gestational obesity group; 8-control group] in umbilical cord using the Infinium Methylation EPIC Bead Chip microarray. Differentially methylated CpGs were identified with Beta Regression Models [false discovery rate (FDR) < 0.05 and an Odds Ratio > 1.5 or < 0.67]. DNA methylation interactions between CpGs of skeletal muscle-specific genes were studied using data from Pearson correlation matrices. In order to quantify the interactions within each network, the number of links was computed. This identification analysis reported 38 differential methylated CpGs within skeletal muscle-specific genes (comprising 4 categories: contractibility, structure, myokines, and myogenesis). Compared to control group, gestational obesity (1) promotes hypermethylation in highly methylated genes and hypomethylation in low methylated genes; (2) CpGs in regions close to transcription sites and with high CpG density are hypomethylated while regions distant to transcriptions sites and with low CpG density are hypermethylated; (3) diminishes the number of total interactions in the co-methylation network. Interestingly, the associations between infant's fasting glucose at age 6 and MYL6, MYH11, TNNT3, TPM2, CXCL2, and NCAM1 were still relevant after correcting for multiple testing. In conclusion, our study showed a complex interaction between gestational obesity and the epigenetic status of muscle-specific genes in umbilical cord tissue. Additionally, gestational obesity may alter the functional co-methylation connectivity of CpG within skeletal muscle-specific genes interactions, our results revealing an extensive reorganization of methylation in response to maternal overweight. Finally, changes in methylation levels of skeletal muscle specific genes may have persistent effects on the offspring of mothers with gestational obesity.

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DNA methylation and differentiation: HOX genes in muscle cells.

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DNA methylation and differentiation: HOX genes in muscle cells.

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