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DNA methylation and differentiation: HOX genes in muscle cells.

Tsumagari K, Baribault C, Terragni J, Chandra S, Renshaw C, Sun Z, Song L, Crawford GE, Pradhan S, Lacey M, Ehrlich M - Epigenetics Chromatin (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hayward Human Genetics Program and Tulane Cancer Center, Tulane Health Sciences Center, New Orleans LA, USA. ehrlich@tulane.edu.

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.

No MeSH data available.


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Myogenic hypermethylation, enrichment in CpG islands and extensive myogenesis-associated transcription localized to the 151-kb HOXC cluster. (a) MyoD binding profiles show that inferred MYOD binding sites form a distant border on both sides of the HOXC cluster. MYOD binding sites were extrapolated and are depicted as in Figure 1. The visualized chromosomal region from the UCSC Genome Browser for this figure is chr12:54,052,006–54,706,150 (654 kb). (b) 119 MbMt-hypermethylated sites and the distribution of CpG islands. (c) Layered RNA-seq track as in Figure 2 with additional layered tracks for H3K4me3, H3K4me1 and H3K27Ac by ChIP-seq (ENCODE/Broad Institute). (d) Chromatin state segmentation analysis as in Figure 1. The pink-highlighted region is the HOXC gene cluster shown in Figure 2.
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Figure 3: Myogenic hypermethylation, enrichment in CpG islands and extensive myogenesis-associated transcription localized to the 151-kb HOXC cluster. (a) MyoD binding profiles show that inferred MYOD binding sites form a distant border on both sides of the HOXC cluster. MYOD binding sites were extrapolated and are depicted as in Figure 1. The visualized chromosomal region from the UCSC Genome Browser for this figure is chr12:54,052,006–54,706,150 (654 kb). (b) 119 MbMt-hypermethylated sites and the distribution of CpG islands. (c) Layered RNA-seq track as in Figure 2 with additional layered tracks for H3K4me3, H3K4me1 and H3K27Ac by ChIP-seq (ENCODE/Broad Institute). (d) Chromatin state segmentation analysis as in Figure 1. The pink-highlighted region is the HOXC gene cluster shown in Figure 2.

Mentions: Not only was HOXD4 silent in most of the examined cells types, including Mb, but this was also the case for the rest of the HOXD cluster, especially the 3′ half of the cluster (Figure 1c). Similarly, there was silencing-associated H3K27me3 throughout the gene cluster in Mb, Mt and most examined nonmyogenic cell types (Figure 1d, PcG, and Additional file 3) as determined by whole-genome chromatin immunoprecipitation/next-generation DNA sequencing (H3K27me3 ChIP-seq; ENCODE/Broad Institute, http://genome.ucsc.edu/). There was an unusually high concentration of CpG islands in the HOXD cluster and the other three HOX clusters (Figures 1, 2, 3, 4, 5 and 6), but this cannot explain the myogenic hypermethylation in HOX gene clusters. For example, there was a much higher density of MbMt-hypermethylated sites in the 3′ half of the HOXD gene cluster relative to the 5′ half, but not a higher density of CpG islands (Figure 1a).


DNA methylation and differentiation: HOX genes in muscle cells.

Tsumagari K, Baribault C, Terragni J, Chandra S, Renshaw C, Sun Z, Song L, Crawford GE, Pradhan S, Lacey M, Ehrlich M - Epigenetics Chromatin (2013)

Myogenic hypermethylation, enrichment in CpG islands and extensive myogenesis-associated transcription localized to the 151-kb HOXC cluster. (a) MyoD binding profiles show that inferred MYOD binding sites form a distant border on both sides of the HOXC cluster. MYOD binding sites were extrapolated and are depicted as in Figure 1. The visualized chromosomal region from the UCSC Genome Browser for this figure is chr12:54,052,006–54,706,150 (654 kb). (b) 119 MbMt-hypermethylated sites and the distribution of CpG islands. (c) Layered RNA-seq track as in Figure 2 with additional layered tracks for H3K4me3, H3K4me1 and H3K27Ac by ChIP-seq (ENCODE/Broad Institute). (d) Chromatin state segmentation analysis as in Figure 1. The pink-highlighted region is the HOXC gene cluster shown in Figure 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750649&req=5

Figure 3: Myogenic hypermethylation, enrichment in CpG islands and extensive myogenesis-associated transcription localized to the 151-kb HOXC cluster. (a) MyoD binding profiles show that inferred MYOD binding sites form a distant border on both sides of the HOXC cluster. MYOD binding sites were extrapolated and are depicted as in Figure 1. The visualized chromosomal region from the UCSC Genome Browser for this figure is chr12:54,052,006–54,706,150 (654 kb). (b) 119 MbMt-hypermethylated sites and the distribution of CpG islands. (c) Layered RNA-seq track as in Figure 2 with additional layered tracks for H3K4me3, H3K4me1 and H3K27Ac by ChIP-seq (ENCODE/Broad Institute). (d) Chromatin state segmentation analysis as in Figure 1. The pink-highlighted region is the HOXC gene cluster shown in Figure 2.
Mentions: Not only was HOXD4 silent in most of the examined cells types, including Mb, but this was also the case for the rest of the HOXD cluster, especially the 3′ half of the cluster (Figure 1c). Similarly, there was silencing-associated H3K27me3 throughout the gene cluster in Mb, Mt and most examined nonmyogenic cell types (Figure 1d, PcG, and Additional file 3) as determined by whole-genome chromatin immunoprecipitation/next-generation DNA sequencing (H3K27me3 ChIP-seq; ENCODE/Broad Institute, http://genome.ucsc.edu/). There was an unusually high concentration of CpG islands in the HOXD cluster and the other three HOX clusters (Figures 1, 2, 3, 4, 5 and 6), but this cannot explain the myogenic hypermethylation in HOX gene clusters. For example, there was a much higher density of MbMt-hypermethylated sites in the 3′ half of the HOXD gene cluster relative to the 5′ half, but not a higher density of CpG islands (Figure 1a).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hayward Human Genetics Program and Tulane Cancer Center, Tulane Health Sciences Center, New Orleans LA, USA. ehrlich@tulane.edu.

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.

No MeSH data available.


Related in: MedlinePlus