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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.


Related in: MedlinePlus

Myogenesis-associated hypermethylation in the 3' half of the HOXD gene cluster, which displayed polycomb silencing in most cell types. (a) Red bars, the 55 CpG sites significantly hypermethylated in Mb plus Mt vs. 16 types of non-muscle-cell cultures and 61 CpG sites significantly hypermethylated in skeletal muscle tissue vs. 14 types of nonmuscle tissues in the chr2:176,921,692 -177,074,604 region. At this scale, many differentially methylated sites cannot be discriminated. (b) Examples of RRBS data (a). Using an 11-color semicontinuous scale (see color guide), these tracks indicate the average DNA methylation levels at each monitored CpG site from the quantitative sequencing data (ENCODE/HudsonAlpha Institute for Biotechnology). Data are shown for only a few of the cell culture samples evaluated for this study. Skin fib, neonatal foreskin fibroblasts. (c) Strand-specific RNA-seq profiling at the HOXD gene cluster for Mb, neonatal foreskin fibroblasts, HUVEC and ESC. Each track displays the signal from RNA-seq (ENCODE/Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) from these cell cultures. The vertical viewing range for the strand-specific RNA-seq was 1 -100 in the UCSC Genome Browser for this and subsequent figures unless otherwise noted. Tan highlighting, the HOXD4 region shown in Additional file 2. (d) The predicted type of chromatin structure in subregions of the HOXD gene cluster is displayed in chromatin state segmentation maps (ENCODE/Broad Institute, Cambridge, MA, USA) based mostly on histone modifications [54]. The predicted local chromatin states are shown with the indicated colors. PcG, polycomb group protein -associated H3K27me3. (e) MyoD binding from C2C12 ChIP-seq [59] and identification of orthologous human sequences. The relative binding strength is indicated, and sites shown in blue overlapped CAGCTG, which is present in approximately 75% of Myod ChIP-seq peaks and is part of the degenerate consensus sequence for MyoD binding [59].
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Figure 1: Myogenesis-associated hypermethylation in the 3' half of the HOXD gene cluster, which displayed polycomb silencing in most cell types. (a) Red bars, the 55 CpG sites significantly hypermethylated in Mb plus Mt vs. 16 types of non-muscle-cell cultures and 61 CpG sites significantly hypermethylated in skeletal muscle tissue vs. 14 types of nonmuscle tissues in the chr2:176,921,692 -177,074,604 region. At this scale, many differentially methylated sites cannot be discriminated. (b) Examples of RRBS data (a). Using an 11-color semicontinuous scale (see color guide), these tracks indicate the average DNA methylation levels at each monitored CpG site from the quantitative sequencing data (ENCODE/HudsonAlpha Institute for Biotechnology). Data are shown for only a few of the cell culture samples evaluated for this study. Skin fib, neonatal foreskin fibroblasts. (c) Strand-specific RNA-seq profiling at the HOXD gene cluster for Mb, neonatal foreskin fibroblasts, HUVEC and ESC. Each track displays the signal from RNA-seq (ENCODE/Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) from these cell cultures. The vertical viewing range for the strand-specific RNA-seq was 1 -100 in the UCSC Genome Browser for this and subsequent figures unless otherwise noted. Tan highlighting, the HOXD4 region shown in Additional file 2. (d) The predicted type of chromatin structure in subregions of the HOXD gene cluster is displayed in chromatin state segmentation maps (ENCODE/Broad Institute, Cambridge, MA, USA) based mostly on histone modifications [54]. The predicted local chromatin states are shown with the indicated colors. PcG, polycomb group protein -associated H3K27me3. (e) MyoD binding from C2C12 ChIP-seq [59] and identification of orthologous human sequences. The relative binding strength is indicated, and sites shown in blue overlapped CAGCTG, which is present in approximately 75% of Myod ChIP-seq peaks and is part of the degenerate consensus sequence for MyoD binding [59].

Mentions: In the HOXD gene cluster, many sites were hypermethylated in the MbMt set vs. nonmuscle cell cultures or in skeletal muscle tissue vs. nonmuscle tissues, as shown in Figure 1a. Figure 1b displays the coverage of RRBS in this region by exhibiting DNA methylation data tracks from the UCSC Genome Browser for representative samples. One of the subregions with the most myogenic hypermethylation in both progenitor cells and tissues was in the vicinity of HOXD4 and had 38 MbMt-hypermethylated sites and 33 skeletal muscle-hypermethylated sites (Figure 1a, tan highlighting, and Additional file 2). The two clusters of MbMt-hypermethylated sites in the HOXD4 upstream region surround a retinoic acid-sensitive mesodermal enhancer [47] and are near the adjacent MIR10B gene (Figure 1), whose methylation was implicated in gene silencing in cis in gastric cancer [32]. Both DNA methylation and H3K27me3 were seen at the MIR10B promoter region in human mammary epithelial cells (HMEC) in a previous study [48] as well as in the present study (Figure 1 and Additional file 2). Our analysis of RNA-seq data (ENCODE/California Institute of Technology; http://genome.ucsc.edu/; [49]) by Cufflinks [50], a program which evaluates RNA-seq profiles to determine steady-state amounts of different RNA isoforms, indicated that human umbilical vein endothelial cells (HUVEC) expressed this gene abundantly, whereas less than 200 times as much HOXD4 RNA was detected in Mb, epidermal keratinocytes (NHEK), lung fibroblasts (NHLF), ESC and an LCL (Additional file 1). Only HUVEC did not have the repressive polycomb group chromatin marks at HOXD4 and throughout most of the HOXD cluster (Figure 1d). However, the predominant, 5.1-kb HUVEC transcript began upstream of HOXD4 near the MIR10B gene and extended past the 3′ end of HOXD4. A second, noncoding transcript was seen in HUVEC (ENST00000465649), whose transcription begins within the single HOXD4 intron. The myogenic intragenic hypermethylated sites in HOXD4 surround or overlap this alternative transcription start site (TSS; pink triangle, Additional file 2). Myogenic hypermethylation of the intron might help suppress the use of a secondary, intronic promoter.


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)

Myogenesis-associated hypermethylation in the 3' half of the HOXD gene cluster, which displayed polycomb silencing in most cell types. (a) Red bars, the 55 CpG sites significantly hypermethylated in Mb plus Mt vs. 16 types of non-muscle-cell cultures and 61 CpG sites significantly hypermethylated in skeletal muscle tissue vs. 14 types of nonmuscle tissues in the chr2:176,921,692 -177,074,604 region. At this scale, many differentially methylated sites cannot be discriminated. (b) Examples of RRBS data (a). Using an 11-color semicontinuous scale (see color guide), these tracks indicate the average DNA methylation levels at each monitored CpG site from the quantitative sequencing data (ENCODE/HudsonAlpha Institute for Biotechnology). Data are shown for only a few of the cell culture samples evaluated for this study. Skin fib, neonatal foreskin fibroblasts. (c) Strand-specific RNA-seq profiling at the HOXD gene cluster for Mb, neonatal foreskin fibroblasts, HUVEC and ESC. Each track displays the signal from RNA-seq (ENCODE/Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) from these cell cultures. The vertical viewing range for the strand-specific RNA-seq was 1 -100 in the UCSC Genome Browser for this and subsequent figures unless otherwise noted. Tan highlighting, the HOXD4 region shown in Additional file 2. (d) The predicted type of chromatin structure in subregions of the HOXD gene cluster is displayed in chromatin state segmentation maps (ENCODE/Broad Institute, Cambridge, MA, USA) based mostly on histone modifications [54]. The predicted local chromatin states are shown with the indicated colors. PcG, polycomb group protein -associated H3K27me3. (e) MyoD binding from C2C12 ChIP-seq [59] and identification of orthologous human sequences. The relative binding strength is indicated, and sites shown in blue overlapped CAGCTG, which is present in approximately 75% of Myod ChIP-seq peaks and is part of the degenerate consensus sequence for MyoD binding [59].
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Related In: Results  -  Collection

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Figure 1: Myogenesis-associated hypermethylation in the 3' half of the HOXD gene cluster, which displayed polycomb silencing in most cell types. (a) Red bars, the 55 CpG sites significantly hypermethylated in Mb plus Mt vs. 16 types of non-muscle-cell cultures and 61 CpG sites significantly hypermethylated in skeletal muscle tissue vs. 14 types of nonmuscle tissues in the chr2:176,921,692 -177,074,604 region. At this scale, many differentially methylated sites cannot be discriminated. (b) Examples of RRBS data (a). Using an 11-color semicontinuous scale (see color guide), these tracks indicate the average DNA methylation levels at each monitored CpG site from the quantitative sequencing data (ENCODE/HudsonAlpha Institute for Biotechnology). Data are shown for only a few of the cell culture samples evaluated for this study. Skin fib, neonatal foreskin fibroblasts. (c) Strand-specific RNA-seq profiling at the HOXD gene cluster for Mb, neonatal foreskin fibroblasts, HUVEC and ESC. Each track displays the signal from RNA-seq (ENCODE/Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) from these cell cultures. The vertical viewing range for the strand-specific RNA-seq was 1 -100 in the UCSC Genome Browser for this and subsequent figures unless otherwise noted. Tan highlighting, the HOXD4 region shown in Additional file 2. (d) The predicted type of chromatin structure in subregions of the HOXD gene cluster is displayed in chromatin state segmentation maps (ENCODE/Broad Institute, Cambridge, MA, USA) based mostly on histone modifications [54]. The predicted local chromatin states are shown with the indicated colors. PcG, polycomb group protein -associated H3K27me3. (e) MyoD binding from C2C12 ChIP-seq [59] and identification of orthologous human sequences. The relative binding strength is indicated, and sites shown in blue overlapped CAGCTG, which is present in approximately 75% of Myod ChIP-seq peaks and is part of the degenerate consensus sequence for MyoD binding [59].
Mentions: In the HOXD gene cluster, many sites were hypermethylated in the MbMt set vs. nonmuscle cell cultures or in skeletal muscle tissue vs. nonmuscle tissues, as shown in Figure 1a. Figure 1b displays the coverage of RRBS in this region by exhibiting DNA methylation data tracks from the UCSC Genome Browser for representative samples. One of the subregions with the most myogenic hypermethylation in both progenitor cells and tissues was in the vicinity of HOXD4 and had 38 MbMt-hypermethylated sites and 33 skeletal muscle-hypermethylated sites (Figure 1a, tan highlighting, and Additional file 2). The two clusters of MbMt-hypermethylated sites in the HOXD4 upstream region surround a retinoic acid-sensitive mesodermal enhancer [47] and are near the adjacent MIR10B gene (Figure 1), whose methylation was implicated in gene silencing in cis in gastric cancer [32]. Both DNA methylation and H3K27me3 were seen at the MIR10B promoter region in human mammary epithelial cells (HMEC) in a previous study [48] as well as in the present study (Figure 1 and Additional file 2). Our analysis of RNA-seq data (ENCODE/California Institute of Technology; http://genome.ucsc.edu/; [49]) by Cufflinks [50], a program which evaluates RNA-seq profiles to determine steady-state amounts of different RNA isoforms, indicated that human umbilical vein endothelial cells (HUVEC) expressed this gene abundantly, whereas less than 200 times as much HOXD4 RNA was detected in Mb, epidermal keratinocytes (NHEK), lung fibroblasts (NHLF), ESC and an LCL (Additional file 1). Only HUVEC did not have the repressive polycomb group chromatin marks at HOXD4 and throughout most of the HOXD cluster (Figure 1d). However, the predominant, 5.1-kb HUVEC transcript began upstream of HOXD4 near the MIR10B gene and extended past the 3′ end of HOXD4. A second, noncoding transcript was seen in HUVEC (ENST00000465649), whose transcription begins within the single HOXD4 intron. The myogenic intragenic hypermethylated sites in HOXD4 surround or overlap this alternative transcription start site (TSS; pink triangle, Additional file 2). Myogenic hypermethylation of the intron might help suppress the use of a secondary, intronic promoter.

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