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Systemic analysis of osteoblast-specific DNA methylation marks reveals novel epigenetic basis of osteoblast differentiation

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ABSTRACT

DNA methylation is an important epigenetic modification that contributes to the lineage commitment and specific functions of different cell types. In this study, we compared ENCODE-generated genome-wide DNA methylation profiles of human osteoblast with 21 other types of human cells in order to identify osteoblast-specific methylation events. For most of the cell strains, data from two isogenic replicates were included, resulting in a total of 51 DNA methylation datasets. We identified 852 significant osteoblast-specific differentially methylated CpGs (DMCs) and 295 significant differentially methylated regions (DMRs). Significant DMCs/DMRs were not enriched in CpG islands (CGIs) and promoters, but more strongly enriched in CGI shores/shelves and in gene body and intergenic regions. The genes associated with significant DMRs were highly enriched in biological processes related to transcriptional regulation and critical for regulating bone metabolism and skeletal development under physiologic and pathologic conditions. By integrating the DMR data with the extensive gene expression and chromatin epigenomics data, we observed complex, context-dependent relationships between DNA methylation, chromatin states, and gene expression, suggesting diverse DNA methylation-mediated regulatory mechanisms. Our results also highlighted a number of novel osteoblast-relevant genes. For example, the integrated evidences from DMR analysis, histone modification and RNA-seq data strongly support that there is a novel isoform of neurexin-2 (NRXN2) gene specifically expressed in osteoblast. NRXN2 was known to function as a cell adhesion molecule in the vertebrate nervous system, but its functional role in bone is completely unknown and thus worth further investigation. In summary, we reported a comprehensive analysis of osteoblast-specific DNA methylation profiles and revealed novel insights into the epigenetic basis of osteoblast differentiation and activity.

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Osteoblastic hypermethylation at SIM2 TSS surrounding region. The following profiles are shown using the UCSC Genome Browser (http://genome.ucsc.edu, version hg19) for the SIM2 gene region: UCSC genes, RefSeq genes, RNA-seq data from ENCODE/Cold Spring Harbor, DNA methylation levels assessed by RRBS from ENCODE/HudsonAlpha, CpG islands, Chromatin state (18-state) annotation from NIH Roadmap Epigenome project. LCL (only GM12878 is shown, but the other LCL samples give similar results), HMEC, HSMM, and osteoblast (HOB) are the only studied cell types having both data in RNA-seq and histone modification tracks available from ENCODE. Methylation status is represented with an 11-color gradient for which red, yellow, and green represent that 100%, 50%, and 0% of molecules sequenced are methylated, respectively. Osteoblast-specific DMRs are indicated in the blue box. At this scale, individual differentially methylated CpG sites cannot be resolved from neighboring sites. The specific non-coding SIM2 isoform (Ensembl transcript ID: ENST00000460783.1) is marked in the red box. The 18 chromatin states are represented with the indicated colors, with E116 representing states for LCL (GM12878), E119 for HMEC, E120 for HSMM, and E129 for osteoblast. The detailed ChIP-seq data for various histone modification marks (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, H3K36me3, H3K79me2, and H4K20me1) are presented in the Supplementary Fig. 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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f0015: Osteoblastic hypermethylation at SIM2 TSS surrounding region. The following profiles are shown using the UCSC Genome Browser (http://genome.ucsc.edu, version hg19) for the SIM2 gene region: UCSC genes, RefSeq genes, RNA-seq data from ENCODE/Cold Spring Harbor, DNA methylation levels assessed by RRBS from ENCODE/HudsonAlpha, CpG islands, Chromatin state (18-state) annotation from NIH Roadmap Epigenome project. LCL (only GM12878 is shown, but the other LCL samples give similar results), HMEC, HSMM, and osteoblast (HOB) are the only studied cell types having both data in RNA-seq and histone modification tracks available from ENCODE. Methylation status is represented with an 11-color gradient for which red, yellow, and green represent that 100%, 50%, and 0% of molecules sequenced are methylated, respectively. Osteoblast-specific DMRs are indicated in the blue box. At this scale, individual differentially methylated CpG sites cannot be resolved from neighboring sites. The specific non-coding SIM2 isoform (Ensembl transcript ID: ENST00000460783.1) is marked in the red box. The 18 chromatin states are represented with the indicated colors, with E116 representing states for LCL (GM12878), E119 for HMEC, E120 for HSMM, and E129 for osteoblast. The detailed ChIP-seq data for various histone modification marks (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, H3K36me3, H3K79me2, and H4K20me1) are presented in the Supplementary Fig. 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mentions: Promoters and 5′end regions are usually constitutively unmethylated, especially when they overlap with CGIs, even in genes with cell type-specific expression (Meissner et al., 2008). Nonetheless, there are notable exceptions as illustrated by gene SIM2. Specifically, we detected multiple osteoblast-specific hypermethylated DMRs (q-value = 8.57 × 10− 19–1.25 × 10− 38, DM% = 32.3–82.4%) at SIM2 promoter and an immediate downstream region of its TSS (Fig. 3). These DMRs were all distributed within a large CGI.


Systemic analysis of osteoblast-specific DNA methylation marks reveals novel epigenetic basis of osteoblast differentiation
Osteoblastic hypermethylation at SIM2 TSS surrounding region. The following profiles are shown using the UCSC Genome Browser (http://genome.ucsc.edu, version hg19) for the SIM2 gene region: UCSC genes, RefSeq genes, RNA-seq data from ENCODE/Cold Spring Harbor, DNA methylation levels assessed by RRBS from ENCODE/HudsonAlpha, CpG islands, Chromatin state (18-state) annotation from NIH Roadmap Epigenome project. LCL (only GM12878 is shown, but the other LCL samples give similar results), HMEC, HSMM, and osteoblast (HOB) are the only studied cell types having both data in RNA-seq and histone modification tracks available from ENCODE. Methylation status is represented with an 11-color gradient for which red, yellow, and green represent that 100%, 50%, and 0% of molecules sequenced are methylated, respectively. Osteoblast-specific DMRs are indicated in the blue box. At this scale, individual differentially methylated CpG sites cannot be resolved from neighboring sites. The specific non-coding SIM2 isoform (Ensembl transcript ID: ENST00000460783.1) is marked in the red box. The 18 chromatin states are represented with the indicated colors, with E116 representing states for LCL (GM12878), E119 for HMEC, E120 for HSMM, and E129 for osteoblast. The detailed ChIP-seq data for various histone modification marks (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, H3K36me3, H3K79me2, and H4K20me1) are presented in the Supplementary Fig. 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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f0015: Osteoblastic hypermethylation at SIM2 TSS surrounding region. The following profiles are shown using the UCSC Genome Browser (http://genome.ucsc.edu, version hg19) for the SIM2 gene region: UCSC genes, RefSeq genes, RNA-seq data from ENCODE/Cold Spring Harbor, DNA methylation levels assessed by RRBS from ENCODE/HudsonAlpha, CpG islands, Chromatin state (18-state) annotation from NIH Roadmap Epigenome project. LCL (only GM12878 is shown, but the other LCL samples give similar results), HMEC, HSMM, and osteoblast (HOB) are the only studied cell types having both data in RNA-seq and histone modification tracks available from ENCODE. Methylation status is represented with an 11-color gradient for which red, yellow, and green represent that 100%, 50%, and 0% of molecules sequenced are methylated, respectively. Osteoblast-specific DMRs are indicated in the blue box. At this scale, individual differentially methylated CpG sites cannot be resolved from neighboring sites. The specific non-coding SIM2 isoform (Ensembl transcript ID: ENST00000460783.1) is marked in the red box. The 18 chromatin states are represented with the indicated colors, with E116 representing states for LCL (GM12878), E119 for HMEC, E120 for HSMM, and E129 for osteoblast. The detailed ChIP-seq data for various histone modification marks (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, H3K36me3, H3K79me2, and H4K20me1) are presented in the Supplementary Fig. 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mentions: Promoters and 5′end regions are usually constitutively unmethylated, especially when they overlap with CGIs, even in genes with cell type-specific expression (Meissner et al., 2008). Nonetheless, there are notable exceptions as illustrated by gene SIM2. Specifically, we detected multiple osteoblast-specific hypermethylated DMRs (q-value = 8.57 × 10− 19–1.25 × 10− 38, DM% = 32.3–82.4%) at SIM2 promoter and an immediate downstream region of its TSS (Fig. 3). These DMRs were all distributed within a large CGI.

View Article: PubMed Central - PubMed

ABSTRACT

DNA methylation is an important epigenetic modification that contributes to the lineage commitment and specific functions of different cell types. In this study, we compared ENCODE-generated genome-wide DNA methylation profiles of human osteoblast with 21 other types of human cells in order to identify osteoblast-specific methylation events. For most of the cell strains, data from two isogenic replicates were included, resulting in a total of 51 DNA methylation datasets. We identified 852 significant osteoblast-specific differentially methylated CpGs (DMCs) and 295 significant differentially methylated regions (DMRs). Significant DMCs/DMRs were not enriched in CpG islands (CGIs) and promoters, but more strongly enriched in CGI shores/shelves and in gene body and intergenic regions. The genes associated with significant DMRs were highly enriched in biological processes related to transcriptional regulation and critical for regulating bone metabolism and skeletal development under physiologic and pathologic conditions. By integrating the DMR data with the extensive gene expression and chromatin epigenomics data, we observed complex, context-dependent relationships between DNA methylation, chromatin states, and gene expression, suggesting diverse DNA methylation-mediated regulatory mechanisms. Our results also highlighted a number of novel osteoblast-relevant genes. For example, the integrated evidences from DMR analysis, histone modification and RNA-seq data strongly support that there is a novel isoform of neurexin-2 (NRXN2) gene specifically expressed in osteoblast. NRXN2 was known to function as a cell adhesion molecule in the vertebrate nervous system, but its functional role in bone is completely unknown and thus worth further investigation. In summary, we reported a comprehensive analysis of osteoblast-specific DNA methylation profiles and revealed novel insights into the epigenetic basis of osteoblast differentiation and activity.

No MeSH data available.


Related in: MedlinePlus