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.

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].

Figure 2

Myogenic hypermethylated sites at both ends of the HOXC gene cluster, which was preferentially transcribed in myogenic cells. (a) 119 CpG sites with significant hypermethylation in Mb plus Mt vs. 16 types of non-muscle-cell cultures at chr12:54,318,064–54,468,880. (b) Representative RRBS tracks with the location of CpG islands beneath them. (c) Strand-specific RNA-seq profiling (as in Figure 1) for the HOXC gene cluster and standard RNA-seq (not strand-specific; ENCODE/California Institute of Technology). The layered RNA-seq shows the superimposed profiles from Mb, LCL, ESC and NHLF cells in the indicated color code. (d) Chromatin state segmentation analysis as in Figure 1. (e) MyoD binding site profiles as in Figure 1 and CTCF binding from ChIP-seq profiling of the indicated cell types (ENCODE/Broad Institute). Arrows and empty boxes denote features mentioned in the text.

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.

Figure 4

Myogenic hypermethylation in the central region of the HOXB gene cluster, which is preferentially transcribed in myogenic cells. (a) 88 MbMt-hypermethylated sites in the chr17:46,602,904–46,814,469 region. (b) Examples of RRBS data. (c) Strand-specific RNA-seq as in Figure 1, except that the vertical viewing ranges were 1–10 for the plus strand was and 1–100 for the minus strand. (d) Chromatin state segmentation analysis. (e) The MyoD binding site track shows no C2C12-extrapolated MYOD sites in this region. Arrows, empty boxes and the triangle denote features mentioned in the text.

Figure 5

Cell type–specific differences in DNA methylation and transcription in the region containing HOXB5, HOXB6 and HOXB-AS3 variant genes. (a) 42 MbMt-hypermethylated sites in a subregion of HOXB (chr17:46,665,998–46,684,371). (b) Chromatin segmentation state maps. (c) Strand-specific RNA-seq as in Figure 4. The pink boxes indicate the RNA-seq evidence for HOXB-AS3 variant 3 as the predominant variant expressed in Mb. (d) RRBS data for two control Mb cell strains and Mt preparations derived from them, as well as two fetal lung fibroblast cell strains analyzed as technical duplicates. Arrows and highlighted subregions are described in the text.

Figure 6

Peripheral myogenic hypermethylation and a central myogenic hypomethylated site in the HOXA gene cluster. (a) 187 MbMt-hypermethylated and 20 muscle-hypermethylated sites as well as one MbMt-hypomethylated site in the chr7:27,116,782–27,273,459 region. (b) Examples of RRBS data. (c) RNA-seq profiles as in Figure 1. (d) Chromatin state segmentation analysis. (e) MyoD binding sites from C2C12.