Hayward Human Genetics Program and Tulane Cancer Center, Tulane Health Sciences Center, New Orleans LA, USA.
Epigenetics Chromatin. 2013 Aug 2;6(1):25. doi: 10.1186/1756-8935-6-25.
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.
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.
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.
同源盒基因的严格调控对于脊椎动物的发育至关重要。在一项全基因组差异甲基化研究中，我们最近发现，同源盒基因，包括 HOX 基因簇中的基因，在骨骼肌谱系中高甲基化的基因中高度富集。通过对出生后成肌细胞、肌管和成体骨骼肌组织以及 30 种非肌肉细胞培养物或组织的简化代表性亚硫酸氢盐测序（RRBS）进行甲基化分析。
在这项研究中，我们发现，所有四个 HOX 基因簇的特定亚区都存在肌源性高甲基化，并且与各种染色质表观遗传特征相关。尽管 HOXD 簇的 3' 端在包括成肌细胞和肌管在内的大多数检查细胞类型中被沉默，并富含多梳抑制相关的 H3 赖氨酸 27 三甲基化，但肌源性样本在该区域也显示出大量 DNA 甲基化，这是不寻常的。相比之下，HOXA 和 HOXC 簇都显示出肌源性高甲基化，边界是一个包含许多在肌源性祖细胞中优先表达的基因的中央区域，主要由这些细胞中启动子和增强子的典型修饰的染色质组成。肌源性高甲基化的一个特别有趣的例子是 HOTAIR，一种 HOXC 非编码 RNA 基因，它可以通过募集多梳蛋白来沉默转录中的 HOXD 基因。在肌源性祖细胞中，HOTAIR 的优先表达与基因下游的高甲基化相关。尽管在肌源性细胞中中度表达，但其他 HOX 基因区域也显示出肌源性 DNA 高甲基化。对代表性肌源性高甲基化位点的 5-羟甲基胞嘧啶进行分析表明，除了骨骼肌组织中 HOXB5 基因的一个内含子位点特别富含这种碱基外，几乎没有或没有这种碱基，而在相同的 CpG 位点，成肌细胞主要含有 5-甲基胞嘧啶。
我们的结果表明，HOX 基因的肌源性高甲基化通过影响 5' 启动子、内含子和基因间增强子以及内部启动子，有助于精细调节 HOX 基因的有义和反义基因表达。肌源性高甲基化也可能影响不同 RNA 异构体的相对丰度，促进转录终止，帮助阻止激活相关染色质结构域的扩散，并稳定抑制性染色质结构。
