Mason Amanda G, Slieker Roderick C, Balog Judit, Lemmers Richard J L F, Wong Chao-Jen, Yao Zizhen, Lim Jong-Won, Filippova Galina N, Ne Enrico, Tawil Rabi, Heijmans Bas T, Tapscott Stephen J, van der Maarel Silvère M
Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands.
Skelet Muscle. 2017 Jun 6;7(1):12. doi: 10.1186/s13395-017-0129-7.
Facioscapulohumeral muscular dystrophy (FSHD) is in most cases caused by a contraction of the D4Z4 macrosatellite repeat on chromosome 4 (FSHD1) or by mutations in the SMCHD1 or DNMT3B gene (FSHD2). Both situations result in the incomplete epigenetic repression of the D4Z4-encoded retrogene DUX4 in somatic cells, leading to the aberrant expression of DUX4 in the skeletal muscle. In mice, Smchd1 regulates chromatin repression at different loci, having a role in CpG methylation establishment and/or maintenance.
To investigate the global effects of harboring heterozygous SMCHD1 mutations on DNA methylation in humans, we combined 450k methylation analysis on mononuclear monocytes from female heterozygous SMCHD1 mutation carriers and unaffected controls with reduced representation bisulfite sequencing (RRBS) on FSHD2 and control myoblast cell lines. Candidate loci were then evaluated for SMCHD1 binding using ChIP-qPCR and expression was evaluated using RT-qPCR.
We identified a limited number of clustered autosomal loci with CpG hypomethylation in SMCHD1 mutation carriers: the protocadherin (PCDH) cluster on chromosome 5, the transfer RNA (tRNA) and 5S rRNA clusters on chromosome 1, the HOXB and HOXD clusters on chromosomes 17 and 2, respectively, and the D4Z4 repeats on chromosomes 4 and 10. Furthermore, minor increases in RNA expression were seen in FSHD2 myoblasts for some of the PCDHβ cluster isoforms, tRNA isoforms, and a HOXB isoform in comparison to controls, in addition to the previously reported effects on DUX4 expression. SMCHD1 was bound at DNAseI hypersensitivity sites known to regulate the PCDHβ cluster and at the chromosome 1 tRNA cluster, with decreased binding in SMCHD1 mutation carriers at the PCDHβ cluster sites.
Our study is the first to investigate the global methylation effects in humans resulting from heterozygous mutations in SMCHD1. Our results suggest that SMCHD1 acts as a repressor on a limited set of autosomal gene clusters, as an observed reduction in methylation associates with a loss of SMCHD1 binding and increased expression for some of the loci.
面肩肱型肌营养不良症（FSHD）在大多数情况下是由4号染色体上D4Z4大卫星重复序列的收缩（FSHD1）或SMCHD1或DNMT3B基因的突变（FSHD2）引起的。这两种情况都会导致体细胞中D4Z4编码的反转录基因DUX4的表观遗传抑制不完全，从而导致DUX4在骨骼肌中异常表达。在小鼠中，Smchd1调节不同位点的染色质抑制，在CpG甲基化的建立和/或维持中发挥作用。
为了研究携带杂合SMCHD1突变对人类DNA甲基化的整体影响，我们将对女性杂合SMCHD1突变携带者和未受影响对照的单核细胞进行的450k甲基化分析与对FSHD2和成肌细胞系对照进行的简化代表性亚硫酸氢盐测序（RRBS）相结合。然后使用ChIP-qPCR评估候选位点的SMCHD1结合情况，并使用RT-qPCR评估表达情况。
我们在SMCHD1突变携带者中鉴定出数量有限的成簇常染色体位点存在CpG低甲基化：5号染色体上的原钙黏蛋白（PCDH）簇、1号染色体上的转运RNA（tRNA）和5S rRNA簇、17号和2号染色体上的HOXB和HOXD簇，以及4号和10号染色体上的D4Z4重复序列。此外，与对照相比，FSHD2成肌细胞中一些PCDHβ簇异构体、tRNA异构体和一种HOXB异构体的RNA表达略有增加，此外还有先前报道的对DUX4表达的影响。SMCHD1结合在已知调节PCDHβ簇的DNA酶I高敏位点和1号染色体的tRNA簇上，在PCDHβ簇位点的SMCHD1突变携带者中结合减少。
我们的研究首次调查了SMCHD1杂合突变对人类整体甲基化的影响。我们的结果表明，SMCHD1作为一组有限的常染色体基因簇的抑制因子，因为观察到的甲基化减少与SMCHD1结合的丧失以及一些位点的表达增加有关。
