Structural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor, which is mutated in > 95% of Facioscapulohumeral dystrophy (FSHD) type 2 cases. In FSHD2,... Show moreStructural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor, which is mutated in > 95% of Facioscapulohumeral dystrophy (FSHD) type 2 cases. In FSHD2, SMCHD1 mutations ultimately result in the presence of the cleavage stage transcription factor DUX4 in muscle cells due to a failure in epigenetic repression of the D4Z4 macrosatellite repeat on chromosome 4q, which contains the DUX4 locus. While binding of SMCHD1 to D4Z4 and its necessity to maintain a repressive D4Z4 chromatin structure in somatic cells are well documented, it is unclear how SMCHD1 is recruited to D4Z4, and how it exerts its repressive properties on chromatin. Here, we employ a quantitative proteomics approach to identify and characterize novel SMCHD1 interacting proteins, and assess their functionality in D4Z4 repression. We identify 28 robust SMCHD1 nuclear interactors, of which 12 are present in D4Z4 chromatin of myocytes. We demonstrate that loss of one of these SMCHD1 interacting proteins, RuvB-like 1 (RUVBL1), further derepresses DUX4 in FSHD myocytes. We also confirm the interaction of SMCHD1 with EZH inhibitory protein (EZHIP), a protein which prevents global H3K27me3 deposition by the Polycomb repressive complex PRC2, providing novel insights into the potential function of SMCHD1 in the repression of DUX4 in the early stages of embryogenesis. The SMCHD1 interactome outlined herein can thus provide further direction into research on the potential function of SMCHD1 at genomic loci where SMCHD1 is known to act, such as D4Z4 repeats, the inactive X chromosome, autosomal gene clusters, imprinted loci and telomeres. Show less
Goossens, R.; Boogaard, M.L. van den; Lemmers, R.J.L.F.; Balog, J.; Vliet, P.J. van der; Willemsen, I.M.; ... ; Maarel, S.M. van der 2019
Background Facioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de... Show moreBackground Facioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of DUX4 in skeletal muscle. The common form of FSHD, FSHD1, is caused by a D4Z4 repeat array contraction. The less common form, FSHD2, is generally caused by heterozygous variants in SMCHD1. Methods We employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic SMCHD1 mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic SMCHD1 variant from patient myoblasts. Results We identified intronic SMCHD1 variants in two FSHD families. In the first family, an intronic variant resulted in partial intron retention and inclusion of the distal 14 nucleotides of intron 13 into the transcript. In the second family, a deep intronic variant in intron 34 resulted in exonisation of 53 nucleotides of intron 34. In both families, the aberrant transcripts are predicted to be non-functional. Deleting the pseudo-exon by CRISPR-Cas9 mediated genome editing in primary and immortalised myoblasts from the index case of the second family restored wild-type SMCHD1 expression to a level that resulted in efficient suppression of DUX4. Conclusions The estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic SMCHD1 variants identified here, emphasises the importance of screening for intronic variants in SMCHD1. Furthermore, the efficient suppression of DUX4 after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies. Show less
