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Changes in gene expression in SMCHD1 knockout clones of HCT116

ABSTRACT: Development of effective therapies for complex Mendelian disorders such as Facioscapulohumeral muscular dystrophy necessitates a clear understanding of the pathology of this disease. The inappropriate expression of DUX4-fl is believed to be responsible for the disease phenotype. We sought to understand the role of SMCHD1, an epigenetic modifier of the D4Z4 array, in DUX4-fl expression, by a TALEN mediated knockout of the gene in a model cell line HCT116. Analysis of SMCHD1 and DNMT knockouts of HCT116, along with patient cell lines highlighted the importance of epigenetic determinants of the disease and revealed a novel DUX4 isoform. We show that while SMCHD1 loss sensitizes cells to DUX4 expression by increasing transcription of unspliced product from D4Z4, splicing of the transcripts to generate disease associated DUX4-fl can only be induced by CpG hypomethylation and loss of heterochromatic histone marker H3K9me3. Additionally, telomere shortening has an effect similar to SMCHD1 loss, suggesting involvement of a telomere position effect in maintaining DUX4 repression. In light of our observations, we have developed a model for expression of DUX4-fl through a simultaneous loss of heterochromatin at D4Z4 and at the telomere, otherwise maintained by SMCHD1 that acts as a bridge between these two regions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE58796 | GEO | 2022/06/20

REPOSITORIES: GEO

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A proteomics study identifying interactors of the FSHD2 gene product SMCHD1 reveals RUVBL1-dependent DUX4 repression.

Project description: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, 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 are well documented, it is unclear how SMCHD1 is recruited to D4Z4, and how it exerts its repressive properties on the 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 47 robust SMCHD1 interactors, of which 19 are present in D4Z4 chromatin. We demonstrate that one of these SMCHD1 interacting proteins in D4Z4, RuvB-like 1 (RUVBL1) is indeed required for maintaining DUX4 silencing in FSHD myocytes. We also confirm the interaction of SMCHD1 with EZH inhibitory protein (EZHIP), 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.

2021-12-15 | PXD023993 | Pride

Small noncoding RNAs in FSHD2 muscle cells reveal both DUX4- and SMCHD1-specific signatures

Project description:Facioscapulohumeral muscular dystrophy (FSHD) is caused by insufficient epigenetic repression of D4Z4 macrosatellite repeat where DUX4, an FSHD causing gene is embedded. There are two forms of FSHD, FSHD1 with contraction of D4Z4 repeat and FSHD2 with chromatin compaction defects mostly due to SMCHD1 mutation. Previous reports showed DUX4-induced gene expression changes as well as changes in microRNA expression in FSHD muscle cells. However, a genome wide analysis of small noncoding RNAs that might be regulated by DUX4 or by mutations in SMCHD1 has not been reported yet. Here, we identified several types of small noncoding RNAs including known microRNAs that are differentially expressed in FSHD2 muscle cells compared to control. Although fewer small RNAs were differentially expressed during muscle differentiation in FSHD2 cells compared to controls, most of the known myogenic microRNAs, such as miR1, miR133a, and miR206 were induced in both FSHD2 and control muscle cells during differentiation. Our small RNA sequencing data analysis also revealed both DUX4- and SMCHD1-specific changes in FSHD2 muscle cells. Six FSHD2 microRNAs were affected by DUX4 overexpression in control myoblasts, whereas increased expression of tRNAs and 5S rRNAs in FSHD2 muscle cells was largely recapitulated in SMCHD1-depleted control myoblasts. Altogether, our studies suggest that the small noncoding RNA transcriptome changes in FSHD2 might be different from those in FSHD1 and that these differences may provide new diagnostic and therapeutic tools specific to FSHD2.

2018-05-29 | GSE113133 | GEO

Identification of SMCHD1 as a Novel Regulator of Alternative Splicing in FSHD2

Project description:Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1) is a non-canonical member of the structural maintenance of chromosomes (SMC) protein family involved in the regulation of chromatin structure, epigenetic regulation and transcription. Mutations in SMCHD1 cause facioscapulohumeral muscular dystrophy type 2 (FSHD2), a rare genetic disorder characterized by progressive muscle weakness and wasting, believed to be caused by aberrant expression of DUX4 in muscle cells. Here we suggest a new role for SMCHD1 as a regulator of alternative splicing, and demonstrate how splicing alteration caused by SMCHD1 mutations lead to DUX4 expression and FSHD pathogenesis. Analyzing RNA-seq data from muscle biopsies of FSHD2 patients and FSHD2 mouse model found that hundreds of genes were alternatively spliced upon SMCHD1 mutation. At least 20% of alternatively spliced genes were associated with abnormalities of the musculature. Moreover, we show that alternatively spliced exons tend to be bound by SMCHD1, and SMCHD1 bound exons demonstrate slower elongation rate, suggesting SMCHD1 binding promotes exon exclusion by slowing RNAPII. Specifically, we discovered that SMCHD1 mutations promotes the splicing of the DNMT3B1 isoform of DNMT3B, which leads to hypomethylation of the D4Z4 region and DUX4 expression. These results suggest that alternative splicing regulated by SMCHD1 may play a major role in FSHD2 pathogenesis by promoting alternative splicing of different targets including DNMT3B, and highlight the potential for targeting alternative splicing as a therapeutic strategy for this disorder.

2024-04-13 | GSE223039 | GEO

Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy [RNA-seq]

Project description:Facioscapulohumeral muscular dystrophy (FSHD), a progressive skeletal muscle disorder, is epigenetically characterized with DNA hypomethylation of D4Z4 repeats in 4q35 region, allowing aberrant DUX4 expression. Sustainable DUX4 suppression is a promising therapeutic clue to prevent disease progression, but most of the supposed methods depend on expression of their mediator biochemical entity, potentially narrowing QoL of individuals with FSHD in the clinical context. In this study, we report that by applying hit-and-run silencing with dCas9-mediated epigenetic editing targeting DNA hypomethylation on D4Z4 repeats, we could achieve suppression of endogenous DUX4 in our FSHD patients-derived iPSC model. Notably, DNA methylation was significantly upregulated in FSHD cells and suppression effect was observed at least two weeks after intervention, which was not the case by transient treatment of typical dCas9-KRAB alone. Off-target analysis showed that despite the potential genome-wide risk in DNA methylation, the impact on transcriptome was limited. We propose that hit-and-run silencing can be a promising option to prevent disease progression with minimum intervention for individuals with FSHD, motivating further study for clinical development.

2024-03-31 | GSE201178 | GEO

Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy [EPIC Methylation]

2024-03-31 | GSE199690 | GEO

DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscle

Project description:Facioscapulohumeral dystrophy (FSHD) is caused by decreased epigenetic repression of the D4Z4 macrosatellite array and recent studies have shown that this results in the expression of low levels of the DUX4 mRNA in skeletal muscle. Several other mechanisms have been suggested for FSHD pathophysiology and it remains unknown whether DUX4 expression can account for most of the molecular changes seen in FSHD. Since DUX4 is a transcription factor, we used RNA-seq to measure gene expression in muscle cells transduced with DUX4, and in muscle cells and biopsies from control and FSHD individuals. We show that DUX4 target gene expression is the major molecular signature in FSHD muscle together with a gene expression signature consistent with an immune cell infiltration. In addition, one unaffected individual without a known FSHD-causing mutation showed expression of DUX4 target genes. This individual has a sibling with FSHD and also without a known FSHD-causing mutation, suggesting the presence of yet unidentified modifier locus for DUX4 expression and FSHD. These findings demonstrate that expression of DUX4 accounts for the majority of the gene expression changes in FSHD skeletal muscle together with an immune cell infiltration. RNA-seq for muscle cells and biopsies from control and FSHD individuals.

2014-07-03 | E-GEOD-56787 | biostudies-arrayexpress

An allele-aware approach identifies altered nuclear associations of the contracted D4Z4 array at the FSHD locus

Project description:Facioscapulohumeral dystrophy (FSHD) is caused by a deletion in a D4Z4 macrosatellite repeat array in the 4q subtelomere that leads to somatic de-repression of the transcription factor DUX4. It is not fully understood how array contractions cause de-repression, but they alter the local chromatin structure of D4Z4, resulting in loss of heterochromatic markers. In order to determine whether pathogenic contractions also alter the nuclear organization of the FSHD locus, we designed an allele-aware, high-throughput circular chromosome conformation capture assay (4C-seq) that distinguishes the two 4q copies of the locus from each other and from a paralogous locus in the 10q subtelomere. Using a short sequence length polymorphism (SSLP) four kilobasepairs from the array as âharacterized the genomic contacts made by the four copies of the FSHD locus in muscle cell nuclei. The SSLP contacts predominate on the same chromosome arm as the bait, decaying rapidly beyond 30-40 Mb. Contacts on other chromosomes were enriched near centromeres and CTCF sites. We also found that the FSHD locus normally contacts regions adjacent to the nuclear lamina and with low overall gene expression. However, we found that the deleted locus in FSHD cells makes more contacts with regions with comparatively lower lamina adjacency and higher overall gene expression than in control cells. Furthermore, association of D4Z4 with components of the nuclear lamina was reduced in FSHD cells relative to control cells. Our results suggest that altered nuclear organization at 4q35 may be one factor in the de-repression of DUX4 in FSHD. 5 primary cells (2 control, 1 FSHD1, 1 FSHD2, and 1 FSHD3) were used to generate 4C libraries in biological duplicate, for a total of 10 4C libraries

2013-08-31 | E-GEOD-40673 | biostudies-arrayexpress

DUX4 activates germline genes, retroelements and immune-mediators: Implications for facioscapulohumeral dystrophy

Project description:Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression. [Overexpression experiment] Quadruplicate total RNA samples were collected from control human primary myoblasts transduced with lentivirus carrying DUX4-fl, DUX4-s or GFP (MOI = 15) for 24 h and from untransduced myoblasts. [Defensin experiment] Quadruplicate samples were also collected from myoblasts and myotubes grown in media containing human beta-defensin 3 peptide or in control media.

2012-01-03 | E-GEOD-33799 | biostudies-arrayexpress

D4Z4-located miRNA

Project description:We have used a tiled microarray based on the D4Z4 sequence, and hybridized amplified transcripts from facioscapulohumeral muscular dystrophy (FSHD) and control (CTRL) samples to detect D4Z4 related expression.

2016-04-01 | GSE12159 | GEO

DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscle

2014-07-03 | GSE56787 | GEO

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