Genetic and Epigenetic Regulation in the Facioscapulohumeral Muscular Dystrophy-associated Genome
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Authors
Chang, Ning
Issue Date
2023
Type
Dissertation
Language
Keywords
Arhinia , BAZ1A , DBE-T , DUX4 , Epigenetics , Facioscapulohumeral muscular dystrophy
Alternative Title
Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies. It initially causes progressive weakness and wasting of the face, shoulder and upper arm muscles, but eventually could also affect all the skeletal muscles. FSHD has a complex etiology characterized by genetic and epigenetic events that combine to cause de-repression of the double homeobox 4 (DUX4) gene in skeletal muscle. DUX4 encodes a transcription factor that is normally expressed in early development and is generally silent in healthy adult somatic tissues. The DUX4 gene is encoded in each repeat unit (RU) of the D4Z4 macrosatellite on the chromosome 4q35 subtelomere. Healthy subjects have ~ 11-100 D4Z4 RUs that provides a condensed structure of chromatin for genome repression and maintains hypermethylated DNAs of the locus to prevent DUX4 gene expression. Importantly, in both types of FSHD (FSHD1 and FSHD2), mis-expression of DUX4 in muscle cells is caused by the loss of normal epigenetic repression and DNA hypomethylation of the D4Z4 region, but caused by different mechanisms. FSHD1 results from large deletions within the D4Z4 array on at least one permissive chromosome 4 leaving between 1-10 RUs. Alternatively, FSHD2 is characterized by mutations in genes encoding epigenetic repressors of the D4Z4 array, most commonly the SMCHD1 gene (structural maintenance of chromosomes flexible hinge domain containing 1). However, it is still unclear how epigenetic repression of DUX4 at the FSHD locus is established or maintained in healthy somatic cells and how DUX4 is activated in the early embryonic stage and in FSHD myogenic cells. Recently, mutations in SMCHD1 have also been observed in arhinia, a rare congenital disorder characterized by the lack of an external nose and often associated with eye and reproductive defects. Interestingly, it has been shown that the D4Z4 DNA methylation profiles of 4q chromosomes in arhinia are characteristic of those found in FSHD2, which prompted us to ask if FSHD2 exists in the arhinia population, or if arhinia patients might be at risk for developing FSHD2 as they age. To explore this potential for FSHD2 comorbidity among arhinia patients (in Chapter 3), we sought to determine if muscle cells from arhinia patients express DUX4. Given that somatic expression of DUX4 is exclusive to skeletal muscle cells in FSHD, and since muscle biopsies are relatively difficult to obtain, we collected skin fibroblasts from arhinia patients. The combined treatment with 5-azacytidine and trichostatin A, to stimulate expression of MyoD and initiate a myogenic program in the cells that is required for DUX4 expression, dramatically induced expression of the DUX4 mRNA in fibroblasts derived from arhinia patients with FSHD2 genetic and epigenetic profiles, but not from those with non-permissive haplotype or healthy relatives. Thus, this study demonstrated that arhinia patients meeting both the genetic and epigenetic criteria for FSHD2 can, in fact, express DUX4 in myogenic cells and therefore might have the potential to develop FSHD. While many viable therapeutic approaches for FSHD have been proposed, the DUX4 gene and the highly repetitive FSHD locus present unique therapeutic challenges. The next study in this dissertation (Chapter 4), we demonstrated an FSHD-specific strategy of drug discovery from pre-identified upstream epigenetic activators. Previous study identified key regulators of the DUX4 gene in FSHD muscle cells using a candidate-based screening approach, including bromodomain adjacent to zinc finger domain protein 1A (BAZ1A), a chromatin remodeling factor. We performed an artificial intelligence-based screening pipeline and searched for potential small molecules predicted to inhibit BAZ1A and validated with FSHD-specific assays in FSHD myocytes. The compound, termed C06, was identified in a screen out of 74 small-molecule candidates, and it was highly specific for inhibiting the expression of DUX4 and DUX4 target genes in FSHD myocytes. In a mechanistic study (Chapter 5), we sought to determine how BAZ1A contributes to regulation of the FSHD region and activation of DUX4 expression using the small-molecule compound C06 for targeting BAZ1A activity. Here, we showed that BAZ1A is enriched at the DUX4 locus in FSHD myocytes compared to healthy myocytes by chromatin immunoprecipitation assay. We found C06 treatment altered the D4Z4 chromatin landscape, reducing levels of DUX4 and its transcriptional targets. Hypothesizing that blocking the binding of BAZ1A would reduce transcription across D4Z4, we also assessed levels of DBE-T, an activating long non-coding RNA produced from the D4Z4 region in FSHD myocytes. DBE-T has been shown to recruit the chromatin-modifying protein ASH1L to the FSHD locus in the disease state, driving H3K36 methylation and DUX4 transcription. Accordingly, we found that C06 reduced DBE-T levels, ASH1L enrichment, and H3K36 methylation in drug-treated FSHD myocytes. Overall, our study demonstrated that the attenuation of epigenetic regulator BAZ1A exerted its effectiveness by two distinct mechanisms: reduction of DUX4 transcription and reduction of DBE-T transcription. Thus, this study sheds light on previously unknown mechanisms of DUX4 activation in FSHD and supports further drug development for FSHD.