Genome Editing of the Disease Locus D4Z4 as a Means to Ameliorate Gene Misregulation in Facioscapulohumeral Muscular Dystrophy

• Other Authors: Das, Sunny (author)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Molecular biology; Cytology; Genetics
• Online Access: http://purl.flvc.org/fsu/fd/2018_Sp_Das_fsu_0071E_14319_comp

D4Z4 is a subtelomeric macrosatellite repeat on chromosome 4q that codes for DUX4, a gene that is causal to the muscle wasting disease Facioscapulohumeral muscular dystrophy (FSHD). DUX4 expression is influenced by a number of genetic and epigenetic modifiers, including variation in D4Z4 copy number, single nucleotide polymorphisms (SNPs) on 4q, DNA and histone methylation changes, modifier genes like SMCHD1 and DNMT3B, and telomeres. The overarching goal of the research presented in this dissertation was to demonstrate the feasibility of targeting the disease locus using genome editing tools. To achieve this, we first needed to identify suitable cellular platforms for subsequent genome editing experiments. Through genotyping analysis, we were able to identify a cell line (HCT116) that was well-suited to studies investigating D4Z4/DUX4 expression, given that it harbors a disease-permissive 4qA allele. Using HCT116 and three of its DNA methyltransferase knockouts (1KO, 3BKO and DKO), we probed for factors that influence DUX4 expression in these cell lines. These experiments revealed that H3K9me3 loss and CpG hypomethylation can independently result in DUX4 expression in non-myogenic cell types. HCT116 and its DNMT KOs offer a new platform for studying DUX4 expression, albeit with some caveats. Importantly, we showed existence of D4Z4 transcripts in a variety of adult human tissues in addition to testis, with notably high expression in the thymus. Using these cell lines, we next explored the mechanism by which modifiers of D4Z4, such as SMCHD1 and telomeres might influence DUX4 expression. We generated several independent SMCHD1 knockout clones in HCT116 using TALENs. Characterization of these KOs revealed that despite no detectable changes in H3K9me3 or CpG methylation at D4Z4, SMCHD1 loss causes expression of unspliced DUX4, a phenomenon that was phenocopied by treatment of cells with a chemical inhibitor of telomerase. Spliced and pathogenic DUX4 was only expressed in these KOs upon treatment with 5-Aza-C, which demethylates DNA and lowers H3K9me3 levels at D4Z4. Given these results and the previously known importance of H3K9me3 enrichment in transcription and splicing, we speculate on a model, where SMCHD1 protein and telomeres, may act in coordination to provide an extra layer of transcriptional repression in addition to H3K9me3, at D4Z4. To achieve the main goal of this project, we took four independent approaches aimed at directly or indirectly repressing DUX4. In the first approach, we successfully deleted the array from a 4q permissive allele (in HCT116 and DKO cells), generating recombinants that likely had a shortened 4q chromosomal end and harbored an exogenously provided telomere seeding construct. Although we were unable to isolate clones of D4Z4-deleted cells, these results showed that the entire array can be deleted, also highlighting adverse side-effects of such targeting on cell viability. For our second approach, we used a CRISPR-based effector system to upregulate SMCHD1 transcript levels. Using a dCas9-VP64 activator construct, we were able to affect >2-fold upregulation in SMCHD1 in 293T cells. This avenue can be explored further to assess the effect of such upregulation on DUX4 and its target gene expression, in clinically relevant cell types. A SNP, resulting in a non-canonical polyadenylation (poly-A) sequence in DUX4 exon 3 is believed to stabilize pathogenic DUX4 transcripts in somatic cells. In a novel third approach, we designed pairs of gRNAs flanking the poly-A and deleted it using Cas9 nuclease recruited by these gRNAs. Significant lowering of DUX4 transcription in a mutant clone of HCT116, which contains a deletion of the pathogenic poly-A highlighted the importance of this sequence in transcript stabilization. Additionally, through lentiviral transduction of patient myoblasts, we showed that this approach can not only repress DUX4 expression, but also alleviate misregulation of a subset of its downstream target genes that are also biomarkers of FSHD. For our final approach, we enriched H3K9me3 at D4Z4 using a the dCas9-KRAB repressor system and a suitable gRNA. In both DKO cells and patient myoblasts, targeting repressed DUX4 expression significantly. Additionally, DUX4 target genes were also repressed in myoblasts and we showed that this repression was a consequence of increase and spread of H3K9me3 at D4Z4. Taken together, the results from these studies have generated promising future directions that can help understand the mechanism of how modifiers regulate D4Z4 expression. More importantly, by demonstrating the feasibility of targeting the disease locus in different cell types (including patient myoblasts), we have laid the foundation for development of future cell-based therapies to alleviate patient suffering in FSHD. === A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Spring Semester 2018. === March 7, 2018. === CRISPR, D4Z4, Epigenetics, FSHD, Genome editing, Macrosatellites === Includes bibliographical references. === Brian P. Chadwick, Professor Directing Dissertation; Michelle N. Arbeitman, University Representative; Thomas C. Keller, Committee Member; David M. Gilbert, Committee Member; Jonathan H. Dennis, Committee Member.