Final Report for grant: RNA-mediated Epigenetic Silencing of D4Z4 repeats: Implications for targeted therapy for FSHD
Jong Won Lim, postdoctoral fellow
Mentors: Galina Filippova, PhD and Stephen Tapscott, MD, PhD
Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete repression of the D4Z4 macrosatellite repeat array on the disease-permissive chromosome 4q that results in aberrant expression of DUX4, the candidate FSHD gene imbedded within the D4Z4 repeat. With support by the Friends of FSH Research Funding, we have previously shown that the small RNA processing machinery including AGO2 and DICER1 is required for maintenance of the D4Z4/DUX4 epigenetic silencing and that exogenous D4Z4 small RNA mimics can trigger the D4Z4 repeat epigenetic silencing and inhibit DUX4 expression in FSHD muscle cells. However, it still remains unclear how insufficient RNA-mediated gene silencing contributes D4Z4/DUX4 de-repression in FSHD and what other factors and D4Z4 noncoding RNAs are implicated in RNA-mediated epigenetic silencing. The manuscript ‘DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD’ has been published in Human Molecular Genetics.
In this project, we have demonstrated that two epigenetic marks of RNA-mediated gene silencing, AGO2 enrichment and H3K9me3 level, are decreased at D4Z4 in FSHD. We have shown that in addition to AGO/DICER, modifying factors are required for D4Z4 silencing. We have also shown that SMCHD1, a gene necessary for silencing the D4Z4 region and that causes FSHD2 when mutated, does not affect AGO recruitment to the D4Z4. By using additional siRNAs targeting either sense or antisense strand, we mapped strand specificity of siRNA-mediated DUX4 silencing and defined which strand should be targeted by exogenous small RNAs to efficiently silence DUX4 expression. These findings will be instrumental for identifying efficient therapeutic targets for long-term epigenetic silencing of DUX4. To identify endogenous small RNAs potentially related to D4Z4/DUX4 silencing and FSHD pathology, we performed whole genome small RNA sequencing, and found several types of small noncoding RNAs including microRNAs that were differentially expressed in FSHD2 muscle cells. These may provide new insight into the mechanisms of FSHD pathobiology and may be used for potential biomarkers and/or therapeutic targets for FSHD.