Periodic Reporting for period 1 - DUX4 (Function of DUX4 in skeletal muscle and non-muscle tissues) Reporting period: 2018-07-01 to 2020-06-30 Summary of the context and overall objectives of the project DUX4 expression in skeletal muscle is associated with the development of facioscapulohumeral muscular dystrophy (FSHD). DUX4 encodes a transcription factor that plays an important role in the initiation of gene expression after fertilization in mammals. As FSHD patients mainly present with muscular symptoms, research in the field has focused on unravelling the consequences of DUX4 expression in skeletal muscle. Research on DUX4 expression in non-muscle tissues is limited, but increasing evidence suggests a biological function for DUX4 in these tissues. As systemic DUX4 suppression is a potential therapy for FSHD, it is important to define the consequences of DUX4 suppression in non-muscle tissues. The action entitled “Function of DUX4 in skeletal muscle and non-muscle tissues” addressed the hypothesis that DUX4 has an underappreciated biological function in non-muscle tissues. The following objectives were formulated:* Objective 1: To establish which non-muscle tissues, and what cell types, express DUX4.* Objective 2: To determine the consequences of DUX4 expression in skeletal muscle and non-muscle tissues and to identify which genes and pathways are direct targets of DUX4 in these tissues.* Objective 3: To establish if systemic treatment with DUX4 antisense oligonucleotides (ASOs) negatively impacts somatic tissues in which DUX4 has a biological function.Upon completion of the action, we now know which non-muscle tissues, and what specific cell types, express DUX4. We also know that systemic treatment with DUX4 ASOs does not have a major impact on non-muscle tissues in which DUX4 has a biological function. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Project 1: I analyzed DUX4 expression in mouse non-muscle tissues, making use of control and FSHD mouse lines available in the host group. I next determined which cell types in these tissues expressed DUX4, focusing on non-muscle tissues in which I found DUX4 expression in all mouse lines and on non-muscle tissues showing a phenotype in FSHD mouse lines. Part of the data was presented during the World Muscle Society Congress and has been published in Skeletal Muscle. I also analyzed DUX4 expression in human non-muscle tissues and found similar results as in mouse; the non-muscle tissues that expressed DUX4 and the cell types expressing DUX4 overlapped.Project 2: I planned single cell RNA sequencing to determine the consequences of DUX4 expression. Data for human myotubes has been published by the host group. I completed all sequencing experiments for mouse non-muscle tissues. Due to the Covid-19 pandemic, I could not establish myotube cultures from the FSHD mice and I could not perform the sequencing experiments for human non-muscle tissues. I also planned DUX4-ChIP sequencing. Data for human myoblasts is available through a collaborator and data for mouse myoblasts has been published by the host group. Because of a delay in obtaining human tissues, I have not performed DUX4-ChIP sequencing in human non-muscle tissues. I performed several experiments to establish DUX4-ChIP sequencing for mouse non-muscle tissues. Even though I successfully isolated chromatin, I could not detect DUX4 enrichment at several mouse DUX4 target genes. As DUX4 may activate different target genes in different tissues, I tested the expression of several “muscle” DUX4 target genes in non-muscle tissues and found that expression of most correlated with DUX4 expression. Thus, DUX4 should bind at these loci and further optimization of the DUX4-ChIP is needed.Project 3: I determined the effect of systemic DUX4 suppression using different FSHD mouse lines. In the first study, I did not find significantly reduced DUX4 transcript levels in the muscles or the non-muscle tissues. However, DUX4 expression in the mouse line was very low, which may explain why I did not see an effect. I used a novel FSHD mouse line, with higher DUX4 expression in skeletal muscles, for two additional studies. Mice were treated for 3 weeks (before muscle pathology occurred) or for 9 weeks (at an age when muscle pathology was already present). In both studies, the DUX4 ASO significantly reduced DUX4 expression in the skeletal muscles. Only in two non-muscle tissues I found a small but significant effect on DUX4 expression. Body weight, organ weight, and serum markers for liver and kidney toxicity were not affected. Taken together, the DUX4 ASO seems relatively specific for the skeletal muscles and does not have a major impact on non-muscle tissues. Because of the Covid-19 pandemic, I added a novel project to the action. Project 4 involved work that could be performed from home when the host laboratory was closed. I performed the DUX4 ASO studies for project 3 together with a PhD student, whose project focuses on the effect of systemic DUX4 suppression on skeletal muscles. Together, we performed all skeletal muscle analyses and found that muscle pathology, but not muscle strength, was improved in mice receiving the DUX4 ASO. During the closure of the laboratory, I also wrote a review paper summarizing developments in therapeutic strategies for FSHD which was published in Current Opinion in Neurology.The LUMC made a video about my research which was shared via their website and has been viewed >500 times. I wrote for the website of the Dutch FSHD Expertise Center. In 2018 and 2019, I was present at the yearly conference for muscular dystrophy patients in the Netherlands and gave a poster presentation. This year, I made a video showing the laboratory while explaining my research for the online version of the conference. I gave several lectures on my research to students, a small groups of students visited the laboratory for two days, and three high school students joined the laboratory for a one-week internship. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Project 1 has been completed; we now know which non-muscle tissues, and what specific cell types, express DUX4, which is important for further establishing the biological function of DUX4 in these tissues. Due to a delay in obtaining human material, I have not yet established the consequences of DUX4 expression in non-muscle tissues in project 2. I will finish the RNA sequencing experiments for the human non-muscle tissues and the mouse myotubes. This is important for establishing the biological function of DUX4 in these tissues and next I will determine whether DUX4 expression in these tissues is higher in FSHD patients. I still expect to identify which genes and pathways are direct targets of DUX4 in human and mouse non-muscle tissues. Unfortunately, DUX4-ChIP sequencing has been more challenging than expected, but I now have access to the necessary human tissues.Project 3 has been completed; we now know that systemic treatment with DUX4 ASOs does not have a major impact on non-muscle tissues in which DUX4 has a biological function and systemic DUX4 suppression by ASOs seems a safe and promising treatment for FSHD patients. The chemistry of the ASOs could be improved to enhance uptake in skeletal muscles and further reduce DUX4 transcript levels and to prevent uptake by non-muscle tissues.