Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei
Autoři:
Shan Jiang aff001; Katherine Williams aff001; Xiangduo Kong aff003; Weihua Zeng aff001; Nam Viet Nguyen aff003; Xinyi Ma aff001; Rabi Tawil aff004; Kyoko Yokomori aff003; Ali Mortazavi aff001
Působiště autorů:
Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America
aff001; Center for Complex Biological Systems, University of California Irvine, Irvine, California, United States of America
aff002; Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, California, United States of America
aff003; Neuromuscular Disease Unit, Department of Neurology, University of Rochester Medical Center, Rochester, New York, United States of America
aff004
Vyšlo v časopise:
Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei. PLoS Genet 16(5): e32767. doi:10.1371/journal.pgen.1008754
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pgen.1008754
Souhrn
FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes (“FSHD-Lo” and “FSHD Hi”, respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression.
Klíčová slova:
Cell cycle and cell division – Cell differentiation – Gene expression – Gene regulation – Myoblasts – RNA sequencing – Transcription factors – Facioscapulohumeral muscular dystrophy
Zdroje
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