Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei
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ABSTRACT:
PROVIDER: PRJNA600755 | ENA |
REPOSITORIES: ENA
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Project description: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.
2020-01-01 | S-EPMC7224571 | BioStudies
Project description: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 first performed pooled RNA-seq on a 6-day differentiation time-course in FSHD2 patient-derived primary myoblasts. We identify a set of 54 FSHD-induced genes upregulated in FSHD2 cells starting at day 2 of differentiation through the end of the time-course. Using single-cell and single-nucleus RNA-seq on FSHD2 myoblasts and day 3 and day 5 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 nuclei upregulate many cell cycle related genes with significant enrichment of E2F target genes and p53 signaling activation. In FSHD-Hi myotube nuclei, multiple DUX4 target genes are co-expressed including a set of transcription factors, such as DUXA, ZSCAN4 and LEUTX. 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 indicate that the DUXA can take over the role of DUX4 and maintain target gene expression. These results may provide explanation as to why it is easier to detect and monitor DUX4 target genes than DUX4 itself in patient cells and suggest a self-sustaining network of gene dysregulation that perpetuates this disease after DUX4 is no longer expressed. Overall design: Full length single-cell and single-nucleus RNA-seq using SmartSeq on the Fluidigm C1 platform for control and FSHD2 myoblast cells and myotube nuclei.
2020-04-07 | GSE143492 | GEO
Project description: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 first performed pooled RNA-seq on a 6-day differentiation time-course in FSHD2 patient-derived primary myoblasts. We identify a set of 54 FSHD-induced genes upregulated in FSHD2 cells starting at day 2 of differentiation through the end of the time-course. Using single-cell and single-nucleus RNA-seq on FSHD2 myoblasts and day 3 and day 5 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 nuclei upregulate many cell cycle related genes with significant enrichment of E2F target genes and p53 signaling activation. In FSHD-Hi myotube nuclei, multiple DUX4 target genes are co-expressed including a set of transcription factors, such as DUXA, ZSCAN4 and LEUTX. 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 indicate that the DUXA can take over the role of DUX4 and maintain target gene expression. These results may provide explanation as to why it is easier to detect and monitor DUX4 target genes than DUX4 itself in patient cells and suggest a self-sustaining network of gene dysregulation that perpetuates this disease after DUX4 is no longer expressed. Overall design: 3' end single-nucleus RNA-seq on 3 and 5 day differentiated primary myoblasts from FSHD2 patient and control biopsies
2020-04-07 | GSE143452 | GEO
Project description: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 first performed pooled RNA-seq on a 6-day differentiation time-course in FSHD2 patient-derived primary myoblasts. We identify a set of 54 FSHD-induced genes upregulated in FSHD2 cells starting at day 2 of differentiation through the end of the time-course. Using single-cell and single-nucleus RNA-seq on FSHD2 myoblasts and day 3 and day 5 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 nuclei upregulate many cell cycle related genes with significant enrichment of E2F target genes and p53 signaling activation. In FSHD-Hi myotube nuclei, multiple DUX4 target genes are co-expressed including a set of transcription factors, such as DUXA, ZSCAN4 and LEUTX. 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 indicate that the DUXA can take over the role of DUX4 and maintain target gene expression. These results may provide explanation as to why it is easier to detect and monitor DUX4 target genes than DUX4 itself in patient cells and suggest a self-sustaining network of gene dysregulation that perpetuates this disease after DUX4 is no longer expressed. Overall design: Time-course of primary FSHD2 myoblasts from tibialis anterior, control myoblasts from tibialis anterior, and control myoblasts from quadricep
2020-04-07 | GSE143453 | GEO
Project description:Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei [Fluidigm]
| PRJNA600756 | ENA
Project description:Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei [ddSeq]
| PRJNA600520 | ENA
Project description:Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei [Time course]
| PRJNA600521 | ENA
Project description:This SuperSeries is composed of the SubSeries listed below. Overall design: Refer to individual Series
2020-04-07 | GSE143493 | GEO
Project description:OBJECTIVE:To compare the clinical features of patients showing a classical phenotype of facioscapulohumeral muscular dystrophy (FSHD) with genetic and epigenetic characteristics of the FSHD1 and FSHD2 loci D4Z4 and SMCHD1. METHODS:This is a national multicenter cohort study. We measured motor strength, motor function, and disease severity by manual muscle testing sumscore, Brooke and Vignos scores, clinical severity score (CSS), and age-corrected CSS, respectively. We correlated these scores with genetic (D4Z4 repeat size and haplotype; SMCHD1 variant status) and epigenetic (D4Z4 methylation) parameters. RESULTS:We included 103 patients: 54 men and 49 women. Among them, we identified 64 patients with FSHD1 and 20 patients with FSHD2. Seven patients had genetic and epigenetic characteristics of FSHD1 and FSHD2, all carrying repeats of 9-10 D4Z4 repeat units (RU) and a pathogenic SMCHD1 variant. In the remaining patients, FSHD was genetically excluded or remained unconfirmed. All clinically affected SMCHD1 mutation carriers had a D4Z4 repeat of 9-16 RU on a disease permissive 4qA haplotype. These patients are significantly more severely affected by all clinical scales when compared to patients with FSHD1 with upper-sized FSHD1 alleles (8-10 RU). CONCLUSION:The overlap between FSHD1 and FSHD2 patients in the 9-10 D4Z4 RU range suggests that FSHD1 and FSHD2 form a disease continuum. The previously established repeat size threshold for FSHD1 (1-10 RU) and FSHD2 (11-20 RU) needs to be reconsidered. CLINICALTRIALSGOV IDENTIFIER:NCT01970735.
2019-01-01 | S-EPMC6537132 | BioStudies
Project description:Nesprins, nuclear envelope spectrin-repeat proteins encoded by the SYNE1 and SYNE2 genes, are involved in localization of nuclei. The short isoform, nesprin-1-alpha2, is required for relocation of the microtubule organizer function from centromeres to the nuclear rim during myogenesis. Using specific antibodies, we now show that both nesprin-1-alpha2 and nesprin-1-giant co-localize with kinesin at the junctions of concatenated nuclei and at the outer poles of nuclear chains in human skeletal myotubes. In adult muscle, nesprin-1-alpha2 was found, together with kinesin, only on nuclei associated with neuromuscular junctions, whereas all adult cardiomyocyte nuclei expressed nesprin-1-alpha2. In a proteomics study, kinesin heavy and light chains were the only significant proteins in myotube extracts pulled down by nesprin-1-alpha2, but not by a mutant lacking the highly-conserved STAR domain (18 amino-acids, including the LEWD motif). The results support a function for nesprin-1-alpha2 in the specific localization of skeletal muscle nuclei mediated by kinesins and suggest that its primary role is at the outer nuclear membrane.
2019-01-01 | S-EPMC6775114 | BioStudies