Facioscapulohumeral dystrophy (FSHD) is associated with the upregulation of the DUX4 transcription factor and its target genes. However, low‐frequency DUX4 upregulation in patient myocytes is difficult to detect and examining the relationship and dynamics of DUX4 and target gene expression has been challenging. Using RNAScope in situ hybridization with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during 13‐day differentiation in vitro. We found that the endogenous DUX4 transcripts primarily localize as foci in one or two nuclei as compared with the accumulation of the recombinant DUX4 transcripts in the cytoplasm. We also found the continuous increase of DUX4 and target gene‐positive myotubes after Day 3, arguing against its expected immediate cytotoxicity. Interestingly, DUX4 and target gene expression become discordant later in differentiation with the increase of DUX4‐positive/target gene‐negative as well as DUX4‐negative/target gene‐positive myotubes. Depletion of DUX4‐activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4‐target gene, KDM4E, later in differentiation, suggesting that after the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, the study provides important new insights into the dynamics of the DUX4 transcriptional network in FSHD patient myocytes.

Facioscapulohumeral dystrophy (FSHD) is linked to misexpression of the transcription factor, DUX4. Although DUX4 target gene expression is often readily detectable, analysis of DUX4 expression has been limited due to its low expression in patient samples. Recently, single cell/nucleus RNA-sequencing was used to detect the native expression of DUX4 for the first time, but important spatial relationships with its target gene expression was missing. Furthermore, dynamics of DUX4 expression during myoblast differentiation has not been fully explored. In order to study the spatiotemporal relationship of DUX4 and key target genes, we performed RNA FISH on immortalized FSHD2 patient skeletal muscle cells. Using two probe sets, DUX4 transcripts were detected in 1-4% of myotubes after 3-day differentiation in vitro. We found that DUX4 transcripts mainly localize as foci in one or two nuclei in a myotube compared to abundant accumulation of the target gene transcripts in the cytoplasm. Over a 13-day differentiation timecourse, DUX4 expression without target gene expression significantly increased and peaked at day 7. Target gene expression correlates better with DUX4 expression early in differentiation while the expression of target genes without detectable DUX4 transcripts increases later. Consistently, shRNA depletion of DUX4-activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4-target gene, KDM4E, later in differentiation, suggesting that following the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, in situ detection of the DUX4 and target gene transcripts provided new insight into dynamics of DUX4 transcriptional network in FSHD patient myocytes. 3 Significance StatementFSHD is the third most common muscular dystrophy and is associated with upregulation of DUX4, a transcription factor, and its target genes. Although target genes are easily detectable in FSHD, low frequency DUX4 upregulation in patient myocytes is difficult to detect, and examining the relationship and dynamics of DUX4 and target gene expression without artificial overexpression of DUX4 has been challenging. Using RNAScope with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during differentiation in vitro. Our study reveals a unique DUX4 expression pattern and its relationship to the expression of target genes, and evidence for self-sustainability of the target gene network. The study provides important new insights into the FSHD disease mechanism.

NIPBL is an essential loader of cohesin to mediate sister chromatid cohesion and chromatin loop organization. NIPBL mutations cause Cornelia de Lange Syndrome. How NIPBL's genomic localization is specified is not fully understood. We found that NIPBL localizes to the nucleolus in an RNA-dependent manner and binds directly to ribosomal RNA (rRNA). We identified two RNA binding domains in NIPBL in vitro, both of which are required for efficient rRNA binding in vivo. NIPBL binds to ribosomal DNA (rDNA) in an RNA-stimulated manner, recruits PAF1 and promotes pre-rRNA transcription. Stress that inhibits rRNA synthesis displaces NIPBL from the nucleolus and rDNA. Interestingly, treacle, mutated in Treacher Collins syndrome, tightly binds to and recruits NIPBL to the nucleolus, nucleolar organizer regions, and the stress-induced nucleolar cap. The results reveal that a subpopulation of NIPBL is recruited to the nucleolus through its interaction with RNA and treacle and regulates pre-rRNA transcription.6 Results NIPBL localizes to the Fibrillar Center of the nucleolusThe nucleolus has a "tripartite structure" consisting of the Fibrillar Center (FC), the Dense Fibrillar Component (DFC), and the Granular Component (GC), each with distinct functions (Fig. 1A) (Boulon et al., 2010a). The FC is the site for pre-rRNA transcription and is enriched in RNA polymerase I (Pol I) machinery. Pre-rRNA is processed in the DFC, which contains many RNA processing factors such as snoRNA and fibrillarin. The GC is the site where pre-ribosome assembly takes place, and contains ribosome assembly factors such as B23 (nucleophosmin). Consistent with cohesin binding to the rDNA region (Zeng et al., 2009a), biochemical fractionation analysis indicates that Nipbl and cohesin are present in both nuclear and nucleolar fractions (Fig. 1B). Using an affinity-purified polyclonal antibody directed against the C-terminus of human NIPBL, we observed clear localization of both mouse Nipbl and human NIPBL in the nucleolus (mouse embryonic fibroblasts (MEFs) in Fig. 1C and D; human 293T cells in Supplemental Fig. S1, respectively). Costaining with antibodies specific for B23 (a marker for the GC) and fibrillarin (a marker for the DFC) revealed that Nipbl tightly clusters to the inside of the DFC corresponding to the FC (Fig. 1C and D). Furthermore, nucleolar Nipbl staining signals were reduced significantly in a Nipbl heterozygous mutant MEFs (modeling NIPBL haploinsufficiency in CdLS (Kawauchi et al., 2009;Newkirk et al., 2017)) and Nipbl siRNAdepleted MEFs compared to the wild type and control siRNA-treated MEFs, respectively (Fig. 1E). These results confirmed the specificity of immunostaining and also indicate that Nipbl in the nucleolus is sensitive to partial protein depletion (haploinsufficiency). NIPBL localizes to the nucleolus in an RNA-dependent manner and binds to preribosomal RNARNA is highly enriched and is an important constituent of nucleoli. Interestingly, RNase treatment dispersed Nipbl nucleolar foci while bulk Nipbl remained in the nucl...

Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of D4Z4 repeats on chromosome 4q (FSHD1). Mutations in the SMCHD1 gene are linked to both minor cases with no prominent repeat loss (FSHD2) and severe cases of FSHD1. Abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, is believed to play a central role in FSHD. However, defining the disease mechanism has been hampered by the heterogeneity of patient-derived cells, difficulty to detect DUX4 in patient myocytes, and limited animal models because D4Z4 repeats are primate-specific. To overcome these limitations, we engineered isogenic human skeletal myoblast lines with D4Z4 and/or SMCHD1 mutations. We found a highly synergistic effect of double mutations on triggering two key disease processes, D4Z4 heterochromatin disruption and cross-stimulation of DUX4 targets, such as histone H3.X/Y and LEUTX transcription factor. Thus, engineered human myocyte models provide unique insights into the molecular mechanisms underpinning FSHD.