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.

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.

The objective of our study is to investigate the modulation of Type I Interferon induction of an antiviral state by Human Immunodeficiency Virus (HIV). Type I Interferons including IFN-α are key innate immune cytokines that activate the JAK/STAT pathway leading to the expression of ISGs (Interferon Stimulated Genes). ISGs consist of a pantheon of antiviral genes whose expression establishes the antiviral state that limits local viral infection in IFN-α stimulated microenvironments. Our previous studies have shown that HIV proteins disrupt the induction of Type I interferon (IFN-α) by degradation of IPS-1 (IFN-β promoter stimulator-1) which acts as an adaptor protein for the upregulation and release of IFN-α into local microenvironment via the RIG-I (retinoic acid-inducible gene 1)-Like Receptor (RLR) signaling pathway. However, IFN-α is still released from other sources such as plasmacytoid dendritic cells (pDCs) via Toll-like Receptor (TLR) dependent recognition of HIV. Here we report that the activation of the JAK/STAT pathway by IFN-α stimulation is disrupted by HIV proteins Vpu and Nef. Vpu and Nef both reduce IFN-α induction of STAT1 phosphorylation. This study shows that regardless of the presence of IFN-α in the local microenvironment, HIV would still be able to avoid antiviral protection induced by IFN-α signaling. These findings provide additional insight into how HIV acts to block multiple signaling points that would lead to the upregulation of ISGs. HIV is allowed to replicate more effectively if there is a reduction in ISG expressions induced by IFN-α.

Mutations in tau proteins are associated with a group of neurodegenerative diseases, termed tauopathies. To investigate whether over-expressing human tau with P301L mutation also affects stroke-induced brain damage, we performed hypoxia/ischemia (H/I) in young adult P301L tau transgenic mice. Surprisingly, brain infarct volume was significantly smaller in transgenic mice compared to wild-type mice 24 h after H/I induction. TUNEL staining also revealed less brain apoptosis in transgenic mice following H/I. H/I resulted in a significant increase in tau fragments generated by caspase activation and a marked decrease in tau phosphorylation at residue T231 in cortex of wild-type but not transgenic mice. Activation of calpain and caspase-3 following H/I was also reduced in transgenic compared to wild-type mice, as reflected by lower levels of the specific spectrin breakdown products generated by calpain or caspase-3. Finally, basal levels of the glial glutamate transporter, GLT-1, were higher in brains of transgenic as compared to wild-type mice. These results support the idea that enhanced levels of GLT-1 in transgenic mice are responsible for reducing H/I-induced brain damage by decreasing extracellular glutamate accumulation and subsequent calpain and caspase activation.

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.