Multiple repeat regions within mouse DUX recruit chromatin regulators to facilitate an embryonic gene expression program.

The embryonic transcription factor DUX regulates chromatin opening and gene expression in totipotent cleavage-stage mouse embryos, and its expression in embryonic stem cells promotes their conversion to 2-cell embryo-like cells (2CLCs) with extraembryonic potential. However, little is known regarding which domains within mouse DUX interact with particular chromatin and transcription regulators. Here, we reveal that the C-terminus of mouse DUX contains five uncharacterized ~100 amino acid (aa) repeats followed by an acidic 14 amino acid tail. Unexpectedly, structure-function approaches classify two repeats as 'active' and three as 'inactive' in cleavage/2CLC transcription program enhancement, with differences narrowed to a key 6 amino acid section. Our proximity dependent biotin ligation (BioID) approach identified factors selectively associated with active DUX repeat derivatives (including the 14aa 'tail'), including transcription and chromatin factors such as SWI/SNF (BAF) complex, as well as nucleolar factors that have been previously implicated in regulating the Dux locus. Finally, our mechanistic studies reveal cooperativity between DUX active repeats and the acidic tail in cofactor recruitment, DUX target opening, and transcription. Taken together, we provide several new insights into DUX structure-function, and mechanisms of chromatin and gene regulation.

p53 convergently activates Dux/DUX4 in embryonic stem cells and in facioscapulohumeral muscular dystrophy cell models.

In mammalian embryos, proper zygotic genome activation (ZGA) underlies totipotent development. Double homeobox (DUX)-family factors participate in ZGA, and mouse Dux is required for forming cultured two-cell (2C)-like cells. Remarkably, in mouse embryonic stem cells, Dux is activated by the tumor suppressor p53, and Dux expression promotes differentiation into expanded-fate cell types. Long-read sequencing and assembly of the mouse Dux locus reveals its complex chromatin regulation including putative positive and negative feedback loops. We show that the p53-DUX/DUX4 regulatory axis is conserved in humans. Furthermore, we demonstrate that cells derived from patients with facioscapulohumeral muscular dystrophy (FSHD) activate human DUX4 during p53 signaling via a p53-binding site in a primate-specific subtelomeric long terminal repeat (LTR)10C element. In summary, our work shows that p53 activation convergently evolved to couple p53 to Dux/DUX4 activation in embryonic stem cells, embryos and cells from patients with FSHD, potentially uniting the developmental and disease regulation of DUX-family factors and identifying evidence-based therapeutic opportunities for FSHD.

DUX4-induced bidirectional HSATII satellite repeat transcripts form intranuclear double-stranded RNA foci in human cell models of FSHD.

The DUX4 transcription factor is normally expressed in the cleavage-stage embryo and regulates genes involved in embryonic genome activation. Misexpression of DUX4 in skeletal muscle, however, is toxic and causes facioscapulohumeral muscular dystrophy (FSHD). We recently showed DUX4-induced toxicity is due, in part, to the activation of the double-stranded RNA (dsRNA) response pathway and the accumulation of intranuclear dsRNA foci. Here, we determined the composition of DUX4-induced dsRNAs. We found that a subset of DUX4-induced dsRNAs originate from inverted Alu repeats embedded within the introns of DUX4-induced transcripts and from DUX4-induced dsRNA-forming intergenic transcripts enriched for endogenous retroviruses, Alu and LINE-1 elements. However, these repeat classes were also represented in dsRNAs from cells not expressing DUX4. In contrast, pericentric human satellite II (HSATII) repeats formed a class of dsRNA specific to the DUX4 expressing cells. Further investigation revealed that DUX4 can initiate the bidirectional transcription of normally heterochromatin-silenced HSATII repeats. DUX4-induced HSATII RNAs co-localized with DUX4-induced nuclear dsRNA foci and with intranuclear aggregation of EIF4A3 and ADAR1. Finally, gapmer-mediated knockdown of HSATII transcripts depleted DUX4-induced intranuclear ribonucleoprotein aggregates and decreased DUX4-induced cell death, suggesting that HSATII-formed dsRNAs contribute to DUX4 toxicity.

DUX4 Suppresses MHC Class I to Promote Cancer Immune Evasion and Resistance to Checkpoint Blockade.

Advances in cancer immunotherapies make it critical to identify genes that modulate antigen presentation and tumor-immune interactions. We report that DUX4, an early embryonic transcription factor that is normally silenced in somatic tissues, is re-expressed in diverse solid cancers. Both cis-acting inherited genetic variation and somatically acquired mutations in trans-acting repressors contribute to DUX4 re-expression in cancer. Although many DUX4 target genes encode self-antigens, DUX4-expressing cancers were paradoxically characterized by reduced markers of anti-tumor cytolytic activity and lower major histocompatibility complex (MHC) class I gene expression. We demonstrate that DUX4 expression blocks interferon-γ-mediated induction of MHC class I, implicating suppressed antigen presentation in DUX4-mediated immune evasion. Clinical data in metastatic melanoma confirmed that DUX4 expression was associated with significantly reduced progression-free and overall survival in response to anti-CTLA-4. Our results demonstrate that cancers can escape immune surveillance by reactivating a normal developmental pathway and identify a therapeutically relevant mechanism of cell-intrinsic immune evasion.

Facioscapulohumeral dystrophy: activating an early embryonic transcriptional program in human skeletal muscle.

Facioscapulohumeral dystrophy (FSHD) is the third most prevalent muscular dystrophy. A progressive disease, it presents clinically as weakness and wasting of the face, shoulder and upper arm muscles, with later involvement of the trunk and lower extremities. FSHD develops through complex genetic and epigenetic events that converge on a common mechanism of toxicity with mis-expression of the transcription factor double homeobox 4 (DUX4). There is currently no treatment available for FSHD. However, the consensus that ectopic DUX4 expression in skeletal muscle is the root cause of FSHD pathophysiology has allowed research efforts to turn toward cultivating a deeper understanding of DUX4 biology and the pathways that underlie FSHD muscle pathology, and to translational studies aimed at developing targeted therapeutics using ever more sophisticated cell and animal-based models of FSHD. This review summarizes recent advances in our understanding of FSHD, including the regulation and activity of DUX4 in its normal developmental roles as well as its pathological contexts. We highlight how these advances raise new questions and challenges for the field as it moves into the next decade of FSHD research.

NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins.

The DUX4 transcription factor is encoded by a retrogene embedded in each unit of the D4Z4 macrosatellite repeat. DUX4 is normally expressed in the cleavage-stage embryo, whereas chromatin repression prevents DUX4 expression in most somatic tissues. Failure of this repression causes facioscapulohumeral muscular dystrophy (FSHD) due to mis-expression of DUX4 in skeletal muscle. In this study, we used CRISPR/Cas9 engineered chromatin immunoprecipitation (enChIP) locus-specific proteomics to characterize D4Z4-associated proteins. These and other approaches identified the Nucleosome Remodeling Deacetylase (NuRD) and Chromatin Assembly Factor 1 (CAF-1) complexes as necessary for DUX4 repression in human skeletal muscle cells and induced pluripotent stem (iPS) cells. Furthermore, DUX4-induced expression of MBD3L proteins partly relieved this repression in FSHD muscle cells. Together, these findings identify NuRD and CAF-1 as mediators of DUX4 chromatin repression and suggest a mechanism for the amplification of DUX4 expression in FSHD muscle cells.

BET bromodomain inhibitors and agonists of the beta-2 adrenergic receptor identified in screens for compounds that inhibit DUX4 expression in FSHD muscle cells.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is a progressive muscle disease caused by mutations that lead to epigenetic derepression and inappropriate transcription of the double homeobox 4 (DUX4) gene in skeletal muscle. Drugs that enhance the repression of DUX4 and prevent its expression in skeletal muscle cells therefore represent candidate therapies for FSHD. METHODS: We screened an aggregated chemical library enriched for compounds with epigenetic activities and the Pharmakon 1600 library composed of compounds that have reached clinical testing to identify molecules that decrease DUX4 expression as monitored by the levels of DUX4 target genes in FSHD patient-derived skeletal muscle cell cultures. RESULTS: Our screens identified several classes of molecules that include inhibitors of the bromodomain and extra-terminal (BET) family of proteins and agonists of the beta-2 adrenergic receptor. Further studies showed that compounds from these two classes suppress the expression of DUX4 messenger RNA (mRNA) by blocking the activity of bromodomain-containing protein 4 (BRD4) or by increasing cyclic adenosine monophosphate (cAMP) levels, respectively. CONCLUSIONS: These data uncover pathways involved in the regulation of DUX4 expression in somatic cells, provide potential candidate classes of compounds for FSHD therapeutic development, and create an important opportunity for mechanistic studies that may uncover additional therapeutic targets.

DUX4-induced dsRNA and MYC mRNA stabilization activate apoptotic pathways in human cell models of facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of DUX4 in skeletal muscle cells. DUX4 is a transcription factor that activates genes normally associated with stem cell biology and its mis-expression in FSHD cells results in apoptosis. To identify genes and pathways necessary for DUX4-mediated apoptosis, we performed an siRNA screen in an RD rhabdomyosarcoma cell line with an inducible DUX4 transgene. Our screen identified components of the MYC-mediated apoptotic pathway and the double-stranded RNA (dsRNA) innate immune response pathway as mediators of DUX4-induced apoptosis. Further investigation revealed that DUX4 expression led to increased MYC mRNA, accumulation of nuclear dsRNA foci, and activation of the dsRNA response pathway in both RD cells and human myoblasts. Nuclear dsRNA foci were associated with aggregation of the exon junction complex component EIF4A3. The elevation of MYC mRNA, dsRNA accumulation, and EIF4A3 nuclear aggregates in FSHD muscle cells suggest that these processes might contribute to FSHD pathophysiology.

Model systems of DUX4 expression recapitulate the transcriptional profile of FSHD cells.

Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of the double-homeodomain transcription factor DUX4 in skeletal muscle cells. Many different cell culture models have been developed to study the pathophysiology of FSHD, frequently based on endogenous expression of DUX4 in FSHD cells or by mis-expression of DUX4 in control human muscle cells. Although results generated using each model are generally consistent, differences have also been reported, making it unclear which model(s) faithfully recapitulate DUX4 and FSHD biology. In this study, we systematically compared RNA-seq data generated from three different models of FSHD­lentiviral-based DUX4 expression in myoblasts, doxycycline-inducible DUX4 in myoblasts, and differentiated human FSHD myocytes expressing endogenous DUX4­and show that the DUX4-associated gene expression signatures of each dataset are highly correlated (Pearson's correlation coefficient, r ∼ 0.75-0.85). The few robust differences were attributable to different states of cell differentiation and other differences in experimental design. Our study describes a model system for inducible DUX4 expression that enables reproducible and synchronized experiments and validates the fidelity and FSHD relevance of multiple distinct models of DUX4 expression.