Abstract 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.

Some tobacco companies claim they are ‘transforming’ by adopting harm reduction goals or even seeking to achieve a ‘smokefree’ world. What characterises transformation and whether companies can or are transforming is unclear. Nevertheless, such claims are gaining traction. We critically investigated tobacco industry transformation by exploring the definition and criteria for evaluating transformation, and assessed whether transformation is occurring and feasible.Companies’ transformation claims centre on increasing sales of new tobacco and nicotine products like e-cigarettes (‘new products’) with little attention to reducing sales of more hazardous smoked and oral products (‘conventional products’).We define a transforming tobacco company as one demonstrating substantial, rapid and verifiable progress towards eliminating the production and sale of conventional tobacco products within 5 years in all markets where it operates.We found no evidence any tobacco company is meeting the three essential criteria of rapidly progressing towards eliminating conventional products, ceasing to obstruct effective tobacco control measures and taking action to minimise smoking uptake and disparities. While some companies are developing new product portfolios, their actions are more consistent with profit maximisation than eliminating conventional product use. This approach is best described as ‘pseudo-transformation’, designed to delay implementation of effective tobacco control policies. In addition, our analysis suggests replacing conventional products with new nicotine products is unlikely to be a viable long-term business model.Public health practitioners should not rely on tobacco industry claims but should lead the transformation debate, establish credible definitions and criteria, and monitor and assess whether transformation is occurring.

19The DUX4 transcription factor is normally expressed in the cleavage stage 20 embryo and regulates genes involved in embryonic genome activation. Mis-expression 21 of DUX4 in skeletal muscle, however, is toxic and causes facioscapulohumeral 22 muscular dystrophy (FSHD). We recently showed DUX4-induced toxicity is due, in part, 23 to the activation of the double-stranded RNA (dsRNA) response pathway and the 24 accumulation of intranuclear dsRNA foci. Here, we determined the composition of 25 DUX4-induced dsRNAs. We found that a subset of DUX4-induced dsRNAs originate 26 from inverted Alu repeats embedded within the introns of DUX4-induced transcripts and 27 from DUX4-induced dsRNA-forming intergenic transcripts enriched for endogenous 28 retroviruses, Alu and LINE-1 elements. However, these repeat classes were also 29 represented in dsRNAs from cells not expressing DUX4. In contrast, pericentric human 30 satellite II (HSATII) repeats formed a class of dsRNA specific to the DUX4 expressing 31 cells. Further investigation revealed that DUX4 can initiate the bidirectional transcription 32 of normally heterochromatin-silenced HSATII repeats. DUX4 induced HSATII RNAs co-33 localized with DUX4-induced nuclear dsRNA foci and with intranuclear aggregation of 34 EIF4A3 and ADAR1. Finally, gapmer-mediated knockdown of HSATII transcripts 35 depleted DUX4-induced intranuclear ribonucleoprotein aggregates and decreased 36 DUX4-induced cell death, suggesting that HSATII formed dsRNAs contribute to DUX4 37 toxicity.38 39 40 41 42The Double Homeobox 4 (DUX4) transcription factor is normally expressed in the 43 testis, likely the germline cells (1), and in the cleavage stage embryo coincident with 44 embryonic genome activation (EGA) (2-5). In contrast, the mis-expression of DUX4 in 45 skeletal muscle causes FSHD (1, 6, 7). In both embryonic stem cells and in skeletal 46 muscle cells, expression of DUX4 activates the transcription of hundreds of genes that 47 are characteristically expressed in the cleavage stage embryo. DUX4 also activates the 48 expression of normally silenced repetitive elements that are transcribed during EGA 49 such as endogenous retroviruses (ERVs) and the pericentric human satellite II (HSATII) 50 repeats (3, 4, 8, 9). The expression of DUX4 in skeletal muscle cells is toxic and causes 51 apoptosis (10-13), however, the specific molecular pathways leading to cell toxicity are 52 not fully understood. Previous studies have suggested multiple, non-mutually exclusive 53 mechanisms for DUX4 toxicity including an increased sensitivity to oxidative stress (12, 54 14), interference with PAX3/PAX7 (12, 15) and the formation of insoluble TDP-43 55 nuclear aggregates due to impaired protein turnover (16). 56More recently, we reported that the expression of DUX4 in skeletal muscle cells 57 resulted in the accumulation of intranuclear foci of dsRNAs (13). The accumulation of 58 DUX4-induced dsRNAs correlated with PKR and eIF2α phosphorylation, both 59 proapoptotic characteristics of the cellular innate immune re...