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

Abstract

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...

Herpesviral induction of germline transcription factor DUX4 is critical for viral gene expression

“…Interestingly, we also confirmed the expression of HSAT-II satellite repeat transcripts. HSAT-II repeats are known DUX4 targets 12,33 and have recently been shown to play a role in HCMV replication 34 . (Fig.…”

CTCF is a barrier for 2C-like reprogramming

“…In support of this idea, additional sources of damage have been associated with the 2C-like state or induced by DUX. In fact, human ortholog DUX4 mediates the accumulation of dsRNA foci and the activation of the dsRNA response contributing to the apoptotic phenotype associated with DUX over-expression 40 . Further work will be needed to understand the exact origin of these DNA breaks and whether the single DNA ends detected are precursor lesions to double strand brakes or are generated due to specific replication or transcriptional mechanisms.…”

“…Data were processed using a modified version of Cut&RunTools 55 (Supplementary Software 1). Reads were adapter trimmed using fastp v.0.20.0 40 . An additional trimming step was performed to remove up to 6 bp adapter from each read.…”