DOI: 10.5061/dryad.k0p2ngfbh
title: DUX4 expression activates JNK and p38 MAP kinases in myoblasts: MAJIQ splicing results

description: Facioscapulohumoral muscular dystrophy (FSHD) is caused by misexpression
 of the DUX4 transcription factor in skeletal muscle that results in
 transcriptional alterations, abnormal phenotypes, and cell death. To gain
 insight into the kinetics of DUX4-induced stresses, we activated DUX4
 expression in myoblasts and performed longitudinal RNA sequencing (GEO
 GSE205421) paired with proteomics and phosphoproteomics
 (MassIVE MSV000089812). This analysis revealed changes in
 cellular physiology including DNA damage and altered mRNA splicing.
 Phosphoproteomic analysis uncovered widespread changes in protein
 phosphorylation rapidly following DUX4 induction indicating that
 alterations in kinase signaling may play a role in DUX4-mediated stress
 and cell death. Indeed, we demonstrate that two stress-responsive MAP
 kinase pathways, JNK and p38, are activated in response to DUX4
 expression. Inhibition of each of these pathways ameliorated DUX4-mediated
 cell death in myoblasts. These findings uncover JNK as a novel pathway
 involved in DUX4-mediated cell death as well as provide additional
 insights into the role of the p38 pathway, a clinical target for the
 treatment of FSHD.
description: <strong>RNAseq:</strong> RNA was isolated using an
 RNeasy mini kit (Qiagen). Total RNA from 24 samples was evaluated for
 quality (RIN scores ≥ 9) by 4200 TapeStation System (Agilent, Santa Clara,
 CA, United States) using RNA ScreenTape (cat. 5067-5576, Agilent, Santa
 Clara, CA, United States). Concentrations were measured by High Lunatic
 plate (cat. 7012000, Unchained Labs, Pleasanton, CA). 200ng total RNA was
 used as input for the mRNA-Seq library preparation using the TruSeq
 Stranded mRNA Library Prep Kit (cat. 20020594, Illumina, San Diego, CA) on
 the Janus G3 Automated Liquid Handler System (PerkinElmer, Akron,
 OH). The protocol includes purification of the poly-A
 containing mRNA using poly-dT beads, fragmentation of RNA, first strand
 and second strand cDNA synthesis, end repairing, A-tailing, adapter
 ligation, followed by PCR amplification to generate the final cDNA
 library. The final libraries were then evaluated for quality by 4200
 TapeStation System (Agilent, Santa Clara, CA, United States) using D1000
 ScreenTape (cat. 5067-5582, Agilent, Santa Clara, CA, United States). The
 concentrations were measured using Qubit 1X dsDNA HS Assay Kit (cat.
 Q33231, Invitrogen- Life Technologies Corporation, Eugene, OR). The
 libraries were then normalized to 4nM, re-quantitated using Qubit 1X dsDNA
 HS Assay Kit (cat. Q33231, Invitrogen- Life Technologies Corporation,
 Eugene, OR) and then pooled for sequencing. The pool was further denatured
 and diluted to 1.8pM and loaded on NextSeq 500 sequencer for 75 Paired End
 run using 150 cycle High Output Sequencing Kit (cat. 20024907, Illumina,
 San Diego, CA). <strong>Splicing
 analysis:</strong> Libraries were demultiplexed using bcl2fastq
 (Illumina, San Diego, CA). Reads were aligned to the genome (GRCh38) using
 STAR 2.7.3a (Dobin et al., 2013). Both annotated and novel splice events
 were quantified from the bam files output from STAR using MAJIQ 2.1 and
 visualized using Voila 2.0.0. The tables here are the output from MAJIQ
 deltapsi. A threshold of 95% posterior probability of a &gt;=20%
 change in percent spliced inclusion (PSI) was used to define significant
 changes in either specific splicing events or LSVs.
description: The file can be opened in Excel or similar.