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DUX4 expression activates JNK and p38 MAP kinases in myoblasts: MAJIQ splicing results

Citation

Hill, Abby et al. (2022), DUX4 expression activates JNK and p38 MAP kinases in myoblasts: MAJIQ splicing results, Dryad, Dataset, https://doi.org/10.5061/dryad.k0p2ngfbh

Abstract

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.

Methods

RNAseq: 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).

Splicing analysis: 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 >=20% change in percent spliced inclusion (PSI) was used to define significant changes in either specific splicing events or LSVs.

Usage Notes

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