DUX4 differentially regulates transcriptomes of human rhabdomyosarcoma and mouse C2C12 cells.

Vishakha Sharma,Naoe Harafuji,Alexandra Belayew,Yi-Wen Chen,Brian P. Chadwick

doi:10.1371/journal.pone.0064691

Vishakha Sharma, Naoe Harafuji + Show 3 more

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https://doi.org/10.1371/journal.pone.0064691

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Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35. Recent studies suggested that aberrant expression of double homeobox 4 (DUX4) from the last D4Z4 repeat causes FSHD. The aim of this study is to determine transcriptomic responses to ectopically expressed DUX4 in human and mouse cells of muscle lineage. We expression profiled human rhabdomyosarcoma (RD) cells and mouse C2C12 cells transfected with expression vectors of DUX4 using the Affymetrix Human Genome U133 Plus 2.0 Arrays and Mouse Genome 430 2.0 Arrays, respectively. A total of 2267 and 150 transcripts were identified to be differentially expressed in the RD and C2C12 cells, respectively. Amongst the transcripts differentially expressed in the RD cells, MYOD and MYOG (2 fold, p<0.05), and six MYOD downstream targets were up-regulated in RD but not C2C12 cells. Furthermore, 13 transcripts involved in germline function were dramatically induced only in the RD cells expressing DUX4. The top 3 IPA canonical pathways affected by DUX4 were different between the RD (inflammation, BMP signaling and NRF-2 mediated oxidative stress) and the C2C12 cells (p53 signaling, cell cycle regulation and cellular energy metabolism). Amongst the 40 transcripts shared by the RD and C2C12 cells, UTS2 was significantly induced by 76 fold and 224 fold in the RD and C2C12 cells, respectively. The differential expression of MYOD, MYOG and UTS2 were validated using real-time quantitative RT-PCR. We further validated the differentially expressed genes in immortalized FSHD myoblasts and showed up-regulation of MYOD, MYOG, ZSCAN4 and UTS2. The results suggest that DUX4 regulates overlapped and distinct groups of genes and pathways in human and mouse cells as evident by the selective up-regulation of genes involved in myogenesis and gametogenesis in human RD and immortalized cells as well as the different molecular pathways identified in the cells.

Highlights

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder and the third most common inherited form of muscular dystrophy
The results showed that top 3 canonical pathways affected by double homeobox 4 (DUX4) expression in RD cells were those involved in Wnt-mediated immune responses, BMP signaling and NRF-2 mediated oxidative stress response, whereas in C2C12 cells were those involved in p53 signaling, cell cycle regulation, and cellular energy metabolism
The results showed that the most significantly affected molecular pathways by DUX4 are distinct in the RD cells and C2C12 cells while some expression changes were shared

Summary

Introduction

FSHD is an autosomal dominant disorder and the third most common inherited form of muscular dystrophy. FSHD2 (OMIM #158901) is not linked to contractions of the D4Z4 repeat array but to mutations in the SMCHD1 protein involved in chromatin structure [5]. DNA hypomethylation of the D4Z4 region is common to both FSHD1 and 2 and causes transcriptional de-repression, which allows the DUX4 gene to be transcribed. The FSHD permissive alleles further present a poly-adenylation signal in the pLAM region distal to the repeat array [6], which allows stabilization of the DUX4 transcripts derived from the last D4Z4 unit and their translation [6,7,8,9,10,11,12,13,14]

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