Post-Translational Modifications of the DUX4 Protein Impact Toxic Function in FSHD Cell Models

Ann Neurol. 2023 Aug;94(2):398-413. doi: 10.1002/ana.26668. Epub 2023 May 19.


Objective: Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the myotoxic transcription factor DUX4. Although the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 post-translational modifications (PTMs) and their impact on the toxic function of DUX4.

Methods: We immunoprecipitated DUX4 protein and performed mass spectrometry to identify PTMs. We then characterized DUX4 PTMs and potential enzyme modifiers using mutagenesis, proteomics, and biochemical assays in HEK293 and human myoblast cell lines.

Results: We identified 17 DUX4 amino acids with PTMs, and generated 55 DUX4 mutants designed to prevent or mimic PTMs. Five mutants protected cells against DUX4-mediated toxicity and reduced the ability of DUX4 to transactivate FSHD biomarkers. These mutagenesis results suggested that DUX4 toxicity could be counteracted by serine/threonine phosphorylation and/or inhibition of arginine methylation. We therefore sought to identify modifying enzymes that could play a role in regulating DUX4 PTMs. We found several enzymes capable of modifying DUX4 protein in vitro, and confirmed that protein kinase A (PKA) and protein arginine methyltransferase (PRMT1) interact with DUX4.

Interpretation: These results support that DUX4 is regulated by PTMs and set a foundation for developing FSHD drug screens based mechanistically on DUX4 PTMs and modifying enzymes. ANN NEUROL 2023;94:398-413.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Regulation
  • HEK293 Cells
  • Homeodomain Proteins / genetics
  • Humans
  • Muscle, Skeletal / metabolism
  • Muscular Dystrophy, Facioscapulohumeral* / genetics
  • Protein Processing, Post-Translational
  • Protein-Arginine N-Methyltransferases / genetics
  • Protein-Arginine N-Methyltransferases / metabolism
  • Repressor Proteins / metabolism


  • Homeodomain Proteins
  • PRMT1 protein, human
  • Protein-Arginine N-Methyltransferases
  • Repressor Proteins
  • DUX4L1 protein, human