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

Details

• Alternative Title:
  Ann Neurol
• Personal Author:
  Knox, Renatta N. ; Eidahl, Jocelyn O. ; Wallace, Lindsay ; Choudury, Sarah ; Rashnonejad, Afrooz ; Daman, Katelyn ; Guggenbiller, Matthew ; Saad, Nizar ; Hoover, Michael E. ; Zhang, Liwen ; Branson, Owen E. ; Emerson, Charles P. ; Freitas, Michael A. ; Harper, Scott Q.
• Description:
  Objective:
  
  Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the myotoxic transcription factor DUX4. While 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.
  
  
• Subjects:
  Gene Expression Regulation HEK293 Cells Homeodomain Proteins Humans Muscle, Skeletal Muscular Dystrophy, Facioscapulohumeral Protein-Arginine N-Methyltransferases Protein Processing, Post-Translational Repressor Proteins
• Keywords:
  DNA Binding Protein Homeobox Mass Spectrometry (MS) Muscular Dystrophy Post-translational Modification (PTM)
• Source:
  Ann Neurol. 94(2):398-413
• Pubmed ID:
  37186119
• Pubmed Central ID:
  PMC10777487
• Document Type:
  Journal Article
• Funding:
  S10 OD018056/OD/NIH HHSUnited States/ ; R01 AR062123/AR/NIAMS NIH HHSUnited States/ ; 1P50AR070604-01/AR/NIAMS NIH HHSUnited States/ ; R21NS101166/NS/NINDS NIH HHSUnited States/ ; P50 AR070604/AR/NIAMS NIH HHSUnited States/ ; P50 HD060848/HD/NICHD NIH HHSUnited States/ ; U54 HD060848/HD/NICHD NIH HHSUnited States/ ; S10 OD018056/CD/ODCDC CDC HHSUnited States/ ; R21 NS101166/NS/NINDS NIH HHSUnited States/
• Volume:
  94
• Issue:
  2
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha-512:985aa525c5089551757ce671a5f9bd70e564d60158dcdd1ac113821be6153d566a8ef045c17ea5f222b17d5aa78d61136969a34b99926ae7c85ced04b9b0b09d
• Download URL:
  https://stacks.cdc.gov/view/cdc/140553/cdc_140553_DS1.pdf
• File Type:
  [PDF - 2.37 MB ]