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

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.

Hypoxic preconditioning protection is eliminated in HIF-1α knockout mice subjected to neonatal hypoxia-ischemia.

BACKGROUND: Hypoxic preconditioning (HPc) protects the neonatal brain in the setting of hypoxia-ischemia (HI). The mechanisms of protection may depend on activation of hypoxia-inducible factor (HIF-1α). This study sought to clarify the role of HIF-1α after HPc and HI. METHODS: To induce HPc, HIF-1α knockout and wild-type (WT) mice were exposed to hypoxia at postnatal day 6. At day 7, the mice underwent HI. Brain injury was determined by histology. HIF-1α, downstream targets, and markers of cell death were measured by western blot. RESULTS: HPc protected the WT brain compared with WT without HPc, but did not protect the HIF-1α knockout brain. In WT, HIF-1α increased after hypoxia and after HI, but not with HPc. The HIF-1α knockout showed no change in HIF-1α after hypoxia, HI, or HPc/HI. After HI, spectrin 145/150 was higher in HIF-1α knockout, but after HPc/HI, it was higher in WT. Lysosome-associated membrane protein was higher in WT early after HI, but not later. After HPc/HI, lysosome-associated membrane protein was higher in HIF-1α knockout. CONCLUSION: These results indicate that HIF-1α is necessary for HPc protection in the neonatal brain and may affect cell death after HI. Different death and repair mechanisms depend on the timing of HPc.