Background: Aberrant expression of the double homeobox transcription factor DUX4 in skeletal muscle is the root cause of Facioscapulohumeral muscular dystrophy (FSHD), where activation of its intracellular downstream targets drives progressive muscle degeneration and disability. Recent reports propose KHDC1L (“cDUX”) as a circulating biomarker for FSHD, offering a less invasive method to monitor muscle DUX4 activity. While the transcriptional regulation of KHDC1L by DUX4 has been reported, its biological function and mechanism of secretion remain poorly understood. In this study, we investigated the temporal relationship between DUX4 and KHDC1L using FSHD-relevant cell models.
Methods: The temporal and dose-dependent relationship between DUX4 and KHDC1L were evaluated in FSHD cell line models, including MB135-iDUX4 myocytes with inducible DUX4 and FSHD patient-derived cell lines. Transcript and protein levels of KHDC1L and other DUX4 target genes were evaluated by RT-qPCR, immunoblots, and ELISA.
Results: In FSHD cell lines, DUX4 expression led to a concordant dose-dependent induction of KHDC1L transcripts, followed by KHDC1L protein production and secretion/release of KHDC1L from cells into the supernatant. The temporal dynamics of induction were validated using RT-qPCR and protein immunoassays, with early and late DUX4 target genes included as controls for DUX4 activity. siRNA-mediated knockdown of DUX4 significantly blunted KHDC1L induction compared to mock transfections.
Conclusions: We demonstrated that KHDC1L transcript and protein levels are tightly tied to DUX4 activation in FSHD model cell lines, with higher levels of DUX4 corresponding to higher KHDC1L secretion. This link between intracellular DUX4 activity and extracellular KHDC1L supports its potential clinical utility as a plasma biomarker for monitoring disease biology and therapeutic response of DUX4 targeted therapies in FSHD.