Elucidating DUX4-mediated molecular mechanisms underlying FSHD pathophysiology using multiomics approaches

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscular disorder caused by aberrant expression of the embryonic transcription factor DUX4 in skeletal muscle. While DUX4 is established as the primary pathogenic driver, its transcriptional effects across heterogeneous muscle cell populations remain incompletely understood.

This thesis investigates DUX4-mediated transcriptional regulation in FSHD using multi-level transcriptomic analyses. Single-nucleus RNA sequencing of primary myotubes revealed distinct DUX4-affected nuclear populations with divergent transcriptional states, including impaired myogenesis and activation of stress- and apoptosis-related pathways, accompanied by reactivation of early embryonic gene programs. Single-fiber RNA sequencing of patient muscle biopsies further demonstrated pronounced transcriptional heterogeneity and focal DUX4 activity at the individual myofiber level. In addition, transcriptomic analysis of 3D tissue-engineered skeletal muscle models derived from FSHD patient iPSCs showed enhanced myogenic maturation and more robust expression of core DUX4 target genes compared to 2D cultures, providing improved molecular resolution for disease modeling. Finally, long-read Iso-Seq analysis revealed that DUX4 extensively increases transcriptomic complexity through alternative splicing, novel isoform generation, and activation of repeat-derived transcripts in a cellular context-dependent manner.

Together, this work delineates how DUX4 reshapes the muscle transcriptome across cellular scales and provides a framework for understanding FSHD pathogenesis.