Stage-dependent chromatin accessibility remodeling defines an architectural state in facioscapulohumeral muscular dystrophy myoblasts

Abstract Facioscapulohumeral muscular dystrophy (FSHD) has been linked to alterations in higher-order genome organization, yet how these structural perturbations shape chromatin accessibility across muscle developmental stages remains unclear. Here, we identify a stage-specific architectural chromatin accessibility state that defines proliferating FSHD myoblasts. Using genome-wide ATAC-seq profiling of primary human myoblasts and differentiated myotubes, analyzed using the telomere-to-telomere (T2T-CHM13) human reference genome, we map the genome-wide distribution and hierarchical organization of this state. We detect extensive chromatin accessibility remodeling, with over 12,000 differentially accessible regions in myoblasts and marked attenuation of this state upon terminal differentiation. Remodeling predominantly affects intronic and distal intergenic regions enriched in non-coding and repeat-proximal sequences, indicating redistribution of regulatory accessibility beyond promoter-centered regulation. At the chromosome scale, accessibility losses cluster within megabase domains enriched in heterochromatin- and nucleolus-associated regions, revealing coordinated reorganization of nuclear architecture. No reproducible accessibility changes occur within the D4Z4 repeat array or canonical DUX4 target loci, demonstrating that early architectural remodeling in FSHD myoblasts is largely uncoupled from sustained DUX4-driven programs. These findings these findings define a transient architectural chromatin accessibility state that emerges in proliferating FSHD myoblasts and is largely resolved upon terminal differentiation. Our work supports a model in which disruption of higher-order genome organization represents an early and stage-restricted determinant of disease susceptibility.