Centromere Instability Drives Chromosome Damage and Autoantigen Exposure in Systemic Sclerosis

Centromere breakage has been associated with anti-centromere antibodies in systemic sclerosis (SSc), yet the origins of centromere damage and its link to immune activation remain unclear. The bleomycin induced fibrosis model is widely used as an experimental model of SSc. Here, we investigated whether bleomycin selectively disrupts centromeres and whether such damage contributes to chromosome instability and immunogenic chromatin mislocalization.

We evaluated centromere integrity and downstream genome damage-response phenotypes across complementary systems: the bleomycin mouse model of skin fibrosis, hTERT-immortalized human fibroblasts, and primary dermal fibroblasts from SSc patients. Centromeric α-satellite copy number, DNA damage and repair markers, micronuclei, nuclear envelope rupture, and colocalization of centromeric chromatin with antigen presentation machinery were assessed by qPCR, RT-qPCR, and immunofluorescence microscopy.

Bleomycin induced selective depletion of α-satellite repeats at centromeres in mouse skin. In human fibroblasts, bleomycin generated double-strand breaks by γH2AX foci that preferentially colocalized with CENP-A-marked active centromeres and produced persistent centromere loss. Damage signaling required ATM and relied mainly on RAD51-mediated homologous-recombination repair, yet centromere restoration remained incomplete. Bleomycin also increased micronuclei, cytoplasmic centromeric foci, and BANF1-marked nuclear-envelope rupture, consistent with mis-segregation-driven chromatin leakage. CENP-B colocalized with HLA-DRB1 in bleomycin-treated fibroblasts and limited cutaneous SSc (lccSSc) fibroblasts, revealing centromere-derived antigen-presentation signatures.

This study identifies active centromeres as selectively vulnerable and incompletely repaired targets of bleomycin-induced DNA damage and demonstrates that centromere instability produces mislocalized chromatin capable of engaging antigen-presentation pathways. These findings provide mechanistic insight into SSc autoantibody specificity and link genome instability to immune activation in fibrotic autoimmunity.