Advisor(s)

Rafael Contreras-Galindo

Committee Member(s)

Arko Sen
Wioletta Czaja

Document Type

Thesis

Date of Award

6-18-2026

Degree Name

Master of Science (MS)

School

Joint Health Sciences (Interdisciplinary)

Department

Joint Health Sciences

Abstract

Centromeres are critical genomic regions responsible for ensuring accurate chromosome segregation during cell division. Disruptions in the structural or functional integrity of centromeric DNA could impair kinetochore assembly, leading to chromosome missegregation and chromosomal instability (CIN), a hallmark of various human diseases, including systemic sclerosis (SSc). This study investigated the contribution of centromeric DNA damage and deletion to CIN and explored potential pathogenic mechanisms relevant to SSc. We utilized hTERT-immortalized fibroblast cell lines (CHON-002 and BJ-5ta) to implement two complementary approaches: (i) treatment with bleomycin, a chemotherapeutic agent known to induce DNA double-strand breaks (DSBs), and (ii) targeted deletion of centromeric alpha-satellite DNA arrays using CRISPR-Cas9 genome editing. Our results demonstrated that bleomycin exposure resulted in significant loss of centromeric DNA and increased formation of micronuclei. Immunofluorescence and colocalization analyses revealed a preferential localization of γ-H2AX, a marker of DSBs, to centromeric regions. DSBs also colocalized with DNA repair proteins, particularly RAD51, indicating activation of homologous recombination. CRISPR-Cas9-mediated deletion of centromeric alpha-satellite sequences induced substantial centromere loss and chromosome missegregation, supporting the critical role of centromere DNA integrity in maintaining genomic stability. Our findings establish a direct mechanistic connection between centromeric DNA instability and CIN, providing novel insights into how centromere dysfunction may drive autoimmune and fibrotic processes in SSc. Furthermore, this study underscores the potential of centromere-targeted genome editing as a valuable tool for modeling and investigating chromosomal instability in human diseases.

Keywords

Alpha-satellite DNA;Bleomycin;Chromosomal instability;CRISPR-Cas9;DNA double-stranded breaks;Micronuclei

Download

Included in

Genetics Commons

Share

COinS