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Abstract

Bloom Syndrome is a recessive genetic disorder characterized by growth deficiency and hyper- recombination. It is caused by mutations in the conserved RecQ helicase gene, BLM. BLM helicase is essential in maintaining genome integrity given its ability to unwind various aberrant DNA structures including DNA G-quadruplexes (G4s). G4s are non-canonical DNA structures formed by G-rich sequences through G-G base pairing. They could regulate processes like replication, transcription, DNA-protein interaction, which enables BLM to exert broad regulatory functions. Previous studies showed a correlation between the occurrence of G4-forming sequences and certain molecular phenotypes in Bloom Syndrome. However, it remains unclear what regulatory role endogenous G4 structures play in Bloom Syndrome. Considering the regulatory potential of G4, I formulated the question if perturbed G4 formation activates in Bloom Syndrome mediate subsequent molecular changes and contributes to its pathogenesis. I addressed this question from three aspects. Paired cell lines of two tissue background derived from age- and sex-matched Bloom Syndrome individuals (BS) or healthy donors (WT) were used. I mapped endogenous G4s via ChIP-seq using a specific antibody against G4s and characterized the chromatin accessibility and gene expression profiles by ATAC-seq and RNA-seq. Firstly, I found that BS cells have disrupted molecular profiles in all data modalities compared to WT cells. Particularly, in BS cells regardless of cell types, differential G4 formation activities positively correlated with both chromatin accessibility and gene expression changes. Secondly, I applied a G4-stabilizing molecule to WT cells to mimic defective G4-resolving abilities in BS. This treatment partially recapitulated the global and differential signals of chromatin accessibility and transcriptome in BS, implying a partial causal relationship between G4 and Bloom Syndrome. Thirdly, I collected data from a BS family and found that differentially accessible chromatin regions in the patients were associated with the prevalence of G4-forming sequences. Regarding possible molecular mechanisms by which G4s may regulate molecular phenotypes in BS, I found that the presence of G4 is associated with wider and more accessible chromatin regions and enhanced gene expression. These results signify a central role of G4 in the molecular etiology of Bloom Syndrome and suggest a novel molecular model: in Bloom Syndrome, persistent G4 formation subsequently cause excessive recombination, persistently accessible chromatin regions and altered gene expression. They also revealed BLM’s regulatory role besides its well-established function as a helicase.