DNA Topoisomerase II, CTCF/cohesin Binding, and Alternative DNA Secondary Structure Alter Genomic Stability and Affect Cancer Susceptibility
Atkin, Naomi, Biochemistry and Molecular Genetics - School of Medicine, University of Virginia
Wang, Yuh-Hwa, MD-BIOC Biochem/Mole Genetics, University of Virginia
DNA double-stranded breaks (DSBs) initiate the formation of oncogenic chromosomal abnormalities such as translocations, amplifications, and deletions. Therefore, identifying the genomic features that contribute to cancer-promoting DNA fragility, as well as sensitively measuring DSBs at these regions is critical to the ability to predict an individual’s propensity to form such abnormalities and thus their subsequent cancer susceptibility. Identifying susceptible individuals prior to cancer formation would allow closer monitoring of patients, thereby facilitating early cancer detection and increasing survival odds. We and others have recently demonstrated individual roles for topoisomerase II (TOP2), CCCTC-binding factor (CTCF)/cohesin binding sites, and alternative DNA secondary structure in mediating genomic instability. To understand the direct effect of these features on genomic instability, we considered how the three features function together within the genomes of both normal and cancer samples. In order to accomplish this, a DNA break mapping method that utilizes low input of carefully purified genomic DNA was developed, making it compatible for use with scarce patient samples. Here we examined genome-wide DNA breakage in both cultured immortal human cells and normal cells from cancer patients as a means to evaluate DNA fragility and cancer susceptibility. We found a direct relationship between CTCF binding strength and DSB incidence, and depletion of CTCF protein levels in non-malignant mammary epithelial cells further increased DSBs at the strongest CTCF binding sites. CTCF’s contributing role in genomic instability was further characterized when we analyzed the end structures of DSBs in cells with reduced CTCF protein. These cells exhibited an accumulation of ends with a blunt-end signature, indicative of less resection occurring at DSBs, agreeing with the previously described role for CTCF in promoting the recruitment of DNA end resection proteins. Importantly, the normal cells of cancer patients whose cancer was driven by a chromosomal abnormality (susceptible individuals) were preferentially susceptible to DSBs at strong CTCF binding sites, suggesting that CTCF binding also contributes to genomic instability in cancer patients. DSBs were also preferentially enriched at TOP2-sensitive and cohesin binding sites in susceptible individuals. Low-dose etoposide exposure further enriched DSBs at TOP2-sensitive and cohesin binding sites, as well as at CTCF binding sites, indicating that TOP2 affected genomic instability at these features. Moreover, TOP2 was found to be directly active at CTCF binding sites in both cultured and patient cells, as demonstrated by a significant reduction of TOP2 cleavage complexes after TOP2 knockout. Interestingly, we found that DNA sequences around strong CTCF binding sites have the potential to form highly stable alternative DNA secondary structures in multiple cell types, and these structure colocalized with TOP2B binding/cleavage sites and cancer patient breakpoint regions. These results indicate that alternative DNA secondary structures at strong CTCF binding sites could act as a recognition signal for TOP2, thereby promoting the formation of DSBs. Together, these results suggest that TOP2, CTCF/cohesin binding sites, and alternative DNA secondary structures alter genomic stability, thus contributing to cancer susceptibility.
PHD (Doctor of Philosophy)
DNA fragility, Chromatin organization, DNA topoisomerase II, Cancer susceptibility
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