Abstract
The primary structure of DNA is the right-handed double helix, termed B-DNA. However, under proper conditions, DNA can form a series of alternative secondary structures (non-B DNA), which are involved in genome regulation and implicated in mutagenic processes associated with cancer and neurodegeneration, such as in amyotrophic lateral sclerosis (ALS). In ALS the leading genetic cause is the expansion of the hexanucleotide repeat G4C2 in the first intron of C9orf72 (C9+ ALS). The G4C2 repeat is an ideal motif to form G-quadruplexes (G4s), one type of non-B DNA, especially in patients whose expansions can contain hundreds to thousands of these repeats. We have previously shown that the G4C2 repeat can stall replication forks, implicating the repeat and its structure in repeat instability. Additionally, we and others have shown that manipulating the stability of the G4 structure reduced the production of toxic dipeptide repeat proteins (DPRs) generated by repeat-associated non-AUG (RAN) translation of both the sense and antisense repeat RNAs. To investigate the safety of using G4 stabilization approaches for therapeutic benefit in ALS, we evaluated the impact of knocking down the G4 helicase DHX36 (aliases G4R1 and RHAU) on the repeat-containing transcripts from the C9orf72 locus and the accumulation of RNA foci. We found that C9+ ALS with intermediate repeats reduced DPRs and reduced transcription of C9orf72, while large expansions showed increased transcription and accumulation of RNA foci. Then we assessed the broader impacts of G4 stabilization in C9+ ALS cells, finding that genes associated with DNA repair and mitosis pathways were upregulated in response to G4 stabilization by DHX36 knockdown and small molecule stabilizing ligands. By mapping DNA double strand breaks (DSBs) following G4 stabilization, we found that the higher G4 burden of C9+ ALS sensitized the cells to DNA damage resulting in the accumulation of DSBs at mapped G4 sites. This revealed that G4 stabilization approaches will need to monitor DNA damage as well as repeat-associated read outs as they are developed. Next, we investigated the nucleosome forming abilities of the G4C2 repeat DNA. We have previously shown that repeats expanded in other neurological diseases have altered nucleosome capabilities, and G-quadruplex forming regions of the genome are usually nucleosome-free. We found that the G4C2 repeats have unfavorable nucleosome formation free energy compared to normal sequences, and longer repeats and CpG methylation confer worse nucleosome formation abilities. The altered nucleosome formation ability could result in changes to the local chromatin structure around the repeat in C9+ ALS. In addition to their role in nucleosome-free regions, G4s are also implicated in the recruitment of CCCTC binding factor (CTCF) to binding sites throughout the genome. We have previously shown that CTCF binding sites are enriched for DSBs and strong non-B DNA secondary structures. Here we investigated how CTCF binding, non-B DNA, and topoisomerase II (TOP2) all interact to drive DNA fragility at CTCF binding sites (CBSs). We determined that TOP2B-mediates breaks preferentially at strong CBSs, which are more likely to form stronger non-B DNA structures. This increased fragility is driven by the topological stress at these sites as they are primarily topologically associated domain (TAD) boundaries. Additionally, we uncovered that in CTCF limiting environments, TAD boundaries that reduce CTCF binding are associated with strong non-B structures, namely G-quadruplexes, supporting a model in which these structures act as back-up boundary insulators. Furthermore, DNA DSBs increased TAD-associated CTCF sites in the CTCF limiting environment, while loop-associated CTCF alter DSBs directionally with CTCF binding changes. Altogether providing mechanistic insight into DNA fragility at CTCF sites, which can further our understand of mutagenic processes associated with cancer development. Overall, the results presented in this dissertation further our understanding of G-quadruplex structures in healthy genome maintenance, DNA fragility, and diseases including ALS and many cancers.
