Molecular Level Insights into the Functional Relationship Between Chromatin Structure and Thymine DNA Glycosylase & L-RNA Inhibitors of the Polycomb Repressive Complex 2
Abstract
Active DNA demethylation by Thymine DNA glycosylase (TDG) is essential for embryogenesis and genetic reprogramming, and like all transactions involving DNA, TDG-dependent demethylation must occur within the structural constraints of chromatin. Although a relationship between TDG and chromatin can sometimes be inferred, a molecular understanding is lacking. To address this knowledge gap we developed a method for introducing DNA modifications into chromatin with single nucleotide resolution and then applied this approach to engineering synthetic chromatin with a substrate for studying TDG catalysis, the demethylation intermediate 5-formylcytosine. Subsequent analysis of TDG reactivity within various chromatin ‘environments' provided the first direct evidence that chromatin structure regulates demethylation by TDG. This platform also enabled the first biochemical investigations into the influence of TDG on chromatin structure. While genomic architectures like chromatin loops and phase separated condensates have been observed at genes regulated by TDG, decoupling the direct contribution of TDG to these structures from the complex milieu of regulatory factors inside the cell remains a significant challenge. Utilizing synthetic chromatin, we discover that TDG destabilizes local fiber compaction while, simultaneously, promoting chromatin oligomerization (i.e. condensation) via bridging interactions mediated by the intrinsically disordered N-terminal domain. Overall, this work establishes a new paradigm for TDG as a dynamic regulator of chromatin structure and organization.
Finally, a novel therapeutic approach for targeting PRC2 based on mirror image RNAs is described. PRC2 is a long non-coding (lnc)RNA-associated chromatin modifying enzyme that is frequently dysregulated in breast, prostate, ovarian, and esophageal cancers and, therefore, is a prime drug target. Inspired by evidence that PRC2 promiscuously (i.e. non-specifically) interacts with RNAs that contain guanine quadruplexes (G4s), we sought to develop inhibitors that mimicked these PRC2-interacting motifs. In this regard we exploited the synthetic enantiomer of naturally occurring D-RNA (L-RNA) and established that PRC2 binds RNA irrespective chemistry and that L-RNA G4s are potent inhibitors of PRC2-substrate interactions.
Citation
Deckard, Charles Edward (2020). Molecular Level Insights into the Functional Relationship Between Chromatin Structure and Thymine DNA Glycosylase & L-RNA Inhibitors of the Polycomb Repressive Complex 2. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /192947.