Date of Award
Summer 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Reiter, Nicholas
Second Advisor
St Maurice, Martin
Third Advisor
Yi, Chae
Abstract
The conserved chromatin-remodeling enzyme, Lysine-Specific histone1/2 Demethylase 1 (LSD1) primarily demethylates Histone H3K4me , acting as a transcriptional repressor. It plays pivotal roles in various physiological processes including cancer and interacts with many regulatory proteins, non-coding RNAs, and small metabolites. However, little biochemical information is known about LSD1’s N-terminal intrinsically disordered region (IDR) and how LSD1 interacts with various biomolecules in the context of nucleosome. I present evidence that the IDR of LSD1, containing multiple post translational modifications (PTMs) and a nuclear localization signal (NLS), can act as a reversible competitive autoinhibitor of LSD1’s activity. This autoinhibition can be relieved by phosphorylation PTMs adjacent to NLS, suggesting synergistic and versatile roles of IDRs and PTMs. The combined results support a new role for phosphorylation mediated NLS regions that may function by fine tuning chromatin-remodeling enzyme activity in an auto regulatory manner. Previous studies show that LSD1-G-quadruplex (GQ) RNA binding coincides with LSD1-nucleosome binding interface and acts as non-competitive inhibitor of LSD1’s peptide substrate demethylation. Here, I present evidence that LSD1 specifically recognizes, and binds GQ-RNA. The GQ-RNA structure preferentially inhibits LSD1 activity on nucleosomal substrates suggesting an RNA structure-based effect on regulatory properties of LSD1. In addition, we propose a higher-order biophysical interaction between GQ TERRA RNA and LSD1, involved in regulating telomere maintenance. The cell localization data and in-vitro fluorescent-labeled studies reveal that TERRA and LSD1 can undergo phase separation. Phase separation appears RNA-structure dependent and suggests a model for how R loop formation and telomere maintenance can be regulated. Lastly, LSD1’s interaction network with anticancer drugs and Ewing-sarcoma oncogenic fusion protein EWS/FLI1 were examined to better understand LSD1’s oncogenic mechanisms. For the first time, we show a direct interaction that occurs between LSD1 and EWS/FLI1 in vitro and in cancer cells. We find that this interaction leads to inhibition of LSD1 catalytic activity on nucleosomes. We examined LSD1’s druggability and demonstrated that Seclidemstat used for treating Ewing-sarcoma is not efficient in inhibiting LSD1’s nucleosomal demethylation in-vitro or in cancer cells highlighting the need for multiple validation experiments in epigenetic pharmacology. Altogether my studies advance an understanding of the wide range of molecular interactions and associated functional roles of LSD1.