STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF LSD1/COREST HISTONE DEMETHYLASE COMPLEX ON THE NUCLEOSOME
Open Access
- Author:
- Kim, Sang-Ah
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 23, 2016
- Committee Members:
- Song Tan, Dissertation Advisor/Co-Advisor
Andrey S. Krasilnikov, Committee Chair/Co-Chair
Lu Bai, Committee Member
David S. Gilmour, Committee Member
Scott Showalter, Outside Member - Keywords:
- nucleosome
chromatin
structural biology
gene regulation
histone demethylase
lsd1 - Abstract:
- Chromatin, a complex of DNA and proteins, is a dynamic structure that can respond to external cues to regulate genes. Posttranslational modification of histones plays a key role in this regulation through modulation of chromatin structure and function. One such modification, methylation of histone H3 lysine 4 (H3K4), has been correlated with transcriptional activation and is enriched at the promoters and enhancers of active genes. Lysine residues can be mono-, di- or trimethylated, and the specific form of methylation is regulated by a synergistic effort of histone methyltransferases and demethylases. The LSD1 (lysine-specific demethylase 1) protein can alone demethylate mono- and dimethylated H3K4 in peptide substrates, but requires the corepressor protein, CoREST, to demethylate nucleosome substrates. The crystal structure of the LSD1/CoREST in complex with the H3-peptide has been solved and a plethora of information exists on how the LSD1/CoREST works on the peptide substrates. However, these studies do not provide insight into how LSD1/CoREST interacts with the nucleosome, the physiological substrate. As such, full elucidation of LSD1/CoREST’s function on the nucleosome is critical to understanding its roles in development, cellular differentiation, embryonic pluripotency, and cancer. In this dissertation, I provide insights into how the LSD1/CoREST complex interacts with the nucleosome via biochemical and structural approaches. My studies of LSD1/CoREST’s enzymatic activity and nucleosome binding show that extranucleosomal DNA dramatically enhances the activity of LSD1/CoREST and that LSD1/CoREST requires DNA beyond the nucleosome core particle for optimal interaction. These results are consistent with the photocrosslinking experiments performed by Nilanjana Chatterjee in collaboration with Blaine Bartholomew’s group, which indicate both LSD1 and CoREST subunits are in close contact with extranucleosomal DNA. I have also determined the molecular replacement solutions of LSD1/CoREST on 161 and 189 bp nucleosome crystal structures at 7.2 Å and 5.3 Å resolution respectively. These studies provide novel insights into how LSD1/CoREST productively engages the nucleosomes with extranucleosomal DNA. Both models suggest that the catalytic domain of LSD1 and the N-terminus of CoREST makes contact with extranucleosomal DNA, while the CoREST SANT2 domain recognizes the nucleosomal disk face. Current work is aimed toward determining the LSD1/CoREST/nucleosome crystal structure at a higher resolution.