Functional Recognition of the Nucleosome by Chromatin Complexes Ran/rcc11 and Lsd1/corest
Open Access
- Author:
- Jennings, Matthew James
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 12, 2104
- Committee Members:
- Song Tan, Dissertation Advisor/Co-Advisor
Benjamin Franklin Pugh, Committee Member
B Tracy Nixon, Committee Member
Tae Hee Lee, Committee Member
Joseph C. Reese, Committee Member - Keywords:
- Nucleosome
RCC1
Ran
LSD1
CoREST
Chromatin - Abstract:
- Chromatin is a combination of protein and nucleic acid whose function includes, but is not limited to, higher order compaction of DNA within eukaryotic cells. The most fundamental subunit of chromatin is the nucleosome. In my studies, I investigate two chromatin complexes, Ran/RCC1 and LSD1/CoREST, to address the fundamental question of how chromatin factors recognize and interact with the nucleosome. Each of these complexes has distinctly different cellular functions ranging from nucleocytoplasmic transport, nuclear envelope formation, and mitotic spindle formation in the case of Ran/RCC1 to transcriptional regulation in the case of LSD1/CoREST. Ran GTPase functions as a molecular switch that is toggled between the active GTP bound and inactive GDP bound states. RCC1 is a chromatin bound, guanine exchange factor for Ran that is responsible for nucleotide exchange from the GDP to GTP states creating a high nuclear concentration of RanGTP. Previous work disclosed that the nucleosome enhances RCC1 mediated nucleotide exchange two-fold. I have identified a hydrophobic face of Ran’s C-terminal α-helix that is required for nucleosomal enhancement of RCC1 mediated nucleotide exchange. The hydrophobic face of Ran’s C-terminal α-helix cross-links to the first 20 amino acids of histone H4 as well as nucleosomal DNA. Given that a nucleosome with deletions of all histone tails still maintained two-fold nucleosomal enhanced Ran nucleotide exchange suggests that enhancement may result from interaction between the Ran C-terminal α-helix and nucleosomal DNA. The LSD1/CoREST (lysine specific demethylase 1/Co-repressor element 1 silencing transcription factor) complex is a histone H3K4 mono- and di- demethylase that is associated with transcriptional repression. Previous work has shown that while LSD1 can demethylate peptide substrates, CoREST is required for LSD1 demethylase activity on the nucleosome. Prior to my work, enzymatic activity of LSD1/CoREST was addressed using colorimetric assays on peptides. I have developed a more sensitive fluorescence based assay using di-methyl lysine analog nucleosomal substrates to investigate LSD1/CoREST demethylase activity on the nucleosome. I have identified that LSD1/CoREST prefers a nucleosomal substrate containing extranucleosomal linker DNA beyond the 147 base pair nucleosome. In addition, symmetrical extensions from both sides of the dyad are preferred over a single extension from one side of the dyad. The results of my work provides further insight into the role of the nucleosome, not just as a structural component that acts to compact the genome, but as a participant in the activity of chromatin factors. My studies expand the chromatin and cellular biology fields by identifying a potential mechanism into how the Ran GTPase interacts with the nucleosome to enhance Ran activity and alter its nucleotide state allowing for the development of the Ran nucleotide gradient responsible for cellular localization of mitotic processes and nucleocytoplasmic transport. My results also demonstrate that LSD1/CoREST activity is enhanced in the presence of extranucleosomal DNA providing a role of extranucleosomal DNA in enzymatic activity.