THE EFFECTS OF INTERNAL HISTONE H3 TRUNCATIONS ON LSD1/COREST DEMETHYLASE ACTIVITY

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Author:
Johnston, James Lakshman
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • Teh-hui Kao, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • James
  • Johnston
  • James Johnston
  • Lakshman
  • LSD1
  • CoREST
  • Histone
  • Chromatin
  • H3
  • Truncations
  • Structure
  • Nucleosome
Abstract:
The LSD1 and CoREST proteins form a complex that specifically demethylates histone H3 at lysines 4 and 9 (D’Oto et al, 2017). Without CoREST, LSD1 cannot act on the nucleosome, though it can act on individual H3 proteins, and several studies have shown that CoREST makes crucial contact with the DNA surrounding the histone octamer (Kim et al, 2015). Our lab is attempting solve the structure of the LSD1/CoREST complex on the nucleosome, both through X-ray crystallography and various enzymatic assays. Previous experiments conducted in our lab demonstrated that 5 and 10 amino acid internal truncations were insufficient to reduce LSD1 activity (Kim et al, 2015). A recent, low-resolution structure of LSD1 on the nucleosome led to the suggestion that our previous structure was inaccurate. Our revised structure suggested an 18 amino acid internal truncation to histone H3 would be sufficient to remove the H3 tail from the LSD1/CoREST active site and eliminate any demethylase activity. To test this hypothesis, I designed histone H3 mutants with 12, 15, 18, and 20 amino acid internal truncations. I incorporated these truncated histone mutants into nucleosomes and was able to demonstrate through a demethylase assay that both 18 and 20 amino acid internal truncations to histone H3 were sufficient to eliminate the activity of LSD1. This finding lends credence to the structure proposed by our laboratory and will help the laboratory continue to fine tune the structure of LSD1 and CoREST on the nucleosome.