Functional recognition of the nucleosome by the chromatin factors Piccolo NuA4 and RCC1/Ran

Open Access
Author:
Huang, Jiehuan
Graduate Program:
Biochemistry and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 21, 2012
Committee Members:
  • Song Tan, Dissertation Advisor
  • David Scott Gilmour, Committee Member
  • B Tracy Nixon, Committee Member
  • Joseph C. Reese, Committee Member
  • Philip C. Bevilacqua, Committee Member
Keywords:
  • nucleosome
  • histone acetyltransferase
  • UV crosslinking
  • chromatin
Abstract:
In this study, I investigated how two chromatin factors, Piccolo NuA4 and RCC1/Ran, recognize the nucleosome. Piccolo NuA4 histone acetyltransferase (HAT) complex acetylates histone H4 and H2A, and controls the global level of histone H4 acetylation in the cell. Piccolo NuA4 contains three subunits: Esa1, Epl1 and Yng2. I identified two key regions in two separate subunits of Piccolo NuA4 that are critical for nucleosomal HAT activity. The Esa1 Tudor domain was previously shown to be important for the nucleosomal acetyltransferase activity of Piccolo NuA4. I identified that residues H55, N60, R62 and D64 within a loop region of the Esa1 Tudor domain are critical for Piccolo NuA4’s nucleosomal HAT activity. The results of pull-down and site-directed UV-crosslinking experiments indicate that this loop region contributes to the binding between Piccolo NuA4 and the nucleosome via interaction with nucleosomal DNA. In vivo studies in yeast show that the point mutations in this loop region affect cell viability and global histone H4 acetylation. Moreover, the Esa1 D64A mutation reduces histone H4 acetylation, recruitment of the HAT complexes to chromatin, and transcription. The N-terminal region of the EPcA domain in Epl1 was shown to be important for Piccolo NuA4 binding to the nucleosome. I further identified that residues R58 and K59 in the N-terminal region of EPcA domain are critical for Piccolo NuA4’s nucleosomal HAT activity. I employed a label transfer assay to determine that the N-terminal region of the EPcA domain is in close proximity to nucleosomal DNA and the histone H2A N-terminal tail. I also investigated how the RCC1/Ran protein complex interacts with the nucleosome. The Ran GTPase regulates important cellular functions by creating a Ran-GTP gradient around the chromosome. This gradient is established through stimulation of Ran nucleotide exchange by the guanine exchange factor RCC1 (regulator of chromosome condensation 1). I developed a competition pull-down binding assay to reveal that the nucleosomal binding site of RCC1 included the histone H2A/H2B acidic patch and overlapped with the binding site of the LANA (latency-associated nuclear antigen) peptide from Kaposi’s sarcoma herpesvirus. I initiated work to solve the structure of Ran/RCC1/nucleosome triple complex by X-ray crystallography. I was able to reconstitute and purify several orthologous Ran/RCC1 complexes for reconstitution with the nucleosome. I have grown crystals of several of these triple complexes, and crystals of at least one complex produce medium resolution diffraction.