The structural analysis of histone H3 lysine 56 acetylation and related histone chaperone complexes

  • Colin Hammond

    Student thesis: Doctoral ThesisDoctor of Philosophy

    Abstract

    Access to the genetic information of eukaryotic organisms is regulated in part by the assembly and disassembly of chromatin. Key regulators of these processes are histone binding proteins known as histone chaperones. The histone chaperone complex Chromatin Assembly Factor 1 (CAF-1) is responsible for depositing H3.1/H4 tetramers in a DNA synthesis dependent manner. During replication independent assembly H3.3/H4 is deposited by Histone Regulator (Hir) complex. In both instances nascent histone H3/H4 dimers are supplied by Anti-Silencing Factor 1 (Asf1).

    In yeast, newly synthesised H3 is acetylated at multiple sites in complex with H4 by the histone acetyl transferase (HAT) Regulator of Ty1 Transposition (Rtt109). The activity of Rtt109 towards H3 tail residues and H3 lysine 56 (K56) is stimulated by the histone chaperones Vacuolar Protein Sorting 75 (Vps75) and Asf1 respectively. In this study, acetylation of K56, located at the C-terminus of the H3 aN helix, was found to promote secondary structure formation in this region by Circular Dichroism. Furthermore, Pulsed Electron Double Resonance experiments demonstrate that the acetylation mimic K56Q stabilises the aN helix in an alternate conformation than observed in the histone octamer. This may have implications in the handover of histones between chaperones or at other stages of de novo nucleosome assembly.

    Vps75 is a member of the Nucleosome Assembly Protein 1 (Nap1) chaperone family. Nap1 family chaperones adopt a characteristic homo-dimeric ‘headphone’ fold and are capable of binding H2A/H2B and H3/H4, the latter in its tetrameric conformation. Experiments described here reveal Vps75 and Nap1 form ring-like tetramers which sequester the highly acidic, putative histone binding, surfaces of the chaperones. Cross-linking analysis shows a H3/H4 tetramer can accommodate two Vps75 dimers. The tetramerisation of Nap1 family chaperones may have an important role in the cooperative assembly or recognition of H3/H4 tetramers allowing the conservation of H3/H4 tetramers evicted from chromatin during nucleosome reassembly. Vps75 and Nap1 were also shown to bind H3/H4 in the presence of Asf1 demonstrating that a Nap1 fold dimer indeed binds a histone fold dimer. These homogeneous complexes are good candidates for future structural analysis of the mode of interaction of Vps75 and Nap1 with H3/H4.
    Date of Award2013
    LanguageEnglish
    Awarding Institution
    • University of Dundee
    SupervisorTom Owen-Hughes (Supervisor)

    Cite this

    The structural analysis of histone H3 lysine 56 acetylation and related histone chaperone complexes
    Hammond, C. (Author). 2013

    Student thesis: Doctoral ThesisDoctor of Philosophy