Histones are a major protein component of chromatin, yet mechanisms which control synthesis, post translational modification, deposition and removal of histones are not fully understood. Although atomic resolution structures have been solved for the nucleosome core particle, there is limited information regarding the conformation of the core histones outside of chromatin. Insight into the structure of soluble histones, and the complexes they form, is likely to further our understanding of important nuclear processes such as transcription, chromatin replication and epigenetic inheritance. In this study we employ novel biochemical and biophysical techniques to address two key questions in the field: the structure of the soluble (H3-H4)2-tetramer, and the conformation of H3 and H4 in complex with histone chaperones.Firstly, we determined the conformation of histones H3 and H4 when in complex with two histone chaperones from S. cerevisaie, Nap1 and Vps75. Within the nucleosome H3 and H4 form a heterotetrameric structure sustained by the interface between two histone H3 proteins. Interestingly, when bound to the histone chaperone Asf1 the H3-H3’ interaction is disrupted, thus Asf1 effectively splits the tetramer binding a single H3-H4 dimer. Using targeted protein crosslinking and pulsed EPR we determine that, unlike Asf1, the Nap1 family of histone chaperones can bind H3-H4 in their tetrameric conformation, analogous to that observed within the nucleosome. The ability to bind H3 and H4 as a tetramer has implications in the prevalence of chromatin states during DNA replication and transcription, and may be in part responsible for the alternate in vivo functions of these two classes of chaperones.Secondly, using site direct spin labelling in conjunction with pulsed EPR we probe in detail the structure of the soluble (H3-H4)2-tetramer. Whilst the core crescent shape of the tetramer surrounding the H3-H3’ interface is retained, discrete regions such as the aN helix of H3 are more structurally heterogeneous than in the histone octamer or nucleosome. Such structural heterogeneity in the aN helix of H3 highlights potential roles in the post translational modification of histones and in their binding to histone-chaperones. These new findings reveal possible modes of interaction between a tetramer of H3-H4 and Nap1 proteins, and highlight the need for further investigation into histone – chaperone complexes.
|Date of Award||2010|
|Supervisor||Tom Owen-Hughes (Supervisor)|