As a centromere-specific protein complex in direct contact with the DNA, CenH3-containing nucleosomes are generally thought to act as the distinguishing epigenetic mark of active centromere location. Confusingly, seemingly disparate models have been proposed for the structure of CenH3 nucleosomes. The most widely supported model is an octameric structure that, like histone H3 nucleosomes, contains two subunits of each histone. Another more contentious, yet persistent model is the hemisome model proposed for fly and human CenH3 nucleosomes. In this case it is suggested that CenH3 nucleosomes contain only single subunit of each histone. One reason for this lack of consensus is that seemingly contradicting models are often proposed, even with material from the same organism, with little overlap in experimental approaches. For example, the proposed hemisome model for fly and human CenH3 nucleosomes is predominantly based on atomic force microscopy (AFM) imaging where the height of nucleosomes on a surface is measured. These AFM measurements are the main data used by protagonists for the hemisome model. However, data supporting an octameric model for human, and other, CenH3 nucleosomes is largely based on biochemical analysis of nucleosomes prepared in vitro, with little cross-over in the methodology used to generate data to support either model. In order to reach a consensus the same analyses needs to be applied to CenH3 nucleosomes assembled in vitro or extracted from cells. Here, recombinant Schizosaccharomyces pombe CENP-ACnp1 and H3 histones expressed and purified from E. coli have been assembled into nucleosomes. To our knowledge this is the first time that recombinant S. pombe nucleosomes have been produced, allowing the stoichiometry and composition of these nucleosomes to be examined in detail by a variety of biochemical and biophysical assays. The application of AFM has enabled the height of these recombinant nucleosomes to be measured and tests the ability of AFM to infer stoichiometry using defined material. The intriguing conclusion is that octameric CenH3 nucleosomes uniquely behave as tetrameric “hemisomes” as defined by AFM. In recent years the contribution of DNA sequence to directing H3 nucleosome location has received a great deal of interest. Since CENP-ACnp1 nucleosomes wrap DNA differently to H3 nucleosomes their preference for sequences that produce a stable nucleosome is expected to be altered. The development of protocols to assemble recombinant CENP-ACnp1 nucleosomes in vitro has also been used here to assess the contribution of primary DNA sequence to CENP-ACnp1 nucleosome positioning. CENP-ACnp1 and H3 nucleosomes were reconstituted on genomic DNA at low density and the resulting nucleosomal DNA from CENP-ACnp1 and H3 particles compared by Illumina sequencing. The stability of CENP-ACnp1 and H3 nucleosomes on specific ‘H3’ and ‘CENP-ACnp1’ sequences was cross-checked. Comparing these data with in vivo CENP-ACnp1 nucleosome positions has allowed the contribution of primary DNA sequence to CENP-ACnp1 nucleosome positioning to be explored.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:586354 |
| Date | January 2013 |
| Creators | Miell, Matthew Daniel David |
| Contributors | Allshire, Robin; Allan, Jim |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/8010 |
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