Characterisation of CenH3 nucleosomes
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Date
29/06/2013Author
Miell, Matthew Daniel David
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Abstract
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.