Abstract
The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.
Highlights
All centromeres are characterized by the presence of unusual centromerespecific nucleosomes in which histone H3 is replaced by the histone H3 variant& 2012 The Authors
We confirmed that CENP-ACnp1 tagged with mEos2 at the N-terminus did not lead to slower growth or lower viability when compared with either an untagged cnp1þ wild-type strain or with other similar strains expressing CENP-ACnp1 tagged with green fluorescent protein (GFP)
Chromatin immunoprecipitation (ChIP) showed that similar levels of CENP-ACnp1 are detected at centromeres
Summary
All centromeres are characterized by the presence of unusual centromerespecific nucleosomes in which histone H3 is replaced by the histone H3 variant& 2012 The Authors. Evidence from Drosophila embryos [5], Drosophila S2 cells [6] and human cells [7] shows that CENP-A (Centromere IDentifier, CID, in flies) is not replenished during DNA replication in S phase, but is instead deposited onto centromeric chromatin during anaphase, metaphase and the late stages of mitosis/ early G1, respectively. This suggests that for a significant part of the cell cycle, from S phase through to the end of mitosis, metazoan centromeres contain half the number of CENPA nucleosomes than are present from G1 until centromere replication. In the fission yeast Schizosaccharomyces pombe, deposition of CENPACnp has been reported to occur both in S phase and in G2 before cells undergo mitosis [9,10]
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