Plasticity of Fission Yeast CENP-A Chromatin Driven by Relative Levels of Histone H3 and H4

Araceli G Castillo,Alison L Pidoux,Georgina L Hamilton,Barbara G Mellone,Robin C Allshire,William Richardson,Janet F Partridge,Asifa Akhtar

doi:10.1371/journal.pgen.0030121

Araceli G Castillo, Alison L Pidoux + Show 6 more

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https://doi.org/10.1371/journal.pgen.0030121

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Abstract

The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-ACnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-ACnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-ACnp1 chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-ACnp1 associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-ACnp1 for assembly into central domain chromatin, resulting in less CENP-ACnp1 and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-ACnp1 influence the extent of DNA at centromeres that is packaged in CENP-ACnp1 chromatin and the composition of this chromatin. Thus, CENP-ACnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-ACnp1 and other core histones.

Highlights

In most eukaryotes, chromosomes contain a centromere that occupies a single locus
The central domain of centromere 1 is composed of the cnt1 element, a portion of which is shared with cen3, and the cen1-specific imr1 repeats that are virtually identical in sequence on the left and right sides [23]
We have found that CENP-ACnp1 in fission yeast can associate with noncentromeric sequences provided that they are placed in an environment with the required contextual cues for CENP-ACnp1 chromatin assembly

Summary

Introduction

Chromosomes contain a centromere that occupies a single locus. The centromere acts as the site for assembly of the kinetochore that mediates the attachment of chromosomes to spindle microtubules and orchestrates their equational segregation to daughter nuclei at mitosis. Long tandem arrays of repetitive satellite DNA, such as alpha-satellite DNA in humans, are found at each centromere [1,2]. Chromosomal DNA is packaged in chromatin composed of nucleosomes containing the four core histones H2A, H2B, H3, and H4. Histone variants can play specific roles in the regulation of gene expression. Kinetochores contain a specific form of chromatin in which canonical histone H3 is replaced by the centromere-specific histone H3 variant known generally as CENP-A [1,2,6,7]

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