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Review
. 2012 Jul;1819(7):776-83.
doi: 10.1016/j.bbagrm.2012.02.008. Epub 2012 Feb 16.

Insights into assembly and regulation of centromeric chromatin in Saccharomyces cerevisiae

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Review

Insights into assembly and regulation of centromeric chromatin in Saccharomyces cerevisiae

John S Choy et al. Biochim Biophys Acta. 2012 Jul.

Abstract

At the core of chromosome segregation is the centromere, which nucleates the assembly of a macromolecular kinetochore (centromere DNA and associated proteins) complex responsible for mediating spindle attachment. Recent advances in centromere research have led to identification of many kinetochore components, such as the centromeric-specific histone H3 variant, CenH3, and its interacting partner, Scm3. Both are essential for chromosome segregation and are evolutionarily conserved from yeast to humans. CenH3 is proposed to be the epigenetic mark that specifies centromeric identity. Molecular mechanisms that regulate the assembly of kinetochores at specific chromosomal sites to mediate chromosome segregation are not fully understood. In this review, we summarize the current literature and discuss results from our laboratory, which show that restricting the localization of budding yeast CenH3, Cse4, to centromeres and balanced stoichiometry between Scm3 and Cse4, contribute to faithful chromosome transmission. We highlight our findings that, similar to other eukaryotic centromeres, budding yeast centromeric histone H4 is hypoacetylated, and we discuss how altered histone acetylation affects chromosome segregation. This article is part of a Special Issue entitled: Chromatin in time and space.

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Figures

Fig. 1.
Fig. 1.
Acetylation pattern of centromeric histone H4 affects chromosome segregation. (A) Centromeric histone H4 N-terminal tails are hypoacetylated, which is associated with proper C-loop (light blue nucleosomes to left of Cse4/H4 nucleosome) and kinetochore (KT) assembly to mediate accurate chromosome segregation. Note that the Cse4 nucleosome illustration does not indicate the actual composition of the centromeric nucleosome (refer to the introduction for various models of centromeric nucleosomes). (B) Phenotypes resulting from hyperacetylated centromeric histone H4 at K16. Increased dosage of histone H4K16 acetylation (Ac) by overexpression of Sas2 histone acetyltransferase activity (Sas), growth on nicotinamide (NAM), an inhibitor of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases that act on H4K16, or use of an H4K16Q acetyl-mimic mutant (Q – glutamine) results in chromosome loss that may be caused by disruption of the C-loop and/or improper kinetochore assembly at the centromere (?). (Far right). We use a visual assay to monitor chromosome loss in a strain engineered with a reporter chromosome. Loss of the reporter chromosome results in red sectors in an otherwise white colony. Chromosome loss within the first cell division results in half-sectored red colonies. Refer to Section 2 for details.
Fig. 2.
Fig. 2.
Mechanisms that prevent non-centromeric localization of Cse4 to maintain genome stability. (A) Cse4 can localize to non-centromeric regions; however, mis-localization is suppressed by Psh1, Snf2, Cac1-Hir1, Spt4, and other proteins (?). Psh1 is an E3 ubiquitin ligase that mediates proteolytic degradation of Cse4 and excludes its incorporation into non-centromeric DNA. Centromeric incorporation of Cse4 and its interaction with Scm3 is thought to protect Cse4 from proteolysis by Psh1. Snf2 is proposed to remove Cse4 from non-centromeric regions. Cse4 eviction by Snf2 may or may not be mutually exclusive from known (Psh1) and unknown (?) proteolytic pathways. The centromeric nucleosome is depicted in blue, and the non-centromeric regions of Cse4 are denoted by the cylinders with a dotted outline. Note that the Cse4 nucleosome illustration does not indicate the actual composition of the centromeric nucleosome (refer to the introduction for various models of centromeric nucleosomes). (B) Overexpression of cse4K16R leads to its mis-localization to non-centromeric regions. Scm3-mediated centromeric localization of cse4K16R is inferred from complementation of a CSE4 deletion strain by cse4K16R. Stable non-centromeric incorporation of cse4K16R leads to its enrichment in chromatin, chromosome loss, and increased protein stability. Refer to Section 3 for details.
Fig. 3.
Fig. 3.
Mis-regulation of Scm3 results in increased chromosome loss in budding yeast. Scm3 forms a stoichiometric complex with Cse4/H4 and mediates the assembly of Cse4 at the centromeric DNA. Depicted is the cell cycle of budding yeast with G1, S, mitotic (M), anaphase (A), and telophase (T) cells. (A). Wild-type cells with a balanced stoichiometry of Scm3 with Cse4/H4 contribute to faithful chromosome segregation. The schematic figure depicts the cell cycle following ChIP experiments using epitope-tagged Scm3 expressed from its endogenous promoter. Scm3 is enriched in S phase and anaphase (A) and shows depletion in early mitotic (M) phase cells. The enrichment and depletion are denoted by the thickness of the lines in each phase. (B) Strains overexpressing SCM3 (OE-SCM3) have an excess of Scm3 compared to the available pools of Cse4 and H4. We propose that centromeric association of Scm3 devoid of Cse4/H4 leads to chromosome loss in strains overexpressing SCM3. Schematic of cell cycle as in (A) shows results of ChIP experiments using epitope-tagged overexpressed SCM3, which fails to show the depletion of centromeric Scm3 in early mitotic cells and constitutively associates with centromeres in all phases of the cell cycle. The thickness of the lines in each phase represents the levels of centromeric Scm3. Figure is adapted from our publication [54]. Refer to Section 4 for details.

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