Biphasic Incorporation of Centromeric Histone CENP-A in Fission Yeast

Abstract

CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In ⌬ams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in ⌬ams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in ⌬ams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged. INTRODUCTIONThe kinetochore is a multiprotein-DNA complex that is indispensable for chromosome segregation and that normally forms on a single chromosomal locus, the centromere (Cleveland et al., 2003). Lack of a kinetochore or formation of multiple kinetochores on a chromosome may have deleterious effects on mitosis (Karpen and Allshire, 1997;Amor and Choo, 2002;Henikoff and Dalal, 2005). CENP-A represents the most likely candidate for the epigenetic "mark" responsible for the maintenance of centromere identity (Black et al., 2004. Because reformation of CENP-A-containing nucleosomes after DNA synthesis is thought to be a prerequisite for mitotic kinetochore assembly, precise targeting of CENP-A into a single, restricted locus on each chromosome before cell division is essential for cell viability . At least three components that affect CENP-A localization, the Mis16 -Mis18 complex (Hayashi et al., 2004;Fujita et al., 2007), the Mis6 -Sim4 complex (Takahashi et al., 2000;Pidoux et al., 2003), and Ams2 GATA-type transcription factor (Chen et al., 2003a), have been identified in the fission yeast Schizosaccharomyces pombe, which is an ideal model organism in which to study complex centromere structure and function (Takahashi et al., 1992;Karpen and Allshire, 1997).Which phase of the cell cycle is used for CENP-A incorporation remains controversial. During S phase, canonical core histones have been suggested to be deposited into duplicated DNAs in a semiconservative manner (Tagami et al., 2004;Natsume et al., 2007). Experiments using fluorescence recovery after photobleaching demonstrated that CENP-A of the budding yeast Saccharomyces cerevisiae is recruited to centromeres coincident with DNA synthesis (Pearson et al., 2004), presumably reflecting disassembly and reassembly of cent...

An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast

“…62 The Δmad2, slp1-mr63, mts3-1, GFP-cnp1, nda3-KM311, cut9-665, slp1-362, his5-303 and lys1-131 are our laboratory stock. 34,42,[49][50][51]63,64 The mutant ubc4-P61S was a generous gift from Dr Hiroaki Seino. 37 Yeast transformations were performed by lithium acetate methods.…”

Temporal control of epigenetic centromere specification

“…In S. pombe, using a temperature sensitive allele of a fluorescently tagged version of CENP-A Cnp1 , it was shown that CENP-A Cnp1 re-incorporation following a shift to the permissive temperature occurs during two discrete cell cycle phases: early S and, to a lesser extent, G2 phase (Takayama et al 2008). While interpretation of experiments based on such ts-alleles is difficult, they suggest that CENP-A is assembled primarily upon mitotic exit (that in fission yeast is immediately followed by S phase), consistent with observations in human cells.…”

“…Limiting CENP-A assembly to outside S phase can therefore help restrict it to the centromere. In fission yeast, CENP-A assembly occurs in early S phase (Takayama et al 2008). However, CENP-A gene synthesis precedes bulk H3 synthesis and centromeres are early replicating, potentially facilitating centromere specific deposition of CENP-A even in a system where assembly is overlapping with S phase (Castillo et al 2007).…”

Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast

“…Two independent investigations have led to somewhat different conclusions for CENP-A/ Cnp1 deposition in fission yeast. One suggests that two redundant pathways operate in S or G 2 phase, respectively (24), whereas a recent study concludes that Cnp1 is replenished exclusively in G 2 phase (25). Overall, a common feature seen in various species is that CENP-A replenishment in many cases may occur without a tight junction with DNA replication (26).…”