The Histone Fold Domain of Cse4 Is Sufficient for
            <i>CEN</i>
            Targeting and Propagation of Active Centromeres in Budding Yeast

Morey, Lisa M.; Barnes, Kelly; Chen, Yinhuai; Fitzgerald-Hayes, Molly; Baker, Richard E.

doi:10.1128/ec.3.6.1533-1543.2004

Cited by 32 publications

(39 citation statements)

References 63 publications

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“…120 min) and similar to 16KR (Figure 1, B and C), showing that K-to-R mutations in the N terminus of Cse4 increase protein stability. Consistent with these results are previous observations showing that Cse4 lacking the N terminus (Δ129) is fivefold more stable than full-length Cse4 (Morey et al 2004). Increased Cse4 stability correlates with chromosome loss, as KC and 16KR strains exhibit fourfold higher chromosome loss than strains expressing CK or wild-type Cse4 ( Figure 1G).…”

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confidence: 79%

A Novel Role of the N Terminus of Budding Yeast Histone H3 Variant Cse4 in Ubiquitin-Mediated Proteolysis

Dawson

Rawson

et al. 2013

Genetics

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Regulating levels of centromeric histone H3 (CenH3) variant is crucial for genome stability. Interaction of Psh1, an E3 ligase, with the C terminus of Cse4 has been shown to contribute to its proteolysis. Here, we demonstrate a role for ubiquitination of the N terminus of Cse4 in regulating Cse4 proteolysis for faithful chromosome segregation and a role for Doa1 in ubiquitination of Cse4.C ENTROMERIC histone H3 (CenH3), an evolutionarily conserved histone H3 variant, is essential for chromosome segregation (Stoler et al. 1995;Meluh et al. 1998;Blower and Karpen 2001;Maddox et al. 2012). Mis-localization and overexpression of CenH3 has been observed in cancers and associated with aneuploidy in Drosophila melanogaster (Tomonaga et al. 2003;Heun et al. 2006;Moreno-Moreno et al. 2006). Studies from budding yeast and fruit flies have shown that proteolysis of CenH3 plays an important role in preventing its mis-localization MorenoMoreno et al. 2006 MorenoMoreno et al. , 2011. Psh1, an E3 ligase, interacts with Cse4, the budding yeast CenH3, and mediates its ubiquitination for proteolysis (Hewawasam et al. 2010;Ranjitkar et al. 2010). Despite ubiquitination of Cse4 by Psh1, Cse4 is only partially stabilized when overexpressed in psh1Δ strains (Hewawasam et al. 2010;Ranjitkar et al. 2010), suggesting that additional factors regulate Cse4 protein stability.In this study, we investigated the role of Cse4 domains in directing Cse4 proteolysis and used a genome-wide screen to identify pathways that regulate Cse4 stability. Since subcellular levels of Cse4 are stringently regulated, strains overexpressing CSE4 or cse4 mutants have been used to identify proteolytic pathways and factors such as Psh1 that mediate its degradation (Hewawasam et al. 2010;Ranjitkar et al. 2010). We initiated our studies with a mutant cse416KR (16KR) and 16KR fusion mutants in which lysines (K) are mutated to arginines (R) (Figure 1, A-C; Supporting Information, Table S1 and File S1). Overexpression of 16KR leads to defects in chromosome segregation, and this correlates with enrichment in chromatin and increased protein stability Au et al. 2008) . Our results show that the stability of CK (fusion of Cse4 N terminus amino acids 1-135 and 16KR C terminus) is low (t 1/2 = 26 6 6 min) and is comparable to that of Cse4 (t 1/2 = 34 6 3 min), indicating that the lack of ubiquitination of the C terminus due to K-to-R mutations does not increase protein stability. In contrast, the stability of KC (a fusion of 16KR N terminus and Cse4 C terminus) is high (t 1/2 . 120 min) and similar to 16KR (Figure 1, B and C), showing that K-to-R mutations in the N terminus of Cse4 increase protein stability. Consistent with these results are previous observations showing that Cse4 lacking the N terminus (Δ129) is fivefold more stable than full-length Cse4 (Morey et al. 2004). Increased Cse4 stability correlates with chromosome loss, as KC and 16KR strains exhibit fourfold higher chromosome loss than strains expressing CK or wild-type Cse4 ( Figure 1G). Based on ...

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confidence: 79%

A Novel Role of the N Terminus of Budding Yeast Histone H3 Variant Cse4 in Ubiquitin-Mediated Proteolysis

Dawson

Rawson

et al. 2013

Genetics

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“…SCM3 is a dosage suppressor of Ts cse4 mutations, and affinity purification and two-hybrid assays demonstrate that Scm3 and Cse4 interact in vivo. The Cse4-Scm3 interaction occurs via the Cse4 HFD, and SCM3 dosage suppression is specific for cse4 HFD alleles; it is the Cse4 HFD where CEN targeting information resides (25). ChIP assays show that Scm3 and Cse4 are colocalized at CEN DNA.…”

Section: Discussionmentioning

confidence: 99%

Scm3, an essential Saccharomyces cerevisiae centromere protein required for G ₂ /M progression and Cse4 localization

Stoler

Rogers

Weitze

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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A universal mark of centromeric chromatin is its packaging by a variant of histone H3 known as centromeric H3 (CenH3). The mechanism by which CenH3s are incorporated specifically into centromere DNA or the specialized function they serve there is not known. In a genetic approach to identify factors involved in CenH3 deposition, we screened for dosage suppressors of a temperaturesensitive cse4 allele in Saccharomyces cerevisiae (Cse4 is the S. cerevisiae CenH3). Independent screens yielded ORF YDL139C, which we named SCM3. Dosage suppression by SCM3 was specific for alleles affecting the histone fold domain of Cse4. Copurification and two-hybrid studies showed that Scm3 and Cse4 interact in vivo, and chromatin immunoprecipitation revealed that Scm3, like Cse4, is found associated with centromere DNA. Scm3 contains two essential protein domains, a Leu-rich nuclear export signal and a heptad repeat domain that is widely conserved in fungi. A conditional scm3 allele was generated to allow us to deplete Scm3. Upon Scm3 depletion, cells undergo a Mad2-dependent G 2/M arrest, and centromere localization of Cse4 is perturbed. We suggest that S. cerevisiae Scm3 defines a previously undescribed family of fungal kinetochore proteins important for CenH3 localization.centromeric H3 ͉ chromosome segregation ͉ kinetochore ͉ yeast ͉ CENP-A

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“…CSE4 was cloned from À575 to 1059 (relative to ATG ¼ 11) into the high-copy 2-mm plasmid YEp351, using the endogenous HindIII site at À575 and a BamHI site added to a PCR primer. We constructed cse4-tailswap and cse4D129 (Morey et al 2004) constructs using the endogenous CSE4 promoter (À575 to 0) by overlapping PCR and cloned them into YEp351 from HindIII to BamHI. These plasmids were transformed into cse4DTkanMX4/CSE4 diploid strains, which were then sporulated.…”

Section: Methodsmentioning

confidence: 99%

“…In S. cerevisiae Cse4p, amino acid residues required for normal function are distributed throughout the histone-fold domain (Keith et al 1999). The N-terminal tail of Cse4p contains an essential region termed the END domain, but overexpression of a Cse4p lacking the tail altogether can rescue a cse4 deletion mutant (Chen et al 2000;Morey et al 2004). In Drosophila melanogaster cells, CENH3/Cid from the distantly related D. bipectinata did not localize to kinetochores unless a specific region of the histone-fold domain, loop 1, was swapped with the corresponding region from D. melanogaster CENH3/ Cid (Vermaak et al 2002).…”

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The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements inArabidopsis thaliana

Kwong²,

et al. 2010

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Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain-CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3's lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP-CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP-CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.

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The Histone Fold Domain of Cse4 Is Sufficient for CEN Targeting and Propagation of Active Centromeres in Budding Yeast

Abstract: Centromere-specific H3-like proteins (CenH3s) are conserved across the eukaryotic kingdom and are required for packaging centromere DNA into a specialized chromatin structure required for kinetochore assembly.

Cited by 32 publications

References 63 publications

A Novel Role of the N Terminus of Budding Yeast Histone H3 Variant Cse4 in Ubiquitin-Mediated Proteolysis

A Novel Role of the N Terminus of Budding Yeast Histone H3 Variant Cse4 in Ubiquitin-Mediated Proteolysis

Scm3, an essential Saccharomyces cerevisiae centromere protein required for G ₂ /M progression and Cse4 localization

The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements inArabidopsis thaliana

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The Histone Fold Domain of Cse4 Is Sufficient for CEN Targeting and Propagation of Active Centromeres in Budding Yeast

Abstract: Centromere-specific H3-like proteins (CenH3s) are conserved across the eukaryotic kingdom and are required for packaging centromere DNA into a specialized chromatin structure required for kinetochore assembly.

Cited by 32 publications

References 63 publications

A Novel Role of the N Terminus of Budding Yeast Histone H3 Variant Cse4 in Ubiquitin-Mediated Proteolysis

A Novel Role of the N Terminus of Budding Yeast Histone H3 Variant Cse4 in Ubiquitin-Mediated Proteolysis

Scm3, an essential Saccharomyces cerevisiae centromere protein required for G 2 /M progression and Cse4 localization

The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements inArabidopsis thaliana

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Scm3, an essential Saccharomyces cerevisiae centromere protein required for G ₂ /M progression and Cse4 localization