Reduced gene dosage of histone H4 prevents CENP-A mislocalization and chromosomal instability in Saccharomyces cerevisiae

Mislocalization of the centromeric histone H3 variant (Cse4 in budding yeast, CID in flies, CENP-A in humans) to noncentromeric regions contributes to chromosomal instability (CIN) in yeast, fly, and human cells. Overexpression and mislocalization of CENP-A have been observed in cancers, however, the mechanisms that facilitate the mislocalization of overexpressed CENP-A have not been fully explored. Defects in proteolysis of overexpressed Cse4 (GALCSE4) lead to its mislocalization and synthetic dosage lethality (SDL) in mutants for E3 ubiquitin ligases (Psh1, Slx5, SCF^Met30, and SCF^Cdc4), Doa1, Hir2, and Cdc7. In contrast, defects in sumoylation of overexpressed cse4K215/216/A/R prevent its mislocalization and do not cause SDL in a psh1Δ strain. Here, we used a genome-wide screen to identify factors that facilitate the mislocalization of overexpressed Cse4 by characterizing suppressors of the psh1Δ GALCSE4 SDL. Deletions of histone H4 alleles (HHF1 or HHF2), which were among the most prominent suppressors, also suppress slx5Δ, cdc4-1, doa1Δ, hir2Δ, and cdc7-4 GALCSE4 SDL. Reduced dosage of H4 leads to defects in sumoylation and reduced mislocalization of overexpressed Cse4, which contributes to suppression of CIN when Cse4 is overexpressed. We determined that the hhf1-20, cse4-102, and cse4-111 mutants, which are defective in the Cse4-H4 interaction, also exhibit reduced sumoylation of Cse4 and do not display psh1Δ GALCSE4 SDL. In summary, we have identified genes that contribute to the mislocalization of overexpressed Cse4 and defined a role for the gene dosage of H4 in facilitating Cse4 sumoylation and mislocalization to noncentromeric regions, leading to CIN when Cse4 is overexpressed.     

An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain

Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 CATD is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation.     

Phosphorylation by Casein Kinase 2 Facilitates Psh1 Protein-assisted Degradation of Cse4 Protein

Cse4 is the centromeric histone H3 variant in budding yeast. Psh1 is an E3 ubiquitin ligase that controls Cse4 levels through proteolysis. Here we report that Psh1 is phosphorylated by the Cka2 subunit of casein kinase 2 (CK2) to promote its E3 activity for Cse4. Deletion of CKA2 significantly stabilized Cse4. Consistent with phosphorylation promoting the activity of Psh1, Cse4 was stabilized in a Psh1 phosphodepleted mutant strain in which the major phosphorylation sites were changed to alanines. Phosphorylation of Psh1 did not control Psh1-Cse4 or Psh1-Ubc3(E2) interactions. Although Cse4 was highly stabilized in a cka2Δ strain, mislocalization of Cse4 was mild, suggesting that Cse4 misincorporation was prevented by the intact Psh1-Cse4 association. Supporting this idea, Psh1 was also stabilized in a cka2Δ strain. Collectively our data suggest that phosphorylation is crucial in Psh1-assisted control of Cse4 levels and that the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation.     

Cdc48Ufd1/Npl4 segregase removes mislocalized centromeric histone H3 variant CENP-A from non-centromeric chromatin

Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48^Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48^Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.     

A SUMO-targeted ubiquitin ligase is involved in the degradation of the nuclear pool of the SUMO E3 ligase Siz1

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5∆ cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase.     

Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination

Evolutionarily conserved histone H3 variant Cse4 and its homologues are essential components of specialized centromere (CEN)-specific nucleosomes and serve as an epigenetic mark for CEN identity and propagation. Cse4 is a critical determinant for the structure and function of the kinetochore and is required to ensure faithful chromosome segregation. The kinetochore protein Pat1 regulates the levels and spatial distribution of Cse4 at centromeres. Deletion of PAT1 results in altered structure of CEN chromatin and chromosome segregation errors. In this study, we show that Pat1 protects CEN-associated Cse4 from ubiquitination in order to maintain proper structure and function of the kinetochore in budding yeast. PAT1-deletion strains exhibit increased ubiquitination of Cse4 and faster turnover of Cse4 at kinetochores. Psh1, a Cse4-specific E3-ubiquitin ligase, interacts with Pat1 in vivo and contributes to the increased ubiquitination of Cse4 in pat1∆ strains. Consistent with a role of Psh1 in ubiquitination of Cse4, transient induction of PSH1 in a wild-type strain resulted in phenotypes similar to a pat1∆ strain, including a reduction in CEN-associated Cse4, increased Cse4 ubiquitination, defects in spatial distribution of Cse4 at kinetochores, and altered structure of CEN chromatin. Pat1 interacts with Scm3 and is required for its maintenance at kinetochores. In conclusion, our studies provide novel insights into mechanisms by which Pat1 affects the structure of CEN chromatin and protects Cse4 from Psh1-mediated ubiquitination for faithful chromosome segregation.     

Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4

Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.     

Distinct SUMO Ligases Cooperate with Esc2 and Slx5 to Suppress Duplication-Mediated Genome Rearrangements

Suppression of duplication-mediated gross chromosomal rearrangements (GCRs) is essential to maintain genome integrity in eukaryotes. Here we report that SUMO ligase Mms21 has a strong role in suppressing GCRs in Saccharomyces cerevisiae, while Siz1 and Siz2 have weaker and partially redundant roles. Understanding the functions of these enzymes has been hampered by a paucity of knowledge of their substrate specificity in vivo. Using a new quantitative SUMO-proteomics technology, we found that Siz1 and Siz2 redundantly control the abundances of most sumoylated substrates, while Mms21 more specifically regulates sumoylation of RNA polymerase-I and the SMC-family proteins. Interestingly, Esc2, a SUMO-like domain-containing protein, specifically promotes the accumulation of sumoylated Mms21-specific substrates and functions with Mms21 to suppress GCRs. On the other hand, the Slx5-Slx8 complex, a SUMO-targeted ubiquitin ligase, suppresses the accumulation of sumoylated Mms21-specific substrates. Thus, distinct SUMO ligases work in concert with Esc2 and Slx5-Slx8 to control substrate specificity and sumoylation homeostasis to prevent GCRs.     

Ectopic Centromere Nucleation by CENP-A in Fission Yeast

The centromere is a specific chromosomal locus that organizes the assembly of the kinetochore. It plays a fundamental role in accurate chromosome segregation. In most eukaryotic organisms, each chromosome contains a single centromere the position and function of which are epigenetically specified. Occasionally, centromeres form at ectopic loci, which can be detrimental to the cell. However, the mechanisms that protect the cell against ectopic centromeres (neocentromeres) remain poorly understood. Centromere protein-A (CENP-A), a centromere-specific histone 3 (H3) variant, is found in all centromeres and is indispensable for centromere function. Here we report that the overexpression of CENP-A^Cnp1 in fission yeast results in the assembly of CENP-A^Cnp1 at noncentromeric chromatin during mitosis and meiosis. The noncentromeric CENP-A preferentially assembles near heterochromatin and is capable of recruiting kinetochore components. Consistent with this, cells overexpressing CENP-A^Cnp1 exhibit severe chromosome missegregation and spindle microtubule disorganization. In addition, pulse induction of CENP-A^Cnp1 overexpression reveals that ectopic CENP-A chromatin can persist for multiple generations. Intriguingly, ectopic assembly of CENP-A^cnp1 is suppressed by overexpression of histone H3 or H4. Finally, we demonstrate that deletion of the N-terminal domain of CENP-A^cnp1 results in an increase in the number of ectopic CENP-A sites and provide evidence that the N-terminal domain of CENP-A prevents CENP-A assembly at ectopic loci via the ubiquitin-dependent proteolysis. These studies expand our current understanding of how noncentromeric chromatin is protected from mistakenly assembling CENP-A.     

R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast

R-loops, the byproduct of DNA–RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric (CEN) chromatin in wild-type budding yeast. Here we used an hpr1∆ strain that accumulates R-loops to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with the CEN-histone H3 variant, Cse4, and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA–RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at CEN chromatin that was reduced by overexpression of RNH1 in hpr1∆ strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at CEN chromatin were observed in hpr1∆ strains. We determined that accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by RNH1 overexpression in hpr1∆ strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to defects in kinetochore integrity and CIN.     

Budding yeast CENP-ACse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

Shugoshin is an evolutionarily conserved protein, which is involved in tension sensing on mitotic chromosomes, kinetochore biorientation, and protection of centromeric (CEN) cohesin for faithful chromosome segregation. Interaction of the C-terminus of Sgo1 with phosphorylated histone H2A regulates its association with CEN and pericentromeric (peri-CEN) chromatin, whereas mutations in histone H3 selectively compromise the association of Sgo1 with peri-CEN but not CEN chromatin. Given that histone H3 is absent from CEN and is replaced by a histone H3 variant CENP-A^Cse4, we investigated if CENP-A^Cse4 interacts with Sgo1 and promotes its association with the CEN chromatin. In this study, we found that Sgo1 interacts with CENP-A^Cse4 in vivo and in vitro. The N-terminus coiled-coil domain of Sgo1 without the C-terminus (sgo1-NT) is sufficient for its interaction with CENP-A^Cse4, association with CEN but not the peri-CEN, and this CEN association is cell cycle dependent with maximum enrichment in mitosis. In agreement with the role of CENP-A^Cse4 in CEN maintenance of Sgo1, depletion of CENP-A^Cse4 results in the loss of Sgo1 and sgo1-NT from the CEN chromatin. The N-terminus of Sgo1 is required for genome stability as a mutant lacking the N-terminus (sgo1-CT) exhibits increased chromosome missegregation when compared to a sgo1-NT mutant. In summary, our results define a novel role for the N-terminus of Sgo1 in CENP-A^Cse4 mediated recruitment of Sgo1 to CEN chromatin for faithful chromosome segregation.     

Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5′ strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.     

Plasticity of fission yeast CENP-A chromatin driven by relative levels of histone H3 and H4

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-A^Cnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A^Cnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A^Cnp1 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-A^Cnp1 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-A^Cnp1 for assembly into central domain chromatin, resulting in less CENP-A^Cnp1 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-A^Cnp1 influence the extent of DNA at centromeres that is packaged in CENP-A^Cnp1 chromatin and the composition of this chromatin. Thus, CENP-A^Cnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A^Cnp1 and other core histones.     

Plasma membrane aminoglycerolipid flippase function is required for signaling competence in the yeast mating pheromone response pathway

The class 4 P-type ATPases (“flippases”) maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MATa cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection (“shmoo”) tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MATa dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2⋅Cdc28-initiated degradation. Similarly, a MATa dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus.     

CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore–microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A–overexpressing cancers.     

The SUMO-targeted ubiquitin ligase subunit Slx5 resides in nuclear foci and at sites of DNA breaks

The Slx5/Slx8 protein complex, a heterodimeric SUMO-targeted ubiquitin ligase, plays an important role in genomic integrity. Slx5/Slx8 is believed to interact with sumoylated proteins that reside in the nuclei of budding yeast cells. In this complex, Slx5, owing to at least two SUMO interacting motifs (SIMs), has been proposed to be the targeting subunit of the Slx8 ubiquitin ligase. However, little is known about the exact subnuclear localization and targets of Slx5/Slx8. In this study we show that Slx5, but not Slx8, forms prominent nuclear foci. The formation of these foci depends on SUMO and a SIM in Slx5. Therefore, we investigated the subnuclear localization and potential chromatin association of Slx5. Using co-localization studies in live cells and fixed chromatin, we were able to localize Slx5 to DNA damage induced foci of Rad52 and Rad9, two proteins involved in the cellular response to DNA damage. Subsequent chromatin immunoprecipitation (ChIP) studies revealed that Slx5 is associated with HO endonuclease induced chromosome breaks. Surprisingly, real-time PCR analysis of Slx5 ChIPs revealed that the level of Slx5 at HO breaks in an slx8 deletion background is reduced about 4-fold. These results indicate that the DNA-damage targeting of Slx5/Slx8 depends on formation of the heterodimer and that this occurs at a subset of nuclear foci also containing DNA damage repair and checkpoint factors.