scholarly journals The ctf13-30/CTF13 Genomic Haploinsufficiency Modifier Screen Identifies the Yeast Chromatin Remodeling Complex RSC, Which Is Required for the Establishment of Sister Chromatid Cohesion

2004 ◽  
Vol 24 (3) ◽  
pp. 1232-1244 ◽  
Author(s):  
Kristin K. Baetz ◽  
Nevan J. Krogan ◽  
Andrew Emili ◽  
Jack Greenblatt ◽  
Philip Hieter
Keyword(s):  
Chromatin Remodeling ◽  
Chromosome Segregation ◽  
High Fidelity ◽  
Chromosome Transmission ◽  
New Genes ◽  
Sister Chromatids ◽  
Chromatin Remodeling Complex ◽  
Chromatid Cohesion ◽  
Spindle Microtubules ◽  
Mitosis And Meiosis

ABSTRACT The budding yeast centromere-kinetochore complex ensures high-fidelity chromosome segregation in mitosis and meiosis by mediating the attachment and movement of chromosomes along spindle microtubules. To identify new genes and pathways whose function impinges on chromosome transmission, we developed a genomic haploinsufficiency modifier screen and used ctf13-30, encoding a mutant core kinetochore protein, as the reference point. We demonstrate through a series of secondary screens that the genomic modifier screen is a successful method for identifying genes that encode nonessential proteins required for the fidelity of chromosome segregation. One gene isolated in our screen was RSC2, a nonessential subunit of the RSC chromatin remodeling complex. rsc2 mutants have defects in both chromosome segregation and cohesion, but the localization of kinetochore proteins to centromeres is not affected. We determined that, in the absence of RSC2, cohesin could still associate with chromosomes but fails to achieve proper cohesion between sister chromatids, indicating that RSC has a role in the establishment of cohesion. In addition, numerous subunits of RSC were affinity purified and a new component of RSC, Rtt102, was identified. Our work indicates that only a subset of the nonessential RSC subunits function in maintaining chromosome transmission fidelity.

scholarly journals The Yeast RSC Chromatin-Remodeling Complex Is Required for Kinetochore Function in Chromosome Segregation

2003 ◽  
Vol 23 (9) ◽  
pp. 3202-3215 ◽  
Author(s):  
Jing-mei Hsu ◽  
Jian Huang ◽  
Pamela B. Meluh ◽  
Brehon C. Laurent
Keyword(s):  
Chromatin Remodeling ◽  
Centromeric Chromatin ◽  
Centromeric Dna ◽  
Chromosome Transmission ◽  
Sister Chromatids ◽  
Chromatin Remodeling Complex ◽  
Histone H3 Variant ◽  
Complex Protein ◽  
Genes Encoding ◽  
Kinetochore Function

ABSTRACT The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus. This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission. Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA. RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin. Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p. Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes. Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission.

scholarly journals Meiotic Cohesion Requires Accumulation of ORD on Chromosomes before Condensation

2002 ◽  
Vol 13 (11) ◽  
pp. 3890-3900 ◽  
Author(s):  
Eric M. Balicky ◽  
Matthew W. Endres ◽  
Cary Lai ◽  
Sharon E. Bickel
Keyword(s):  
Chromosome Segregation ◽  
Wild Type ◽  
Chromatin Compaction ◽  
Meiotic Chromosomes ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Meiotic Nondisjunction ◽  
Males And Females ◽  
Mitosis And Meiosis

Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic nondisjunction in males and females because cohesion is absent by the time that sister kinetochores make stable microtubule attachments. We provide evidence that ORD is concentrated within the extrachromosomal domains of the nuclei ofDrosophila primary spermatocytes during early G2, but accumulates on the meiotic chromosomes by mid to late G2. Moreover, using fluorescence in situ hybridization to monitor cohesion directly, we show that cohesion defects first become detectable inord null spermatocytes shortly after the time when wild-type ORD associates with the chromosomes. After condensation, ORD remains bound at the centromeres of wild-type spermatocytes and persists there until centromeric cohesion is released during anaphase II. Our results suggest that association of ORD with meiotic chromosomes during mid to late G2 is required to maintain sister-chromatid cohesion during prophase condensation and that retention of ORD at the centromeres after condensation ensures the maintenance of centromeric cohesion until anaphase II.

scholarly journals A novel mechanism for the establishment of sister chromatid cohesion by the ECO1 acetyltransferase

2015 ◽  
Vol 26 (1) ◽  
pp. 117-133 ◽  
Author(s):  
Vincent Guacci ◽  
Jeremiah Stricklin ◽  
Michelle S. Bloom ◽  
Xuánzōng Guō ◽  
Meghna Bhatter ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Current Model ◽  
S Phase ◽  
High Fidelity ◽  
In Vivo Assays ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Cohesin Subunit ◽  
Mutant Cells

Cohesin complex mediates cohesion between sister chromatids, which promotes high-fidelity chromosome segregation. Eco1p acetylates the cohesin subunit Smc3p during S phase to establish cohesion. The current model posits that this Eco1p-mediated acetylation promotes establishment by abrogating the ability of Wpl1p to destabilize cohesin binding to chromosomes. Here we present data from budding yeast that is incompatible with this Wpl1p-centric model. Two independent in vivo assays show that a wpl1∆ fails to suppress cohesion defects of eco1∆ cells. Moreover, a wpl1∆ also fails to suppress cohesion defects engendered by blocking just the essential Eco1p acetylation sites on Smc3p (K112, K113). Thus removing WPL1 inhibition is insufficient for generating cohesion without ECO1 activity. To elucidate how ECO1 promotes cohesion, we conducted a genetic screen and identified a cohesion activator mutation in the SMC3 head domain (D1189H). Smc3-D1189H partially restores cohesion in eco1∆ wpl1∆ or eco1 mutant cells but robustly restores cohesion in cells blocked for Smc3p K112 K113 acetylation. These data support two important conclusions. First, acetylation of the K112 K113 region by Eco1p promotes cohesion establishment by altering Smc3p head function independent of its ability to antagonize Wpl1p. Second, Eco1p targets other than Smc3p K112 K113 are necessary for efficient establishment.

scholarly journals Kre28-Spc105 interaction is essential for their recruitment at the kinetochore

2021 ◽  
Author(s):  
Babhrubahan Roy ◽  
Janice Sim ◽  
Simon J. Y. Han ◽  
Ajit P. Joglekar
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
High Fidelity ◽  
Protein Assemblies ◽  
Sister Chromatids ◽  
Exact Function ◽  
Interaction Interface ◽  
Macromolecular Protein ◽  
Spindle Microtubules ◽  
Turn Over

Kinetochores are macromolecular protein assemblies that attach sister chromatids to spindle microtubules and mediate accurate chromosome segregation during mitosis. The outer kinetochore consists of the KMN network, a protein super complex made of Knl1 (yeast Spc105), Mis12 (yeast Mtw1) and Ndc80 (yeast Ndc80), which harbors sites for microtubule binding. Within the KMN network, Spc105 acts as interaction hub of components involved in spindle assembly checkpoint (SAC) signaling. It is known that Spc105 forms a complex with kinetochore component Kre28. However, where Kre28 physically localizes in the budding yeast kinetochore is not clear. The exact function of Kre28 at the kinetochore is also unknown. Here, we reveal how Spc105 and Kre28 interact and how they are organized within bioriented yeast kinetochores using genetics and cell biological experiments. We also identify the interaction interface between the two proteins and show that this interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the kinetochores. We created several truncation mutants of kre28 that do not localize at the kinetochores and so cannot mediate Spc105 loading at the kinetochores. When we over-expressed these mutants, they could sustain the cell viability even though failed to facilitate proper SAC activation and/or error correction. Thus, we inferred that Kre28 indirectly contributes to chromosome biorientation and high-fidelity segregation by regulating Spc105 localization at the kinetochores.

scholarly journals Determinants of Sir2-Mediated, Silent Chromatin Cohesion

2016 ◽  
Vol 36 (15) ◽  
pp. 2039-2050 ◽  
Author(s):  
Yu-Fan Chen ◽  
Chia-Ching Chou ◽  
Marc R. Gartenberg
Keyword(s):  
Chromatin Remodeling ◽  
Molecular Basis ◽  
Charged Surface ◽  
Silent Chromatin ◽  
Chromatin Domains ◽  
Content Type ◽  
Chromatin Remodelers ◽  
Sister Chromatids ◽  
Chromatin Remodeling Complex ◽  
Chromatid Cohesion

Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeastSaccharomyces cerevisiaerepresent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. WhenESC8was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lackingESC8or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.

scholarly journals Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

Open Biology ◽  
2016 ◽  
Vol 6 (2) ◽  
pp. 150236 ◽  
Author(s):  
Yahui Liu ◽  
Arsen Petrovic ◽  
Pascaline Rombaut ◽  
Shyamal Mosalaganti ◽  
Jenny Keller ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Chromosome Segregation ◽  
Functional Organization ◽  
Histone H3 Variant ◽  
Centromeric Protein ◽  
Spindle Microtubules ◽  
Specialized Region ◽  
Structural Methods ◽  
Mitosis And Meiosis ◽  
Shed Light

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.

scholarly journals Identification of four unconventional kinetoplastid kinetochore proteins KKT22–25 in Trypanosoma brucei

Open Biology ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 190236 ◽  
Author(s):  
Olga O. Nerusheva ◽  
Patryk Ludzia ◽  
Bungo Akiyoshi
Keyword(s):  
Trypanosoma Brucei ◽  
Chromosome Segregation ◽  
Rna Binding ◽  
Affinity Purification ◽  
Potential Interaction ◽  
Interacting Proteins ◽  
Interaction Partners ◽  
Spindle Microtubules ◽  
Acetyltransferase Domain ◽  
Mitosis And Meiosis

The kinetochore is a multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromere DNA and interacts with spindle microtubules during mitosis and meiosis. Although most eukaryotes have canonical kinetochore proteins, kinetochores of evolutionarily divergent kinetoplastid species consist of at least 20 unconventional kinetochore proteins (KKT1–20). In addition, 12 proteins (KKT-interacting proteins 1–12, KKIP1–12) are known to localize at kinetochore regions during mitosis. It remains unclear whether KKIP proteins interact with KKT proteins. Here, we report the identification of four additional kinetochore proteins, KKT22–25, in Trypanosoma brucei . KKT22 and KKT23 constitutively localize at kinetochores, while KKT24 and KKT25 localize from S phase to anaphase. KKT23 has a Gcn5-related N -acetyltransferase domain, which is not found in any kinetochore protein known to date. We also show that KKIP1 co-purifies with KKT proteins, but not with KKIP proteins. Finally, our affinity purification of KKIP2/3/4/6 identifies a number of proteins as their potential interaction partners, many of which are implicated in RNA binding or processing. These findings further support the idea that kinetoplastid kinetochores are unconventional.

scholarly journals ConnectingGCN5’s centromeric SAGA to the mitotic tension-sensing checkpoint

2018 ◽  
Vol 29 (18) ◽  
pp. 2201-2212 ◽  
Author(s):  
Emily L. Petty ◽  
Masha Evpak ◽  
Lorraine Pillus
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Spindle Assembly Checkpoint ◽  
Binding Function ◽  
Chromatid Cohesion ◽  
Histone H3 Variant ◽  
Spindle Microtubules ◽  
Low Tension ◽  
Centromere Histone H3 ◽  
Segregation Of Chromosomes

Multiple interdependent mechanisms ensure faithful segregation of chromosomes during cell division. Among these, the spindle assembly checkpoint monitors attachment of spindle microtubules to the centromere of each chromosome, whereas the tension-sensing checkpoint monitors the opposing forces between sister chromatid centromeres for proper biorientation. We report here a new function for the deeply conserved Gcn5 acetyltransferase in the centromeric localization of Rts1, a key player in the tension-sensing checkpoint. Rts1 is a regulatory component of protein phopshatase 2A, a near universal phosphatase complex, which is recruited to centromeres by the Shugoshin (Sgo) checkpoint component under low-tension conditions to maintain sister chromatid cohesion. We report that loss of Gcn5 disrupts centromeric localization of Rts1. Increased RTS1 dosage robustly suppresses gcn5∆ cell cycle and chromosome segregation defects, including restoration of Rts1 to centromeres. Sgo1’s Rts1-binding function also plays a key role in RTS1 dosage suppression of gcn5∆ phenotypes. Notably, we have identified residues of the centromere histone H3 variant Cse4 that function in these chromosome segregation-related roles of RTS1. Together, these findings expand the understanding of the mechanistic roles of Gcn5 and Cse4 in chromosome segregation.

scholarly journals Suppressor screening reveals common kleisin–hinge interaction in condensin and cohesin, but different modes of regulation

2019 ◽  
Vol 116 (22) ◽  
pp. 10889-10898 ◽  
Author(s):  
Xingya Xu ◽  
Mitsuhiro Yanagida
Keyword(s):  
Chromatin Remodeling ◽  
Chromosome Segregation ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Whole Genome ◽  
Chromatid Cohesion ◽  
Suppression Mechanism ◽  
Elimination Pathway ◽  
Extragenic Suppressors ◽  
Structural Maintenance Of Chromosomes

Cohesin and condensin play fundamental roles in sister chromatid cohesion and chromosome segregation, respectively. Both consist of heterodimeric structural maintenance of chromosomes (SMC) subunits, which possess a head (containing ATPase) and a hinge, intervened by long coiled coils. Non-SMC subunits (Cnd1, Cnd2, and Cnd3 for condensin; Rad21, Psc3, and Mis4 for cohesin) bind to the SMC heads. Here, we report a large number of spontaneous extragenic suppressors for fission yeast condensin and cohesin mutants, and their sites were determined by whole-genome sequencing. Mutants of condensin’s non-SMC subunits were rescued by impairing the SUMOylation pathway. Indeed, SUMOylation of Cnd2, Cnd3, and Cut3 occurs in midmitosis, and Cnd3 K870 SUMOylation functionally opposes Cnd subunits. In contrast, cohesin mutants rad21 and psc3 were rescued by loss of the RNA elimination pathway (Erh1, Mmi1, and Red1), and loader mutant mis4 was rescued by loss of Hrp1-mediated chromatin remodeling. In addition, distinct regulations were discovered for condensin and cohesin hinge mutants. Mutations in the N-terminal helix bundle [containing a helix–turn–helix (HTH) motif] of kleisin subunits (Cnd2 and Rad21) rescue virtually identical hinge interface mutations in cohesin and condensin, respectively. These mutations may regulate kleisin’s interaction with the coiled coil at the SMC head, thereby revealing a common, but previously unknown, suppression mechanism between the hinge and the kleisin N domain, which is required for successful chromosome segregation. We propose that in both condensin and cohesin, the head (or kleisin) and hinge may interact and collaboratively regulate the resulting coiled coils to hold and release chromosomal DNAs.

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