Prolyl isomerization of the CENP-A N-terminus regulates centromeric integrity in fission yeast

Tan, Hwei Ling; Lim, Kieron; Yang, Qiaoyun; Fan, Jing‐Song; Sayed, Ahmed M.; Low, Liy Sim; Ren, Beibei; Lim, Teck Kwang; Lin, Qingsong; Mok, Yu Keung; Liou, Yih‐Cherng; Chen, Ee Sin

doi:10.1093/nar/gkx1180

Cited by 12 publications

(35 citation statements)

References 54 publications

(72 reference statements)

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“…In our previous investigation into the role of the SpCENP-A NTD, we assessed the importance of the proline residues residing in the GRANT motif by overexpressing a mutant SpCENP-A cnp1-4PA bearing alanine mutations at four proline residues (P10AP13AP15AP17A). We found that the SpCENP-A cnp1-4PA mutant was almost as stable as the wild-type protein in a protein turnover assay [ 28 ]. These observations suggested that the ‘GRANT-proline’ residues have no effect on the proteasome-associated turnover of SpCENP-A protein.…”

Section: Resultsmentioning

confidence: 99%

“…The localization of CENP-A to centromeric regions is dependent on a CENP-A-targeting domain (CATD) nested within the histone fold domain of CENP-A, which interacts with the CENP-A loading factor Holliday Junction Recognition Protein (HJURP) and stabilizes CENP-A–histone H4 interaction [ 36 , 37 ]. Recently, it has been shown by us and others that the flexible NTD tail of CENP-A directs CENP-A centromeric targeting independently of the CATD via binding with CENP-A loading factors such as Sim3/Nuclear Autoantigen Sperm Protein (NASP) [ 28 , 38 ], the CCAN component CENP-T [ 39 ], and CENP-B [ 28 , 40 ]. We have further shown that the Sim3–SpCENP-A interaction is mediated via prolyl cis – trans isomerization of a proline-rich GRANT motif within the SpCENP-A NTD [ 28 ].…”

Section: Discussionmentioning

confidence: 99%

“…Recently, we interrogated the contribution of individual amino acid residues in the NTD of Schizosaccharomyces pombe CENP-A (SpCENP-A) [ 28 ]. We uncovered a proline-rich GRANT (Genomic stability-Regulating site within CENP-A N -Terminus) motif that undergoes cis – trans prolyl isomerization to regulate the association of the SpCENP-A NTD with the loading chaperone Sim3, which functions to target SpCENP-A to the centromere.…”

Section: Introductionmentioning

confidence: 99%

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N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A

Tan

Zeng

Chen

2020

IJMS

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Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (N)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in Saccharomyces cerevisiae CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of Schizosaccharomyces pombe CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (Δclr4), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A

Tan

Zeng

Chen

2020

IJMS

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“…could not be identified when Cse4 was used as a query in BLAST search. In fission yeast, the proline-rich motif GRANT ( g enomic stability- r egulating site within CENP- A N - t erminus) is essential for centromeric targeting of Cnp1 [ 29 ]. A proline-rich GRANT motif was found in the N-terminal region of CENP-A L.s.…”

Section: Resultsmentioning

confidence: 99%

Identification of Genes Encoding CENP-A and Heterochromatin Protein 1 of Lipomyces starkeyi and Functional Analysis Using Schizosaccharomyces pombe

Takayama

2020

Genes

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Centromeres function as a platform for the assembly of multiple kinetochore proteins and are essential for chromosome segregation. An active centromere is characterized by the presence of a centromere-specific histone H3 variant, CENP-A. Faithful centromeric localization of CENP-A is supported by heterochromatin in almost all eukaryotes; however, heterochromatin proteins have been lost in most Saccharomycotina. Here, identification of CENP-A (CENP-AL.s.) and heterochromatin protein 1 (Lsw1) in a Saccharomycotina species, the oleaginous yeast Lipomyces starkeyi, is reported. To determine if these proteins are functional, the proteins in S. pombe, a species widely used to study centromeres, were ectopically expressed. CENP-AL.s. localizes to centromeres and can be replaced with S. pombe CENP-A, indicating that CENP-AL.s. is a functional centromere-specific protein. Lsw1 binds at heterochromatin regions, and chromatin binding is dependent on methylation of histone H3 at lysine 9. In other species, self-interaction of heterochromatin protein 1 is thought to cause folding of chromatin, triggering transcription repression and heterochromatin formation. Consistent with this, it was found that Lsw1 can self-interact. L. starkeyi chromatin contains the methylation of histone H3 at lysine 9. These results indicated that L. starkeyi has a primitive heterochromatin structure and is an attractive model for analysis of centromere heterochromatin evolution.

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“…Previously published procedures were followed closely [ 55 , 56 ]. Briefly, log-phase growing cultures (OD 600nm = 0.5) were fixed with 3% paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA) before homogenization with glass beads.…”

Section: Methodsmentioning

confidence: 99%

Fission Yeast Methylenetetrahydrofolate Reductase Ensures Mitotic and Meiotic Chromosome Segregation Fidelity

Lim

Teo

Tan

et al. 2021

IJMS

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Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway, and its loss of function through polymorphisms is often associated with human conditions, including cancer, congenital heart disease, and Down syndrome. MTHFR is also required in the maintenance of heterochromatin, a crucial determinant of genomic stability and precise chromosomal segregation. Here, we characterize the function of a fission yeast gene met11+, which encodes a protein that is highly homologous to the mammalian MTHFR. We show that, although met11+ is not essential for viability, its disruption increases chromosome missegregation and destabilizes constitutive heterochromatic regions at pericentromeric, sub-telomeric and ribosomal DNA (rDNA) loci. Transcriptional silencing at these sites were disrupted, which is accompanied by the reduction in enrichment of histone H3 lysine 9 dimethylation (H3K9me2) and binding of the heterochromatin protein 1 (HP1)-like Swi6. The met11 null mutant also dominantly disrupts meiotic fidelity, as displayed by reduced sporulation efficiency and defects in proper partitioning of the genetic material during meiosis. Interestingly, the faithful execution of these meiotic processes is synergistically ensured by cooperation among Met11, Rec8, a meiosis-specific cohesin protein, and the shugoshin protein Sgo1, which protects Rec8 from untimely cleavage. Overall, our results suggest a key role for Met11 in maintaining pericentromeric heterochromatin for precise genetic inheritance during mitosis and meiosis.

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Prolyl isomerization of the CENP-A N-terminus regulates centromeric integrity in fission yeast

Cited by 12 publications

References 54 publications

N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A

N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A

Identification of Genes Encoding CENP-A and Heterochromatin Protein 1 of Lipomyces starkeyi and Functional Analysis Using Schizosaccharomyces pombe

Fission Yeast Methylenetetrahydrofolate Reductase Ensures Mitotic and Meiotic Chromosome Segregation Fidelity

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