Methylation of CENP-A/Cse4 on arginine 143 and lysine 131 regulates kinetochore stability in yeast.

Post-translational modifications on histones are well known to regulate chromatin structure and function, but much less information is available on modifications of the centromeric histone H3 variant and their effect at the kinetochore. Here, we report two modifications on the centromeric histone H3 variant CENP-A/Cse4 in the yeast Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me), that affect centromere stability and kinetochore function. Both R143me and K131me lie in the core region of the centromeric nucleosome, near the entry/exit sites of the DNA from the nucleosome. Unexpectedly, mutation of Cse4-R143 (cse4-R143A) exacerbated the kinetochore defect of mutations in components of the NDC80 complex of the outer kinetochore (spc25-1) and the MIND complex (dsn1-7). The analysis of suppressor mutations of the spc25-1 cse4-R143A growth defect highlighted residues in Spc24, Ndc80, and Spc25 that localize to the tetramerization domain of the NDC80 complex and the Spc24-Spc25 stalk, suggesting that the mutations enhance interactions among NDC80 complex components and thus stabilize the complex. Furthermore, the Set2 histone methyltransferase inhibited kinetochore function in spc25-1 cse4-R143A cells, possibly by methylating Cse4-K131. Taken together, our data suggest that Cse4-R143 methylation and Cse4-K131 methylation affect the stability of the centromeric nucleosome, which is detrimental in the context of defective NDC80 tetramerization and can be compensated for by strengthening interactions among NDC80 complex components.

Queuosine salvage in fission yeast by Qng1-mediated hydrolysis to queuine.

Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside that is found at position 34, also known as the wobble position, of tRNAs with a GUN anticodon, and Q ensures faithful translation of the respective C- and U-ending codons. While Q is present in tRNAs in most eukaryotes, only bacteria can synthesize it denovo. In contrast, eukaryotes rely on external sources like their food and the gut microbiome in order to Q-modify their tRNAs, and Q therefore can be regarded as a micronutrient. The eukaryotic tRNA guanine transglycosylase (eTGT) uses the base queuine (q) as a substrate to replace G34 by Q in the tRNAs. Eukaryotic cells can uptake both q and Q, raising the question how the Q nucleoside is converted to q for incorporation into the tRNAs. Here, we identified Qng1 (also termed Duf2419) as a queuosine nucleoside glycosylase in Schizosaccharomyces pombe. S. pombe cells with a deletion of qng1+ contained Q-modified tRNAs only when cultured in the presence of the nucleobase q, but not with the nucleoside Q, indicating that the cells are proficient at q incorporation, but not in Q hydrolysis. Furthermore, purified recombinant Qng1 hydrolyzed Q to q in vitro. Qng1 displays homology to DNA glycosylases and has orthologs across eukaryotes, including flies, mice and humans. Qng1 therefore plays an essential role in allowing eukaryotic cells to salvage Q from bacterial sources and to recycle Q from endogenous tRNAs.

Dynamics of SAS-I mediated H4 K16 acetylation during DNA replication in yeast.

The acetylation of H4 lysine 16 (H4 K16Ac) in Saccharomyces cerevisiae counteracts the binding of the heterochromatin complex SIR to chromatin and inhibits gene silencing. Contrary to other histone acetylation marks, the H4 K16Ac level is high on genes with low transcription, whereas highly transcribed genes show low H4 K16Ac. Approximately 60% of cellular H4 K16Ac in S. cerevisiae is provided by the SAS-I complex, which consists of the MYST-family acetyltransferase Sas2, Sas4 and Sas5. The absence of SAS-I causes inappropriate spreading of the SIR complex and gene silencing in subtelomeric regions. Here, we investigated the genome-wide dynamics of SAS-I dependent H4 K16Ac during DNA replication. Replication is highly disruptive to chromatin and histone marks, since histones are removed to allow progression of the replication fork, and chromatin is reformed with old and new histones after fork passage. We found that H4 K16Ac appears in chromatin immediately upon replication. Importantly, this increase depends on the presence of functional SAS-I complex. Moreover, the appearance of H4 K16Ac is delayed in genes that are strongly transcribed. This indicates that transcription counteracts SAS-I-mediated H4 K16 acetylation, thus "sculpting" histone modification marks at the time of replication. We furthermore investigated which acetyltransferase acts redundantly with SAS-I to acetylate H4 K16Ac. esa1Δ sds3Δ cells, which were also sas2Δ sir3Δ in order to maintain viability, contained no detectable H4 K16Ac, showing that Esa1 and Sas2 are redundant for cellular H4 K16 acetylation. Furthermore, esa1Δ sds3Δ sas2Δ sir3Δ showed a more pronounced growth defect compared to the already defective esa1Δ sds3Δ sir3Δ. This indicates that SAS-I has cellular functions beyond preventing the spreading of heterochromatin.

Division of labour: tRNA methylation by the NSun2 tRNA methyltransferases Trm4a and Trm4b in fission yeast.

Enzymes of the cytosine-5 RNA methyltransferase Trm4/NSun2 family methylate tRNAs at C48 and C49 in multiple tRNAs, as well as C34 and C40 in selected tRNAs. In contrast to most other organisms, fission yeast Schizosaccharomyces pombe carries two Trm4/NSun2 homologs, Trm4a (SPAC17D4.04) and Trm4b (SPAC23C4.17). Here, we have employed tRNA methylome analysis to determine the dependence of cytosine-5 methylation (m5C) tRNA methylation in vivo on the two enzymes. Remarkably, Trm4a is responsible for all C48 methylation, which lies in the tRNA variable loop, as well as for C34 in tRNALeuCAA and tRNAProCGG, which are at the anticodon wobble position. Conversely, Trm4b methylates C49 and C50, which both lie in the TΨC-stem. Thus, S. pombe show an unusual separation of activities of the NSun2/Trm4 enzymes that are united in a single enzyme in other eukaryotes like humans, mice and Saccharomyces cerevisiae. Furthermore, in vitro activity assays showed that Trm4a displays intron-dependent methylation of C34, whereas Trm4b activity is independent of the intron. The absence of Trm4a, but not Trm4b, causes a mild resistance of S. pombe to calcium chloride.

The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A.

Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from Saccharomyces cerevisiae regulate molecular interactions at the inner kinetochore. Importantly, we found that the Cse4 N-terminus binds with high affinity to the Ctf19 complex subassembly Okp1/Ame1 (CENP-Q/CENP-U in higher eukaryotes), and that this interaction is inhibited by R37Me and K49Ac modification on Cse4. In vivo defects in cse4-R37A were suppressed by mutations in OKP1 and AME1, and biochemical analysis of a mutant version of Okp1 showed increased affinity for Cse4. Altogether, our results demonstrate that the Okp1/Ame1 heterodimer is a reader module for posttranslational modifications on Cse4, thereby targeting the yeast CCAN complex to centromeric chromatin.

A role for CENP-A/Cse4 phosphorylation on serine 33 in deposition at the centromere.

Centromeres are the sites of assembly of the kinetochore, which connect the chromatids to the microtubules for sister chromatid segregation during cell division. Centromeres are characterized by the presence of the histone H3 variant CENP-A (termed Cse4 in Saccharomyces cerevisiae). Here, we investigated the function of serine 33 phosphorylation of Cse4 (Cse4-S33ph) in S. cerevisiae, which lies within the essential N-terminal domain (END) of the extended Cse4 N-terminus. Significantly, we identified histone H4-K5, 8, 12R to cause a temperature-sensitive growth defect with mutations in Cse4-S33 and sensitivity to nocodazole and hydroxyurea. Furthermore, the absence of Cse4-S33ph reduced the levels of Cse4 at centromeric sequences, suggesting that Cse4 deposition is defective in the absence of S33 phosphorylation. We furthermore identified synthetic genetic interactions with histone H2A-E57A and H2A-L66A, which both cause a reduced interaction with the histone chaperone FACT and reduced H2A/H2B levels in chromatin, again supporting the notion that a combined defect of H2A/H2B and Cse4 deposition causes centromeric defects. Altogether, our data highlight the importance of correct histone deposition in building a functional centromeric nucleosome and suggests a role for Cse4-S33ph in this process.