Mahito Sadaie

Autophagy mediates the mitotic senescence transition

As a stress response, senescence is a dynamic process involving multiple effector mechanisms whose combination determines the phenotypic quality. Here we identify autophagy as a new effector mechanism of senescence. Autophagy is activated during senescence and its activation is correlated with negative feedback in the PI3K-mammalian target of rapamycin (mTOR) pathway. A subset of autophagy-related genes are up-regulated during senescence: Overexpression of one of those genes, ULK3, induces autophagy and senescence. Furthermore, inhibition of autophagy delays the senescence phenotype, including senescence-associated secretion. Our data suggest that autophagy, and its consequent protein turnover, mediate the acquisition of the senescence phenotype.

Cellular senescence and its effector programs

Cellular senescence is a stress response that accompanies stable exit from the cell cycle. Classically, senescence, particularly in human cells, involves the p53 and p16/Rb pathways, and often both of these tumor suppressor pathways need to be abrogated to bypass senescence. In parallel, a number of effector mechanisms of senescence have been identified and characterized. These studies suggest that senescence is a collective phenotype of these multiple effectors, and their intensity and combination can be different depending on triggers and cell types, conferring a complex and diverse nature to senescence. Series of studies on senescence-associated secretory phenotype (SASP) in particular have revealed various layers of functionality of senescent cells in vivo. Here we discuss some key features of senescence effectors and attempt to functionally link them when it is possible.

A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast

The chromodomain is a conserved motif that functions in the epigenetic control of gene expression. Here, we report the functional characterization of a chromodomain protein, Chp1, in the heterochromatin assembly in fission yeast. We show that Chp1 is a structural component of three heterochromatic regions—centromeres, the mating‐type region, and telomeres—and that its localization in these regions is dependent on the histone methyltransferase Clr4. Although deletion of the chp1+ gene causes centromere‐specific decreases in Swi6 localization and histone H3‐K9 methylation, we show that the role of Chp1 is not exclusive to the centromeres. We found that some methylation persists in native centromeric regions in the absence of Chp1, which is also true for the mating‐type region and telomeres, and determined that Swi6 and Chp2 are critical to maintaining this residual methylation. We also show that Chp1 participates in the establishment of repressive chromatin in all three chromosomal regions. These results suggest that different heterochromatic regions share common structural properties, and that centromeric heterochromatin requires Chp1‐mediated establishment steps differently than do other heterochromatic regions.

Telomere Binding Protein Taz1 Establishes Swi6 Heterochromatin Independently of RNAi at Telomeres

BACKGROUND The telomere is a specialized heterochromatin conserved among eukaryotes. However, it remains unknown how heterochromatin protein 1 (HP1) is recruited to telomeres and how telomere heterochromatin is formed. In fission yeast, the RNAi (RNA interference)-RITS (RNA-induced initiation of transcriptional silencing) pathway initiates heterochromatin formation at the centromeres and the silent mat locus by using common DNA sequences, the dg and dh repeats, as the templates for small interfering RNA (siRNA). RESULTS We found that telomeric repeats are sufficient for the establishment of Swi6 (a fission-yeast HP1 homolog) heterochromatin, and the establishment requires Taz1, a telomere binding protein of the TRF family. Additionally, Swi6 heterochromatin is established by a part of the subtelomere that contains sequences highly homologous to that of the dh repeat, and it is strikingly destabilized by the deletion of both Taz1 and RNAi-RITS. Transcripts from the telomeric dh-homologous region were specifically associated with RITS, and deletion of the telomeric dh-homologous region showed the phenotype similar to that of the rnai mutant in terms of the telomeric silencing, indicating that the RNAi-RITS pathway acts at the telomeric dh-homologous region to establish Swi6 heterochromatin. Furthermore, we found that Taz1 establishes Swi6 heterochromatin independently of the telomeric repeats and the RNAi-RITS pathway at the subtelomeres. CONCLUSION The telomere heterochromatin is regulated by at least two factors: One is Taz1, which is telomere specific, and the other is RNAi-RITS, which is commonly used at the constitutive heterochromatin regions.

Redistribution of the Lamin B1 genomic binding profile affects rearrangement of heterochromatic domains and SAHF formation during senescence

Senescence is a stress-responsive form of stable cell cycle exit. Senescent cells have a distinct gene expression profile, which is often accompanied by the spatial redistribution of heterochromatin into senescence-associated heterochromatic foci (SAHFs). Studying a key component of the nuclear lamina lamin B1 (LMNB1), we report dynamic alterations in its genomic profile and their implications for SAHF formation and gene regulation during senescence. Genome-wide mapping reveals that LMNB1 is depleted during senescence, preferentially from the central regions of lamina-associated domains (LADs), which are enriched for Lys9 trimethylation on histone H3 (H3K9me3). LMNB1 knockdown facilitates the spatial relocalization of perinuclear H3K9me3-positive heterochromatin, thus promoting SAHF formation, which could be inhibited by ectopic LMNB1 expression. Furthermore, despite the global reduction in LMNB1 protein levels, LMNB1 binding increases during senescence in a small subset of gene-rich regions where H3K27me3 also increases and gene expression becomes repressed. These results suggest that LMNB1 may contribute to senescence in at least two ways due to its uneven genome-wide redistribution: first, through the spatial reorganization of chromatin and, second, through gene repression.

Balance between Distinct HP1 Family Proteins Controls Heterochromatin Assembly in Fission Yeast

ABSTRACT Heterochromatin protein 1 (HP1) is a conserved chromosomal protein with important roles in chromatin packaging and gene silencing. In fission yeast, two HP1 family proteins, Swi6 and Chp2, are involved in transcriptional silencing at heterochromatic regions, but how they function and whether they act cooperatively or differentially in heterochromatin assembly remain elusive. Here, we show that both Swi6 and Chp2 are required for the assembly of fully repressive heterochromatin, in which they play distinct, nonoverlapping roles. Swi6 is expressed abundantly and plays a dose-dependent role in forming a repressive structure through its self-association property. In contrast, Chp2, expressed at a lower level, does not show a simple dose-dependent repressive activity. However, it contributes to the recruitment of chromatin-modulating factors Clr3 and Epe1 and possesses a novel ability to bind the chromatin-enriched nuclear subfraction that is closely linked with its silencing function. Finally, we demonstrate that a proper balance between Swi6 and Chp2 is critical for heterochromatin assembly. Our findings provide novel insight into the distinct and cooperative functions of multiple HP1 family proteins in the formation of higher-order chromatin structure.

Phosphorylation of Swi6/HP1 regulates transcriptional gene silencing at heterochromatin

Heterochromatin protein 1 (HP1) recruits various effectors to heterochromatin for multiple functions, but its regulation is unclear. In fission yeast, a HP1 homolog Swi6 recruits SHREC, Epe1, and cohesin, which are involved in transcriptional gene silencing (TGS), transcriptional activation, and sister chromatid cohesion, respectively. We found that casein kinase II (CK2) phosphorylated Swi6. Loss of CK2-dependent Swi6 phosphorylation alleviated heterochromatic TGS without affecting heterochromatin structure. This was due to the inhibited recruitment of SHREC to heterochromatin, accompanied by an increase in Epe1. Interestingly, loss of phosphorylation did not affect cohesion. These results indicate that CK2-dependent Swi6 phosphorylation specifically controls TGS in heterochromatin.

Telomeric DNA Ends Are Essential for the Localization of Ku at Telomeres in Fission Yeast

The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway. Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated. In fission yeast,pku70 +, the gene encoding the Ku70 homologue, has been reported. Here we report the identification and characterization of pku80 +, the gene encoding Ku80. Both pku70 + andpku80 + are essential for efficient non-homologous end-joining. We also found that the pku70and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. Thepku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70mutant. By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences. Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein. We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes. These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.

A conserved SET domain methyltransferase, Set11, modifies ribosomal protein Rpl12 in fission yeast.

SET domain-containing methyltransferases post-translationally modify a variety of cellular proteins, such as histones, cytochrome c, ribulose-bisphosphate carboxylase/oxygenase, and ribosomal proteins. In the fission yeast Schizosaccharomyces pombe, at least 13 SET domain-containing proteins have been identified in the genome, four of which are involved in transcriptional regulation through their modification of histone tails. However, the roles played by the other SET domain proteins in cellular processes and their physiological substrates remain unresolved. We show here that S. pombe Set11, a SET domain-containing protein encoded by SPCC1223.04c, specifically modifies Rpl12 (ribosomal protein L12). Recombinant Set11 prepared from Escherichia coli had catalytic activity and methylated a 17-kDa polypeptide in cellular extracts of set11 mutant cells. The methylated protein was isolated by two-dimensional gel electrophoresis or by reverse-phase chromatography and was identified as Rpl12 by mass spectrometry. In vitro methylation experiments using wild-type and mutant Rpl12 proteins verified that Set11 modified recombinant Rpl12 and suggested that its potential target site was lysine 3. The methylation site modified by Set11 was also confirmed by mass spectrometric analysis, which also revealed other unique methylation sites of Rpl12. Finally, we found that Set11 predominantly localized to the nucleolus and that the overproduction of Set11 caused a severe growth defect. These results suggest that Rpl12 methylation occurs during the ribosomal assembly processes and that control of the Set11 expression level is important for its cellular function.

Cell-based screen for altered nuclear phenotypes reveals senescence progression in polyploid cells after Aurora kinase B inhibition

Compound screening for altered nuclear phenotypes identifies several promiscuous kinase inhibitors that trigger progression of senescence during a polyploid G1. Their common target is AURKB. More-specific inhibition of AURKB phenocopies these compounds, demonstrating a causative role for AURKB defects in a unique mode of senescence development.