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.Supplemental material is available at http://www.genesdev.org.Received December 24, 2008; revised version accepted February 11, 2009. Cellular senescence is a state of stable cell cycle arrest with active metabolism. Similar to apoptosis, senescence can be a failsafe program against a variety of cellular insults. In contrast to apoptosis, however, in which the cytotoxic signals converge to a common mechanism, senescence is typically a delayed stress response involving multiple effector mechanisms. These effector mechanisms include epigenetic regulation (Narita et al. 2006;Adams 2007), the DNA damage response (Bartkova et al. 2006;Di Micco et al. 2006;Mallette et al. 2007), and the senescence-associated secretion phenotype (Kortlever et al. 2006;Acosta et al. 2008;Coppé et al. 2008;Kuilman et al. 2008;Wajapeyee et al. 2008). The relative contribution of these effectors varies depending on the trigger and cell type.Oncogene-induced senescence (OIS) illustrates well the tumor-suppressive role of senescence (Collado and Serrano 2006). The initial phenotype of oncogene induction is a highly proliferative state, which mimics transformation. However, this mitotic burst is gradually replaced by senescence. Although it has been proposed that global and progressive epigenetic alterations play a crucial role in OIS (Narita et al. 2006), the precise mechanism by which cells can achieve such a dramatic change is still unclear.Autophagy is a genetically regulated program responsible for the turnover of cellular proteins and damaged or superfluous organelles. This evolutionarily conserved process is characterized by the formation of doublemembrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic content and deliver it to lysosomes (Ohsumi 2001;Klionsky et al. 2007;Mizushima et al. 2008). Autophagy is often associated with acute metabolic changes and rapid protein replacement. For example, autophagy is required for preimplantation development, where maternal proteins are recycled by autophagy (Tsukamoto et al. 2008). Autophagy is also required for survival in the early neonatal starvation period (Kuma et al. 2004;Komatsu et al. 2005). In addition to these physiological conditions, cytotoxic stimuli can also activate autophagy, but its precise role as a stress response...

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.

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.

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 þ gene causes centromerespecific 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.

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.

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.

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.Supplemental material is available at http://www.genesdev.org.

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 ؉ and pku80 ؉ are essential for efficient non-homologous end-joining. We also found that the pku70 and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. The pku80 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 pku70 mutant. 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 DNAbinding 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. DNA double strand breaks (DSBs)1 cause serious damage to genetic materials in living cells. Indeed, a single chromosomal break is lethal if left unrepaired (1). DSBs are introduced by a variety of DNA-damaging agents, such as ionizing radiation and radiomimetic chemicals, or even spontaneously. DSB results in the loss of the continuity of DNA and the formation of two physical ends. These non-physiological ends of DNA are repaired in eukaryotes by two major pathways, the error-prone NHEJ pathway and the more accurate homologous recombination (HR) pathway (reviewed in Ref. 2). NHEJ ligates two broken DNA ends with little or no requirement for sequence homology between the two ends. In contrast, HR requires an intact DNA that is homologous with the DNA to be repaired and that serves as a template for the DNA synthesis to repair the break. The HR pathway requires products of the RAD52 epistasis group genes, including Rad51 and Rad54 in Saccharomyces cerevisiae. On the other hand, a different set of gene products is necessary for the NHEJ pathway, which includes the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV proteins, and the recently described Artemis protein in human cells (3). Artemis possesses nuclease activity that functions to process double-stranded DNA ends and is regulated by DNA-PKcs (4). The DNA-PK holoenzyme is a serinethreonine protein kinase consisting of DNA-PKcs and the Ku70-Ku80 heterodimer. The Ku70-Ku80 complex binds to the very end of broken DNA and then recruits and activates the DNA-PKcs (reviewed in Ref. 5).The function of Ku proteins is not limited to the repair of non-physiological DNA ends. Ku is a bona fide component of telomeres, the physiological end of li...