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an age of fewer histonesphilipp oberdoerffer

Changes in chromatin structure are a conserved hallmark of ageing, and the mechanism driving these changes, as well as their functional significance, are heavily investigated. Loss of core histones is now observed in aged cells and may contribute to this phenomenon. Histone loss is coupled to cell division and seems to be triggered by telomeric dna damage.

Histones are the basic building blocks of eukaryotic chromatin, a complex DNA pack-aging system that helps organize the genome in three-dimensional nuclear space. This so-called epigenetic organization is critical to normal cell function and failure to maintain it can result in genomic instability and deregu-lated gene expression, features that have been tightly linked to the ageing process across spe-cies1. Now, the groups of Tyler and Karlseder report that ageing may be directly related to the loss of the core histones H3 and H4 (refs 2, 3). It has been previously reported that old yeast cells show a significant drop in the protein lev-els and chromatin incorporation of these his-tones2,4. Karsleder and colleagues now extend this observation to human fibroblasts and pro-pose DNA damage as a driving force for histone loss3. Most notably, Tyler and colleagues were able to demonstrate that increasing histone levels is sufficient to extend lifespan in yeast2. Together, these data provide a compelling, evo-lutionarily conserved explanation for the loss of epigenomic integrity with age, hitting the very core of chromatin organization.

Much effort in recent ageing research has been directed towards understanding the role of chromatin in cellular, and ultimately organ-ismal, decline. Perhaps the most heavily inves-tigated questions ask what drives age-related epigenomic changes and how can we exploit their — at least theoretically — reversible nature to forestall the ageing process. Recent work from several groups collectively identi-fies DNA damage as a key mediator of age-related chromatin reorganization. However, the epigenetic manifestations and conse-quences of DNA damage are manifold (Fig. 1). Chronic DNA damage has been reported to induce a senescence-associated secretory phenotype (SASP) affecting the secretion of inflammatory cytokines, presumably to sig-nal their compromised state to surrounding

cells5. A progeria syndrome associated with defects in transcription-coupled DNA repair produces transcriptional changes that are usu-ally found in aged mice6. DNA damage further induces transcriptional noise, a phenomenon also observed in single cells isolated from age-ing tissues7. Lastly, genotoxic stress can cause the genome-wide reorganization of chroma-tin through the redistribution of a chromatin modifier (RCM), SIRT1, from certain pro-moters and repetitive DNA to sites of DNA damage8. All of these DNA damage-induced epigenetic changes correlate with, or mimic, transcriptional deregulation observed with ageing. However, these findings also demon-strate the many ways in which DNA damage can alter the transcriptional profiles of cells. At this point, it is unclear if these pathways are connected, and only some have been shown to be relevant across species.

Histones have a central role in all of these processes, and the loss of core histones with age is evolutionarily conserved (Fig. 1). Histone biosynthesis has previously been shown to be sensitive to genomic stress, such as DNA dam-age or replication stress9,10. This observation prompted Karlseder and colleagues to inves-tigate if the chronic genomic stress observed with age may also negatively affect histone

production3. The authors compared early and late passage human diploid fibroblasts as an approximation for cellular ageing, where tel-omere attrition is a driving force for chronic DNA damage1. Using a careful analysis of his-tone biosynthesis by stable isotope labelling with amino acids in cell culture (SILAC), the authors convincingly demonstrate a drop in histone H3 and H4 production in late-passage cells, as well as in fibroblasts from older indi-viduals. The authors also observed changes in epigenetic marks and activation of the DNA-damage response. These changes were depend-ent on the cell cycle and loss of histones could further be observed when fibroblasts were treated with a DNA damaging agent, bleomy-cin. To test the impact of endogenous genomic stress, the authors then asked if the population-doubling-related telomeric DNA damage was critical for this drop in H3 levels. Indeed, over-expression of telomerase reverse transcriptase (hTERT), which inhibits telomere attrition and the associated DNA damage response11, restored some of the histone-related defects observed in old cells (Fig. 2a). However, it remains to be seen if this effect is a direct con-sequence of reduced DNA damage signalling or indirectly related to the overall phenotypically youthful state inferred by hTERT re-expression

Philipp Oberdoerffer is in the Mouse Cancer Genetics Program, Center for Cancer Research, NCI‑Frederick, Fort Detrick, Frederick, MD 21702, USA.e‑mail: oberdoerfferp@mail.nih.gov

Histone loss

RCM response

DNA damage

SASP

Genomerearrangements

Transcription-coupledrepair defects

Age

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Transcriptional noise

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Epigenomic dysfunction

Figure 1 Mediators of DNA‑damage induced deregulation of the epigenome. Double‑arrows indicate possible cross‑talk between individual pathways. SASP; senescence‑associated secretory phenotype; RCM: redistribution of chromatin modifiers.

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in fibroblasts12. Ablation of bona fide DNA damage mediators is one way to conclusively address this issue, similarly to experiments in fibroblasts and mice where telomere attri-tion could be rescued by inactivation of DNA-damage-response genes, such as p21 or p53 (refs 11, 13).

In the search for a mechanism by which increased population doubling and telomere attrition may cause the loss of core histones, Kalrseder and colleagues observed modest changes in the H3/H4 chaperones, ASF1 and CAF1, as well as in the histone pre-mRNA processing factor stem loop binding protein (SLBP). How telomere attrition is linked to these observations is at this point unclear and a direct link to histone biosynthesis remains to be demonstrated. It is of note that genetic inactivation of ASF1 and CAF1 was shown to be involved in the histone loss seen with age-ing in yeast2, suggesting that histone main-tenance with age depends on evolutionarily conserved pathways.

Based on the findings by Karlseder and colleagues alone, it is unclear if histone loss is a causal factor for the functional decline observed with age, or a consequence of age-related chromatin re-organization. Tyler and colleagues provide support for the former by showing that overexpression of H3 and H4 can extend yeast replicative lifespan, sug-gesting that rescue of histone production

maintains epigenomic integrity in old cells. (Fig. 2b)2. The authors initially observed that yeast cells lacking the histone chaperone, ASF1, lost the potential for regrowth with age. Using an impressive array of genetic manipulations, Feser et al. were able to pinpoint this defect to reduced incorporation of H3 and H4 into chro-matin in old cells, which was directly affected by ASF1. The function of ASF1 is known to depend on the tightly regulated acetylation and deacetylation of H3 at Lys 56 (H3K56; ref. 14), a process that occurs primarily during DNA replication and DNA repair, and the authors showed that manipulation of H3K56 acetyla-tion resulted in shortened lifespan and a pre-mature reduction in chromatin-associated H3. Indeed, a single point mutation at H3K56 can reduce yeast lifespan to almost the same extent as deletion of ASF1 (refs 2, 4). A similar effect has been previously reported for the acetylation of histone H4 at Lys 16 (ref. 4). Furthermore, the histone acetyltransferase RTT109, loss of which negatively affects yeast lifespan, can tar-get both lysine residues, suggesting that H3 and H4 levels are controlled by similar pathways2,4. Underlining the importance of efficient histone incorporation, overexpression of H3 and H4 was able to significantly extend yeast lifespan. Interestingly, deleting a transcriptional repres-sor of histones (HIR) also extended lifespan. However, downregulation of histone protein levels appears to occur post-transcriptionally

as their mRNA levels increase with age2. It is therefore not entirely obvious how loss of HIR promotes lifespan extension and more work is needed to dissect the complex regulation of histone biosynthesis.

Given the marked effect of histone over-expression on yeast ageing, it is tempting to speculate that increasing histone production or incorporation will have similar benefits in human cells, thereby opening new possibilities for targeted intervention in the quest to reset the ageing epigenome. Karlseder and colleagues suggest that this may indeed be the case based on altered ASF1 and SLBP levels in late passage cells, although the functional consequences of this observation remain to be demonstrated3. Vice versa, it is unclear if the drop in yeast his-tone levels is influenced by the DNA damage response, as it appears to be in humans. H3K56 acetylation and H3 incorporation through ASF1 have recently been implicated in DNA-break repair14,15, suggesting that DNA dam-age and replication may affect histone levels through similar pathways.

Finally, it is important to note that age-related histone loss, in yeast or human cells, appears to be tightly linked to cell division. Tyler and colleagues studied replicative lifespan, a system that determines the replica-tive potential of a given mother cell, but do not address how chronological ageing of a single cell affects histone levels2. Similarly, O’Sullivan et al. studied population-doubling-related defects in replicating fibroblasts and propose telomere shortening during cell division as a driving force behind histone depletion3. However, (mammalian) ageing affects all organs, and indeed, one of major interest to ageing research, the ageing brain, is largely post-mitotic but shows abundant signs of DNA damage and epigenomic deregulation16. Unlike histone loss, most of the DNA damage effects on the ageing epigenome highlighted in Figure 1 are independent of cell division and, therefore, have the potential to affect all cell types and tissues. If, and how, histone depletion occurs over the lifetime of a single cell remains to be determined. Nevertheless, the direct link between histone biosynthe-sis and lifespan regulation provides perhaps the most basic evidence for the importance of chromatin in ageing and further fuels the hope of some day being able to reverse age-related nuclear decline.

CompeTing finanCial inTereSTSThe author declares no competing interests.

Reduced H3/H4 Production

Histones H3/H4

Histones H3/H4

Replication (telomeric) DNA damage

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ReplicationDNA damage?

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Young Old

Reduced H3/H4 incorporation

Human �broblasts

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Figure 2 Replicative ageing causes histone loss in yeast and human fibroblasts. a and b highlight similarities and possible differences between human cells and yeast. Only nuclei are depicted. Blue ovals represent telomere‑associated silent‑ or hetero‑chromatin, generally seen at the nuclear periphery, grey ovals represent facultative heterochromatin, which can be found throughout the nuclei. Loss of histone H3/H4 may, at least in part, explain age‑related heterochromatin loss. In old cells, green areas indicate aberrant gene regulation, a possible consequence of histone loss and its concomitant chromatin alterations.

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1. Sinclair, D. A. & Oberdoerffer, P. Ageing Res. Rev. 8, 189–198 (2009).

2. Feser, J. et al. Mol. Cell 39, 724–735 (2010). 3. O’Sullivan, R. J., Kubicek, S., Schreiber, S. L. &

Karlseder, J. Nat. Struct. Mol. Biol. 17, 1218–1225 (2010).

4. Dang, W. et al. Nature 459, 802–807 (2009).5. Rodier, F. et al. Nat. Cell Biol. 11, 973–979

(2009).

6. Niedernhofer, L. J. et al. Nature 444, 1038–1043 (2006).

7. Bahar, R. et al. Nature 441, 1011–1014 (2006).8. Oberdoerffer, P. et al. Cell 135, 907–918 (2008).9. Su, C. et al. Embo J. 23, 1133–1143 (2004).10. Hoek, M. & Stillman, B. Proc. Natl Acad. Sci. USA

100, 12183–12188 (2003).11. Campisi, J. & d’Adda di Fagagna, F. Nat. Rev. Mol.

Cell Biol. 8, 729–740 (2007).

12. Bodnar, A. G. et al. Science 279, 349–352 (1998).13. Choudhury, A. R. et al. Nat Genet. 39, 99–105

(2007).14. Das, C., Lucia, M. S., Hansen, K. C. & Tyler, J. K.

Nature 459, 113–117 (2009).15. Tjeertes, J. V., Miller, K. M. & Jackson, S. P. Embo J.

28, 1878–1889 (2009).16. Yankner, B. A., Lu, T. & Loerch, P. Annu. Rev. Pathol.

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Bending the path to TORBrian m. Wiczer, adem Kalender and george Thomas

Cells sense and respond to physical stresses through mechanotransduction, a process that converts mechanical stimuli into biochemical signals. The bending of primary cilia has now been shown to modulate TOR signalling to negatively regulate cell size.

Our bodies have developed multiple sys-tems to respond to many different types of mechanical stimuli. When a person shouts from across a room, the sound waves bend the stereocilia on the surface of hair cells in the inner ear, converting those waves into electri-cal impulses that are interpreted by our brains as sound. Similarly, stretching after exercise activates a number of biochemical pathways in our muscle cells, including those involved in growth. Even the compression of cartilage during a walk alters pathways involved in matrix synthesis and maintenance. On page 1115 of this issue, Boehlke et al.1 demonstrate that the bending of primary cilia that line the kidney epithelium inhibits cell growth path-ways to reduce cell size (Fig. 1).

Primary cilia are non-motile, microtubule-based sensory organelles that extend from the surface of nearly every cell type in vertebrates. They are segregated into two basic compart-ments — the axoneme, which is composed of organized sets of microtubules containing acetylated tubulin, and the basal body, which consists of γ-tubulin-containing microtu-bules. The basal body is located at the base of the axoneme and is required for ciliogenesis. Although primary cilia were originally thought to be an evolutionary leftover, our knowledge of this functionally enigmatic piece of cell machinery, has exploded over the last decade. Primary cilia have been reported to be the base

of operation of vital developmental pathways, such as the hedgehog- and Wnt-signalling cas-cades2. Primary cilia are essential for develop-ment and cellular differentiation2, during which they are thought to aid in the maintenance of cell polarity. Disruption of primary cilia can lead to disorders including polydactyly, cogni-tive impairment, sterility and even obesity.

Polycystic kidney disease (PKD), a genetic disorder resulting in fluid-filled cysts and enlarged kidneys, is also thought to be the result of disrupted cilia-mediated signalling. The dominant form of PKD arises because of mutations in the genes encoding polycystin 1 (a G protein-coupled receptor) and polycystin 2 (a mechanosensitive cation channel)3. These polycystins are localized at the primary cilium (Fig. 1), and studies have since revealed that these polycystins mediate the calcium signal elicited in response to the bending of primary cilia2,3. This suggests that defects in calcium sig-nalling due to the lack of functional primary cilia might instigate the formation of cysts.

Interestingly, polycystin 1 was also found to co-localize with the mammalian target of rapamycin (mTOR) and to interact with tuberin (TSC2), a component of the tumour suppressor Rheb GTPase-activating protein complex, termed TSC1–TSC2 (ref. 4). This protein complex negatively regulates the nutrient/growth factor-sensitive mTOR com-plex 1 (mTORC1) by increasing the GTPase activity of Rheb, thereby preventing it from functioning as a positive effector of mTORC1. Polycystin 1 is also able to negatively regulate mTORC1 signalling5, presumably through its association with TSC2. Furthermore, mTORC1 signalling is increased in PKD cysts,

and treating animals manifesting PKD with the mTORC1 inhibitor, rapamycin, reduces cyst formation4.

Armed with this knowledge, and given the role of downstream effectors of mTORC1, such as S6K1 (refs 6, 7), in controlling cell growth, Boehlke et al.1 hypothesized that primary cilia are regulators of cell size. Using PKD as a starting point, they focused their attention on the kidney and found that mice harbouring a mutation in the kinesin subunit, Kif3a, which disrupts the formation of primary cilia, have larger renal collecting duct cells than their wild-type counterparts. However, inducible disruption of cilia in cultured kidney epithe-lial cells, showed no cell-size difference under normal conditions. This led the authors to hypothesize that it is the action of primary cilia bending, which would normally occur in the presence of fluid flow that is important for the regulation of cell size. Taking advantage of cell- culture flow chambers, Boehlke et al. found that fluid flow did, in fact, decrease the cell size of ciliated kidney epithelial cells and that cilia disruption through two independent mechanisms ablated this effect.

The authors went on to test whether primary cilia regulate cell size through the mTORC1 signalling cascade, and discovered that this pathway is indeed negatively regulated by fluid flow, an effect that was abolished in the absence of cilia. Additionally, ectopic expression of GTPase-inactive Rheb constitutively acti-vated the mTORC1 pathway in ciliated kidney epithelial cells, and resulted in increased cell size, an effect that was insensitive to fluid flow. Conversely, inhibition of the mTORC1 path-way, either through RNAi depletion of raptor,

Brian M. Wiczer, Adem Kalender and George Thomas are in the University of Cincinnati, Department of Cancer and Cell Biology, Metabolic Diseases Institute, College of Medicine, 2180 East Galbraith Road, Building D/Room 284, Cincinnati, OH 45237, USA. e‑mail: thomasg4@ucmail.uc.edu

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