Summary Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging and the accompanying changes to the chromatin are unclear. Here we report a mechanism whereby altering chromatin structure regulates lifespan. We show that normal aging is accompanied by a profound loss of histone proteins from the genome. Indeed, yeast lacking the histone chaperone Asf1 or acetylation of histone H3 on lysine 56 are short lived and this appears to be at least partly due to their having decreased histone levels. Conversely, increasing the histone supply by inactivation of the Hir (histone information regulator) complex or overexpression of histones dramatically extends lifespan, via a pathway that is distinct from previously known pathways of lifespan extension. This study indicates that maintenance of the fundamental chromatin structure is critical for slowing down the aging process and reveals that increasing the histone supply extends lifespan.

The hydrophilic amino-terminal sequences of histones H3 and H4 extend from the highly structured nucleosome core. Here we examine the importance of the amino termini and their position in the nucleosome with regard to both nucleosome assembly and gene regulation. Despite previous conclusions based on nonphysiological nucleosome reconstitution experiments, we find that the histone amino termini are important for nucleosome assembly in vivo and in vitro. Deletion of both tails, a lethal event, alters micrococcal nuclease-generated nucleosomal ladders, plasmid superhelicity in whole cells, and nucleosome assembly in cell extracts. The H3 and H4 amino-terminal tails have redundant functions in this regard because the presence of either tail allows assembly and cellular viability. Moreover, the tails need not be attached to their native carboxy-terminal core. Their exchange re-establishes both cellular viability and nucleosome assembly. In contrast, the regulation of GALl and the silent mating loci by the H3 and H4 tails is highly disrupted by exchange of the histone amino termini.

Abstract. The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.

Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase-9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase-8. Based on these data, we propose a mechanism underlying a potent and selective anti-tumor effect of LCA in cultured human NB cells. Moreover, our finding that LCA kills cultured human breast cancer and rat glioma cells implies that it has a broad anti-tumor effect on cancer cells derived from different tissues and organisms.

Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan lifespan. A key function in this process involves the regulation of the cell cycle and stress responses including free radical scavenging. We employed yeast chronological and replicative lifespan assays, as well as oxidative stress assays, to explore the potential evolutionary conservation of function between the FOXOs and the yeast forkhead box transcription factors FKH1 and FKH2. We report that the deletion of both FKH genes impedes normal lifespan and stress resistance, particularly in stationary phase cells, which are non-responsive to caloric restriction. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress response. Here we show the Anaphase-Promoting Complex (APC) genetically interacts with the Fkh pathway, likely working in a linear pathway under normal conditions, as fkh1Δ fkh2Δ post-mitotic survival is epistatic to that observed in apc5CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5CA fkh1Δ fkh2Δ, while increased expression of either FKH rescues APC mutant growth defects. This study establishes the FKHs role as evolutionarily conserved regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism under certain conditions, likely through combined regulation of stress response, genomic stability, and cell cycle regulation.