Showing papers by "Leonard Guarente published in 2000"

Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase

Abstract: Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci, telomeres and the ribosomal DNA, and that also suppresses recombination in the rDNA and extends replicative life span. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3. Here we show that yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Our analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes.

Requirement of NAD and SIR2 for Life-Span Extension by Calorie Restriction in Saccharomyces cerevisiae

Abstract: Calorie restriction extends life-span in a wide variety of organisms. Although it has been suggested that calorie restriction may work by reducing the levels of reactive oxygen species produced during respiration, the mechanism by which this regimen slows aging is uncertain. Here, we mimicked calorie restriction in yeast by physiological or genetic means and showed a substantial extension in life-span. This extension was not observed in strains mutant for SIR2 (which encodes the silencing protein Sir2p) or NPT1 (a gene in a pathway in the synthesis of NAD, the oxidized form of nicotinamide adenine dinucleotide). These findings suggest that the increased longevity induced by calorie restriction requires the activation of Sir2p by NAD.

Genetic pathways that regulate ageing in model organisms

Abstract: Searches for genes involved in the ageing process have been made in genetically tractable model organisms such as yeast, the nematode Caenorhabditis elegans, Drosophila melanogaster fruitflies and mice. These genetic studies have established that ageing is indeed regulated by specific genes, and have allowed an analysis of the pathways involved, linking physiology, signal transduction and gene regulation. Intriguing similarities in the phenotypes of many of these mutants indicate that the mutations may also perturb regulatory systems that control ageing in higher organisms.

Sir2 links chromatin silencing, metabolism, and aging

Abstract: Aging is manifested by a progressive decline in vitality over time leading to death. Studies in budding yeast allow aging to be followed in individual pedigrees of cells, that is, those of mother cells, consequent to many rounds of cell division (Mortimer and Johnston 1959). These studies have led to the general conclusion that the silencing protein Sir2 is a limiting component of longevity; deletions of SIR2 shorten life span and an extra copy of this gene increases life span (Kaeberlein et al. 1999). Recent studies have spurred interest in Sir2 as a candidate longevity factor in a broad spectrum of eukaryotic organisms. SIR2 gene homologs have been found in a very wide range of organisms ranging from bacteria to humans (Brachmann et al. 1995). Moreover, a biochemical activity of Sir2 likely responsible for chromatin silencing, nicotinamide–adenine dinucleotide (NAD)-dependent histone deacetylase, has recently been discovered and shown to be broadly conserved (Imai et al. 2000). In this review, I will briefly discuss silencing as it pertains to SIR2 and its relationship to aging. I will then trace the studies that led to the discovery of the NADdependent histone deacetylase. I will next speculate how the regulation of Sir2 by NAD could represent the link between caloric intake and the pace of aging, which is widely observed in many organisms (Weindruch et al. 1986). Finally, I will present a speculative model of how a gradual disruption in chromatin silencing may occur and how such a change may cause aging.

Nuclear structure in normal and Bloom syndrome cells

Abstract: Bloom syndrome (BS) is a rare cancer-predisposing disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. BLM, the protein altered in BS, is a RecQ DNA helicase. This report shows that BLM is found in the nucleus of normal human cells in the nuclear domain 10 or promyelocytic leukemia nuclear bodies. These structures are punctate depots of proteins disrupted upon viral infection and in certain human malignancies. BLM is found primarily in nuclear domain 10 except during S phase when it colocalizes with the Werner syndrome gene product, WRN, in the nucleolus. BLM colocalizes with a select subset of telomeres in normal cells and with large telomeric clusters seen in simian virus 40-transformed normal fibroblasts. During S phase, BS cells expel micronuclei containing sites of DNA synthesis. BLM is likely to be part of a DNA surveillance mechanism operating during S phase.

Mutations in the WRN Gene in Mice Accelerate Mortality in a p53-Null Background

Abstract: Werner's Syndrome (WS) is a recessive genetic disease which shows premature onset of many pathologies normally associated with old age (18). Patients with WS appear normal during the first decade of life. The first manifestation of this disease is typically growth failure during adolescence. Subsequently, these patients suffer prematurely from a variety of age-related disorders: skin changes, osteoporosis, diabetes, accelerated atherosclerosis, and cancer, particularly sarcomas. Fibroblasts derived from individuals with WS divide many fewer times prior to senescence than do fibroblasts from age-matched control individuals (13). Genomic instability has been observed in WS cells, as chromosomal rearrangements (5, 19, 21) and as mutations within the hypoxanthine phosphoribosyltransferase gene (HPRT); in vivo, an increased frequency of HPRT mutant cells has been observed in patients with WS (2, 3, 14). The gene defective in WS, WRN, encodes a protein of 1,432 amino acids with similarity to the RecQ subfamily of DNA helicases (26). Although mutations throughout the WRN gene have been observed in the homozygous state, homozygosity for a mutation very near the 3′ end of the WRN open reading frame is sufficient to lead to the disease (15). A mouse knockout (KO) of the WRN gene has been described (10). Lebel and Leder deleted exons III and IV in the catalytic helicase domain of the WRN locus, a mutation predicted to eliminate catalytic function. Cells containing this mutation express an internally deleted, nearly full-length WRN protein. Homozygous mutant mice are viable, indicating that this particular mutation is not lethal. However Lebel and Leder showed a decreased embryonic survival of their mutant: on a C57BL/6-129/SvEv outbred background and on a 129/SvEv inbred background, the ratios of +/+:+/−:−/− mice born are 1:2.0:0.8 and 1:1.9:0.6, respectively. Mutant embryonic stem (ES) cells have an approximately sixfold increased mutation rate at the HPRT locus. They are also 10-fold more sensitive to camptothecin, a topoisomerase I inhibitor, and are two- to threefold more sensitive to etoposide, a topoisomerase II inhibitor. Late-passage mutant embryonic fibroblasts also show decreased saturation density in culture, although this was not evident in early-passage cells. The mice themselves, however, are healthy and fertile, showing no signs of premature organismic aging or increased rates of tumor formation. Thus, this KO does not recapitulate many of the phenotypes of human WS. Here, the generation and characterization of a WRN-null mouse mutant is described. Most phenotypes in the mutant are remarkably similar to the wild type. Cells from these animals are not hypersensitive to camptothecin, unlike those of Lebel and Leder. Most interestingly, the WRN−/− homozygous animal displays a shorter life span in the p53−/− background. We discuss this shortening with respect to a possible aging phenotype.

Association of the Bloom Syndrome Protein with Topoisomerase IIIα in Somatic and Meiotic Cells

Abstract: Bloom syndrome (BS) is characterized by genomic instability and cancer susceptibility caused by defects in BLM, a DNA helicase of the RecQ-family (J. German and N. A. Ellis, The Genetic Basis of Human Cancer, pp. 301–316, 1998). RecQ helicases and topoisomerase III proteins interact physically and functionally in yeast (S. Gangloff et al., Mol. Cell. Biol., 14: 8391–8398, 1994) and in Escherichia coli can function together to enable passage of double-stranded DNA (F. G. Harmon et al., Mol. Cell, 3: 611–620, 1999). We demonstrate in somatic and meiotic human cells an association between BLM and topoisomerase IIIα. These proteins colocalize in promyelocytic leukemia protein nuclear bodies, and this localization is disrupted in BS cells. Thus, mechanisms by which RecQ helicases and topoisomerase III proteins cooperate to maintain genomic stability in model organisms likely apply to humans.

Nijmegen Breakage Syndrome Disease Protein and MRE11 at PML Nuclear Bodies and Meiotic Telomeres

Abstract: Nijmegen breakage syndrome is a disease characterized by immunodeficiency, genomic instability, and cancer susceptibility. The gene product defective in Nijmegen breakage syndrome, p95, associates with two other proteins, MRE11 and RAD50. Here we demonstrate that in the absence of DNA damage, a portion of p95 and MRE11 is concentrated in PML nuclear bodies (NBs); MRE11 localization to the NBs is p95-dependent. In mammalian meiocytes, these proteins are specifically found at the telomeres. These results implicate the NBs in the maintenance of genomic stability and suggest that p95 and MRE11 may have roles in telomere maintenance in mammals, analogous to the role their homologues play in yeast.

A common muscarinic pathway for diapause recovery in the distantly related nematode species Caenorhabditis elegans and Ancylostoma caninum

Abstract: Converging TGF-β and insulin-like neuroendocrine signaling pathways regulate whether Caenorhabditis elegans develops reproductively or arrests at the dauer larval stage. We examined whether neurotransmitters act in the dauer entry or recovery pathways. Muscarinic agonists promote recovery from dauer arrest induced by pheromone as well as by mutations in the TGF-β pathway. Dauer recovery in these animals is inhibited by the muscarinic antagonist atropine. Muscarinic agonists do not induce dauer recovery of either daf-2 or age-1 mutant animals, which have defects in the insulin-like signaling pathway. These data suggest that a metabotropic acetylcholine signaling pathway activates an insulin-like signal during C. elegans dauer recovery. Analogous and perhaps homologous cholinergic regulation of mammalian insulin release by the autonomic nervous system has been noted. In the parasitic nematode Ancylostoma caninum, the dauer larval stage is the infective stage, and recovery to the reproductive stage normally is induced by host factors. Muscarinic agonists also induce and atropine potently inhibits in vitro recovery of A. caninum dauer arrest. We suggest that host or parasite insulin-like signals may regulate recovery of A. caninum and could be potential targets for antihelminthic drugs.

Methods for identifying agents that alter NAD-dependent deacetylation activity of a SIR2 protein

Abstract: Methods of identifying agents which alter the NAD-dependent deacetylation activity of a Sir2 protein or a fragment of a Sir2 protein are disclosed. The acetylated protein can be a nuclear protein, such as a histone protein, or a cytoplasmic protein. The Sir2 protein employed in the methods can include at least a core domain of a Sir2 protein, such as a human Sir2 protein.