The yeast Sir2p protein has an essential role in maintaining telomeric and mating type genes in their transcriptionally inactive state. Mammalian cells have a very large proportion of their genome inactive and also contain seven genes that have regions of homology with the yeast sir2 gene. One of these mammalian genes, sir2␣, is the presumptive mammalian homologue of the yeast sir2 gene. We set out to determine if sir2␣ plays a role in mammalian gene silencing by creating a strain of mice carrying a null allele of sir2␣. Animals carrying two null alleles of sir2␣ were smaller than normal at birth, and most died during the early postnatal period. In an outbred background, the sir2␣ null animals often survived to adulthood, but both sexes were sterile. We found no evidence for failure of gene silencing in sir2␣ null animals, suggesting that either SIR2␣ has a different role in mammals than it does in Saccharomyces cerevisiae or that its role in gene silencing in confined to a small subset of mammalian genes. The phenotype of the sir2␣ null animals suggests that the SIR2␣ protein is essential for normal embryogenesis and for normal reproduction in both sexes.The irreversible inactivation of genes occurs in the mammalian genome in the context of X chromosome inactivation, genomic imprinting, and allelic exclusion. Sporadic silencing of certain tumor suppressor genes also occurs during the development of some cancers (reviewed in reference 31). The events responsible for initiating gene silencing and maintaining silent genes in their inactive form are widely believed to rely on DNA methylation and nucleosome modifications (reviewed in references 9, 19, 22, and 39). The molecular events comprising histone modifications and their relationship to gene inactivation have been intensively investigated in the budding yeast Saccharomyces cerevisiae, in which telomere-dependent gene silencing and maintenance of the silent mating type loci have indicated that the Sir2p protein plays a central role in maintaining genes in their inactive configuration (reviewed in reference 17).Sir2p has NAD ϩ -dependent histone deacetylase activity (18,24,43,45), and the catalytic domain of Sir2p is present in four other genes in S. cerevisiae (10). This domain is also present in genes encoded by the genomes of the most primitive organisms (7) as well as in mammals (13). Seven mammalian genes carry the SIR2 catalytic domain, and this domain can substitute for the same domain in yeast Sir2p (42).Yeast Sir2p plays an important role not only in gene silencing but also in a variety of other biological processes. These include regulation of the cell cycle, DNA repair, DNA recombination, and aging (reviewed in references 14, 17, and 33).Given the extraordinary conservation of the SIR2 catalytic domain and the multitude of biological functions served by the yeast Sir2p, it seems likely that the related proteins in mammalian cells also have important functions. We were particularly interested in determining whether the mammalian sir2 homologues play ro...

Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.

The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. We have studied mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, and found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, CR did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction.

The function of the class III histone deacetylase, Sir2, in promoting lifespan extension is well established in small model organisms. By analogy, SirT1, the mammalian orthologue of Sir2, is a candidate gene to slow down aging and forestall the onset of age-associated diseases. We have used SirT1-null mice to study the function of SirT1 in susceptibility to tumorigenesis. The number of intestinal polyps induced in mice carrying the Apc min mutation was unaffected by the SirT1 genotype although the average polyp size was slightly smaller in the SirT1-null animals. Similarly, the presence or absence of SirT1 had no effect on incidence and tumor load of skin papillomas induced by the classical two-stage carcinogenesis protocol. We found that resveratrol topically applied to the skin profoundly reduced tumorigenesis. This chemoprotective effect was significantly reduced but not ablated in SirT1-null mice, suggesting that part of the protection afforded by resveratrol requires the SirT1-encoded protein. Thus, our results suggest that SirT1 does not behave like a classical tumor-suppressor gene but the antitumor activity of resveratrol is mediated at least in part by SirT1.

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The Pgk-1 gene encodes the housekeeping enzyme, 3-phosphoglycerate kinase, and is ubiquitously expressed. This gene resides on the X chromosome in mammals and is always expressed except where it is silenced along with most other genes on the inactive X chromosome of female somatic cells or male germ cells. The Pgk-1 promoter is in a region rich in nucleotides G and C. This promoter can efficiently drive high levels of expression of reporter genes such as E. coli lacZ and neo. We have determined that the 120 bp upstream of the transcription start site functions as a core promoter. Upstream of this is a 320 bp region which enhances transcription from the core promoter in an orientation and position independent fashion. This 320 bp region does not enhance transcription from the core promoter of the SV40 early region. Nuclear proteins bind to this 320 bp fragment although the restricted regions to which binding can be demonstrated with gel mobility shift assays suggests that the activity of the enhancer may be mediated by factors which bind at multiple sites each with low affinity.

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The protein encoded by the sirt1 gene is an enzyme, SirT1, that couples the hydrolysis of NAD(+) to the deacetylation of acetyl-lysine residues in substrate proteins. Mutations of the sirt1 gene that fail to encode protein have been introduced into the mouse germ line, and the animals homozygous for these null mutations have various physiological abnormalities. To determine which of the characteristics of these sirt1(-/-) mice are a consequence of the absence of the catalytic activity of the SirT1 protein, we created a mouse strain carrying a point mutation (H355Y) that ablates the catalytic activity but does not affect the amount of the SirT1 protein. Mice carrying point mutations in both sirt1 genes, sirt1(Y/Y), have a phenotype that is overlapping but not identical to that of the sirt1-null animals. The sirt1(Y/Y) phenotype is significantly milder than that seen in the sirt1(-/-) animals. For example, female sirt1(Y/Y) animals are fertile, while sirt1(-/-) females are sterile. On the other hand, both sirt1(-/-) and sirt1(Y/Y) male mice are sterile and hypermetabolic. We report that sirt1(Y/Y) mice respond aberrantly to caloric restriction, although the effects are more subtle than seen in sirt1(-/-) mice. Thus, the SirT1 protein has functions that can be attributed to the catalytic activity of the protein, as well as other functions that are conferred by the protein itself.