The NAD-dependent deacetylase SIR2 and the forkhead transcription factor DAF-16 regulate lifespan in model organisms, such as yeast and C. elegans. Here we show that the mammalian SIR2 ortholog SIRT1 deacetylates and represses the activity of the forkhead transcription factor Foxo3a and other mammalian forkhead factors. This regulation appears to be in the opposite direction from the genetic interaction of SIR2 with forkhead in C. elegans. By restraining mammalian forkhead proteins, SIRT1 also reduces forkhead-dependent apoptosis. The inhibition of forkhead activity by SIRT1 parallels the effect of this deacetylase on the tumor suppressor p53. We speculate how down-regulating these two classes of damage-responsive mammalian factors may favor long lifespan under certain environmental conditions, such as calorie restriction.

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic β cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In β cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in β cells to affect insulin secretion.

SummaryWe generated mice that overexpress the sirtuin, SIRT1. Transgenic mice have been generated by knocking in SIRT1 cDNA into the β β β β -actin locus. Mice that are hemizygous for this transgene express normal levels of β β β β -actin and higher levels of SIRT1 protein in several tissues. Transgenic mice display some phenotypes similar to mice on a calorierestricted diet: they are leaner than littermate controls; are more metabolically active; display reductions in blood cholesterol, adipokines, insulin and fasted glucose; and are more glucose tolerant. Furthermore, transgenic mice perform better on a rotarod challenge and also show a delay in reproduction. Our findings suggest that increased expression of SIRT1 in mice elicits beneficial phenotypes that may be relevant to human health and longevity.

Gene expression is controlled by gene-specific trans-acting factors and general transcription proteins recognizing discrete elements in promoters-enhancers and operating in the context of chromatin structures (reviewed in reference 45). The fundamental chromatin unit is the nucleosome, a DNA-protein complex formed by 146 bp of DNA that wraps around core histones, H2A, H2B, H3, and H4. Histones are among the most conserved proteins in evolution, having in their C-terminal regions a 65-amino-acid histone fold motif (HFM) with low sequence identity (14 to 18%) and high structural resemblance (2). It is minimally composed of three ␣ helices, ␣1, ␣2, and ␣3. The long central ␣2 (28 amino acids) is flanked by two short ones separated by loop-strand regions; this structure enables histone-histone interactions and contacts with the DNA (2, 29). Histones also possess N-terminal tails that are acetylated at specific lysine residues. This process is highly regulated by several acetylases, including some transcriptional coactivators, and is thought to contribute to regulation of gene expression in multiple ways: facilitation of transcription factors binding (43) and of RNA polymerase progression (42) and chromatin solubility (38). Formation of nucleosomes is a stepwise phenomenon initiated by tetramerization of H3-H4 through H3-H3 interactions and binding of the tetramer to DNA; this nucleates wrapping of DNA, but formation of a stable complex requires subsequent association of two H2A-H2B dimers, mainly through H2B-H4 contacts (12,(16)(17)(18) (15); (iii) an hTAF II 80-like subunit (PAF65␣) of the P/CAF histone acetylase complex (37); and (iv) two subunits (NF-YB and NF-YC) of NF-Y, a ubiquitous CCAAT-binding heterotrimeric complex (3).The CCAAT box is present in 25% of eukaryotic promoters, with a strong position preference at Ϫ60 to Ϫ80 (33). In vivo footprinting of several promoters invariably found this element protected, and functional experiments indicate that it plays an important and sometimes essential role in transcription. NF-Y, originally identified as the protein binding to the major histocompatibility complex (MHC) class II Ea promoter Y box, has an almost absolute requirement for the five CCAAT nucleotides and has been implicated in the activation of most, if not all, CCAAT-containing promoters (reviewed in references 31 and 33). It is composed of three different subunits, NF-YA, NF-YB, NF-YC, each containing evolutionarily conserved domains. NF-YB and NF-YC belong to the H2A-H2B subfamily; their dimerization, elicited through strong HFM interactions, is required for NF-YA binding. For this function, a complex surface resulting from heterodimerization and comprising specific residues in NF-YC ␣1, in NF-YB ␣2, and at the C terminus of ␣3 is necessary (21,40). Detailed mutational analysis of NF-YA and of the yeast homologue HAP2 identified a 56-* Corresponding author. Mailing address:

NF-Y is a sequence-specific evolutionary conserved activator binding to CCAAT boxes with high affinity and specificity. It is a trimer formed by NF-YA and two putative histone-like subunits, NF-YB and NF-YC, showing similarity to histones H2B and H2A, respectively. We investigated the relationships between NF-Y and chromatin using an Artemia franciscana chromatin assembly system with plasmids containing the Major HistoCompatibility complex class II Ea promoter. The NF-Y trimer, but not single subunits, protects the Y box in the presence of reconstituted chromatin, and it can bind the target sequence during and after assembly. Using reconstitution assays with purified chicken histones, we show that NF-Y associates with preformed nucleosomes. Translational analysis of various Ea fragments of identical length in which the CCAAT box is at different positions indicated that the lateral fragment was slightly more prone to NF-Y binding. In competition experiments, NF-Y is able to prevent formation of nucleosomes significantly. These data support the idea that NF-Y is a gene-specific activator with a built-in capacity to interface with chromatin structures.Regulation of gene expression is a complex set of events controlled by gene-specific trans-acting factors and general transcription proteins recognizing cis-acting elements in promoters and enhancers and operating in the context of higher order chromatin structures (1). The fundamental chromatin unit is the nucleosome, a DNA-protein complex formed by core histones (H2A, H2B, H3, H4) wrapped around 146 base pairs of DNA. Histones are among the most conserved proteins in evolution; analysis of their quasi-invariant COOH-terminal sequences revealed a 65-amino acid histone fold motif shared by all histone proteins, with low sequence identity, 14/18%, and high structural resemblance (2). It is composed of three ␣-helices: a long central one of 28 amino acids flanked by two short ones separated by short loops/strand regions; this structure enables histones to dimerize with the companion subunit and to make non-sequence-specific contacts with the DNA (3). The latter function results from tetramerization of H3-H4, which first nucleates the wrapping of DNA and subsequently promotes the association of two H2A-H2B dimers. Recent computational analysis of protein data banks identified additional polypeptides involved in the process of transcriptional regulation which contain putative histone fold domains (4) The CCAAT box is a widely distributed regulatory sequence, present in 25% of promoters and enhancers, very often at position Ϫ60/Ϫ80 (8). Functional experiments indicate that the CCAAT box plays an important role in essentially all such promoters. NF-Y, originally identified as the protein binding to the major histocompatibility complex class II Ea promoter Y box, has an almost absolute requirement for these five nucleotides (9) and has been implicated in the activation of more than 100 promoters (10). NF-Y genes have been cloned in different species, including yeast, plants,...