Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic doublestranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells. D ouble-stranded RNA (dsRNA) can trigger silencing of homologous gene expression by a mechanism termed RNAi (for RNA-mediated interference) (1). RNAi is an evolutionarily conserved phenomenon and a multistep process that involves generation of active small interfering RNA (siRNA) in vivo through the action of an RNase III endonuclease, Dicer. The resulting 21-to 23-nt siRNA mediates degradation of the complementary homologous RNA (reviewed in refs. 2-4). RNAi has been used as a reverse genetic tool to study gene function in multiple model organisms, including plants, Caenorhabditis elegans, and Drosophila where large dsRNAs efficiently induce gene-specific silencing (1, 5-7).One obstacle to achieving RNAi in mammals is that dsRNAs longer than 30 nt will activate an antiviral response, leading to the nonspecific degradation of RNA transcripts and a general shutdown of host cell protein translation (8, 9). As a result, the long dsRNA, with a few exceptions (10, 11), does not produce RNAi activity, and RNAi therefore is not a general method for silencing specific genes in mammalian cells. This obstacle has been recently overcome by Tuschl and colleagues (12) who found that gene-specific suppression in mammalian cells can be achieved by vitro-synthesized siRNA that are 21 nt in length, long enough to induce gene-specific suppression, but short enough to evade the host interferon response.In this article, we describe a DNA vector-based approach to achieve RNAi in mammalian cells. With this approach, small RNAs are predicted to be synthesized from a DNA template under the control of an RNA polymerase III (Pol III) promoter in transfected cells. Pol III has the advantage of directing the synthesis of small, noncoding transcripts whose 3Ј ends are defined by termination within a stretch of 4-5 thymidines (Ts) (13). These properties make it possible to use DNA templates to synthesize, in vivo, small RNAs with structural features close to what has been found to be required for active siRNAs synthesized in vitro (14).Using this DNA vector-based RNAi approach, we show that transfected as well as endogenous genes can be efficiently inhibited. We have examined the effects of the in vivosynthe...

A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.

The transcriptional co-repressor CtBP (C-terminal binding protein) is implicated in tumorigenesis because it is targeted by the adenovirus E1A protein during oncogenic transformation. Genetic studies have also identified a crucial function for CtBP in animal development. CtBP is recruited to DNA by transcription factors that contain a PXDLS motif, but the detailed molecular events after the recruitment of CtBP to DNA and the mechanism of CtBP function in tumorigenesis are largely unknown. Here we report the identification of a CtBP complex that contains the essential components for both gene targeting and coordinated histone modifications, allowing for the effective repression of genes targeted by CtBP. Inhibiting the expression of CtBP and its associated histone-modifying activities by RNA-mediated interference resulted in alterations of histone modifications at the promoter of the tumour invasion suppressor gene E-cadherin and increased promoter activity in a reporter assay. These findings identify a molecular mechanism by which CtBP mediates transcriptional repression and provide insight into CtBP participation in oncogenesis.

Yin Yang 2 (YY2) is a multifunctional zinc-finger transcription factor that belongs to YY family. Unlike the well-characterized YY1, our understanding regarding the biological functions of YY2 is still very limited. Here we found for the first time that in contrast to YY1, which had been reported to be oncogenic, the expression level of YY2 in tumor cells and/or tissues was downregulated compared with its expression level in the normal ones. We also demonstrated that YY2 exerts biological function contrary to YY1 in cell proliferation. We elucidated that YY2 positively enhances p21 expression, and concomitantly, its silencing promotes cells to enter G2/M phase and enhances cell proliferation. Furthermore, we found that YY2 regulation on p21 occurs p53-dependently. Finally, we identified a novel YY2 binding site in the promoter region of tumor suppressor p53. We found that YY2 binds to the p53 promoter and activates its transcriptional activity, and subsequently, regulates cell cycle progression via p53/p21 axis. Taken together, our study not only identifies YY2 as a novel tumor suppressor gene that plays a pivotal role in cell cycle regulation, but also provides new insights regarding the regulatory mechanism of the conventional p53/p21 axis.