Sirt1 is an NAD+-dependent deacetylase that plays a role in cellular processes such as transcriptional regulation, stress response, longevity and apoptosis. Sirt1 deacetylates histone proteins and certain transcription factors such as p53, CTIP2 (chicken ovalbumin upstream promoter-transcription factor-interacting protein 2), FOXO (forkhead box O) and NF-kappaB (nuclear factor kappaB). To identify potential Sirt1-interacting factors, we performed a yeast two-hybrid screen. The screen identified TLE1 (transducin-like enhancer of split-1) as a possible Sirt1-interacting factor, which was then confirmed by co-immunoprecipitation. TLE1 is a non-DNA binding co-repressor for several transcriptional factors including NF-kappaB. We have demonstrated using co-transfection assays that Sirt1 and TLE1 repress NF-kappaB activity. The catalytic mutant of Sirt1, Sirt1-H363Y, and the N-terminal Sirt1 fragment (amino acids 1-270) also show similar repression activity, suggesting that the deacetylase activity of Sirt1 may not be critical for its effect on NF-kappaB activity. Furthermore, analysis in Sirt1-null MEFs (murine embryonic fibroblasts) and HeLa cells stably expressing siRNA (small interfering RNA) specific to Sirt1 or TLE1 demonstrate that both Sirt1 and TLE1 are required for negative regulation of NF-kappaB activity. Taken together, these results suggest that the interaction between Sirt1 and TLE1 is important for mediating repression of NF-kappaB activity.

The TATA-binding protein (TBP) nucleates the assembly and determines the position of the preinitiation complex at RNA polymerase II-transcribed genes. We investigated the importance of two conserved residues on the DNA binding surface of Saccharomyces cerevisiae TBP to DNA binding and sequence discrimination. Because they define a significant break in the twofold symmetry of the TBP-TATA interface, Ala100 and Pro191 have been proposed to be key determinants of TBP binding orientation and transcription directionality. In contrast to previous predictions, we found that substitution of an alanine for Pro191 did not allow recognition of a reversed TATA box in vivo; however, the reciprocal change, Ala100 to proline, resulted in efficient utilization of this and other variant TATA sequences. In vitro assays demonstrated that TBP mutants with the A100P and P191A substitutions have increased and decreased affinity for DNA, respectively. The TATA binding defect of TBP with the P191A mutation could be intragenically suppressed by the A100P substitution. Our results suggest that Ala100 and Pro191 are important for DNA binding and sequence recognition by TBP, that the naturally occurring asymmetry of Ala100 and Pro191 is not essential for function, and that a single amino acid change in TBP can lead to elevated DNA binding affinity and recognition of a reversed TATA sequence.Transcription of protein-encoding genes in eukaryotes requires the assembly of a large, multiprotein complex at the promoter. This preinitiation complex contains RNA polymerase II, six different general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), and a large number of accessory proteins that mediate the response to transcriptional activators and repressors (46). Of these proteins, TFIID is unique in its ability to bind DNA in a sequence-specific manner in the absence of other members of the preinitiation complex. Sequence recognition by TFIID is directed by the TATA binding protein (TBP) subunit. As the initial step in the assembly of the preinitiation complex, the binding of TFIID to the TATA box is a highly regulated event. In vivo and in vitro studies have shown that many transcriptional activators stimulate transcription by facilitating the rate-limiting binding of TFIID to the TATA box (35,41,48,50,67,79), while certain transcriptional repressors, including histones, impede this step (4,36,41,83).As the central component of the general transcription machinery, the TBP-TATA box complex has been the subject of intense study. Computational studies have identified an 8-bp consensus sequence for the TATA box [TATA(A/T)A(A/T)N] (9). The importance of this sequence for transcriptional activity has been confirmed by systematic mutational studies (14,25,82). These previous experiments, along with crystallographic analyses (62), have demonstrated that base substitutions in the 5Ј half of the consensus sequence are particularly detrimental to binding by TBP. X-ray crystal structures have been solved for TBP-TATA complexes from divers...