Fig. 4.

Sirt-1 promotes CREB-dependent gene expression. (A) Forskolin-inducible physical association of CREB with Sirt-1 in PC12 cells. mycCREB or mycCREB-ΔLZ were transfected in PC12 cells and immunoprecipitated from protein lysates untreated or stimulated with Fsk for 30 min; the presence of Sirt-1 in immunocomplexes was verified by immunoblotting (Upper); anti-Ser133 CREB and anti-myc immunoblotting were used to confirm CREB phosphorylation by Fsk and assess the expression level of transfected CREB isoforms, respectively (Lower). (Ba) ChIP assays showing parallel interaction of CREB and Sirt-1 with the CRE-containing promoter regions of nNOS and PGC-1α in hippocampal neurons treated with NGF. Promoter amplification from total chromatin input is also reported as control. (b) CREB mediates Sirt-1 interaction with CRE-containing promoter regions. NGF-inducible binding of Sirt-1 to nNOS (Upper) and Sirt-1 promoter (Lower) are drastically reduced in hippocampal neurons lacking CREB. Binding of CREB to the same promoter regions confirms severe reduction of CREB binding in Cre-infected cells. rIgG (rabbit IgG) is a negative control for ChIP. Total chromatin input was equal throughout the lanes. (C) RT-PCR analysis showing reduced induction of nNOS and PGC-1α mRNA by NGF in cortical neurons treated with the Sirt-1 inhibitor Nicotinamide (NAM). (D) (a and b) Representative RT-PCR analysis of PGC-1α and nNOS mRNA expression in whole brains from WT and Sirt-1-deficient mice under both AL and CR (6 mo) feeding. Each lane represents a pool of two mice. Actin was used as loading control. (c) Western blot analysis of whole brain protein homogenates from WT and Sirt-1 KO mice (fed AL) indicating reduced expression of nNOS but normal levels of immunoreactive CREB and total histone H4. Anti-AcH4K16 and antiactin immunostaining confirm, respectively, reduced deacetylase activity in the SirtKO sample and equal protein loading in the two lanes.