Fig. 3.
Transcriptional regulation of Sirt-1 by CREB in neuronal cells. (Aa) Immunoblot analysis of whole-cell lysates from primary neurons (hippocampal or cortical) exposed to CREB-activating stimuli. NGF, 50 ng/mL; Fsk, 10 μM. Relevant bands are indicated by arrows. Relative densitometric values for the Sirt-1 band are indicated. CREB phosphorylation and Sirt-1 expression were assayed at different times (30 min and 16 h, respectively). (b) RT PCR analysis of Sirt-1 mRNA in cortical neurons treated with Fsk for 6 h. Actin was amplified as an internal loading control. Panels representative of several independent experiments. (Ba) Scheme displaying several putative CRE elements within the mouse Sirt-1 gene (MGSCv37 C57BL/6J, locus NC_000076). The segment inserted in the Sirt-1-Luc reporter gene, and primers used in ChIP studies (red arrows) are indicated. Two CRE half-sites internal to the segment are also highlighted; numbers are positions relative to the annotated TSS. Exons refer to transcript variant 1. The exon 2 box is shaded because this exon is absent in transcript variants 2 and 3. (b) Luciferase reporter assay confirming responsiveness of the 1738–2180 genomic fragment to Fsk and PKA in PC12 cells. Bars are fold-induction ± SD of triplicate samples; picture representative of two independent experiments. (c) ChIP assay showing NGF-induced binding of CREB to the 1824–2090 Sirt-1 region in hippocampal neurons. Minutes of stimulation are indicated. Sirt-1 promoter was amplified from the total chromatin input as quantitative control (Lower). (Ca) Deletion of CREB exon 10 in cultured CREBloxP/loxP hippocampal neurons 2 or 5 d after adenoviral delivery of Cre recombinase (Ad-Cre). Genomic DNA was amplified with two primers external to the recombination sites. Bands corresponding to undeleted (Upper) and deleted (Lower) alleles are indicated by arrows. (b) RT-PCR analysis revealing defective induction of Sirt-1 mRNA by NGF and Fsk (6 h) in CREB-deleted hippocampal neurons. Actin was amplified as loading control. Bands corresponding to deleted (ΔCREB) and residual undeleted CREB mRNA are indicated by arrows. Picture is representative of several independent experiments. (c) Western blot analysis of whole cell lysates from mock (Ad-GFP) and Cre-infected cells indicating reduced expression of Sirt-1 in the latter cell population. Actin band confirms equal protein loading.