SIRT1 Controls Circadian Histone Acetylation

The finding that CLOCK is a HAT revealed that chromatin remodeling is intimately connected to circadian physiology (Doi et al., 2006; Grimaldi et al., 2007). The remarkable effect of SIRT1 on clock gene expression (Figures 2 and and3)3) suggested that this effect may be mediated by changes in histone acetylation at specific sites. We analyzed the acetylation at Lys9 and Lys14 of histone H3 because we had previously found these to be preferential sites of CLOCK’s HAT activity (Doi et al., 2006), and also because SIRT1 deacetylase function was described to be targeted to these lysines (Imai et al., 2000). We performed chromatin immunoprecipitation (ChIP) assays using WT MEFs stimulated by a serum shock. Acetylation of H3 at the Dbp transcription start site (TSS) follows a circadian profile (Figure 3B and Ripperger and Schibler, 2006), presumably due to the concerted action of CLOCK and of an HDAC. Pharmacological treatment with the SIRT1 inhibitors (splitomicin and NAM) induced a loss in the circadian oscillation of acetylation, generating a noncyclic, high level of Lys9/Lys14 acetylation (Figures 3C and 3D).
Next, we extended this analysis to the MEFs from the Sirt1−/− mice. Again, histone H3 displays a robust cyclic acetylation at the Dbp promoter in WT MEFs, while genetic ablation of Sirt1 results in a constitutive, high acetylation at Lys9/Lys14 (Figures 4A and 4B). Thus, SIRT1 plays a critical role in maintaining a controlled rhythmicity in histone acetylation, thereby contributing to circadian chromatin remodeling.