We found that the endogenous liver SIRT1 obtained by immunoprecipitation from entrained mice, although constant in levels, displays circadian HDAC activity that peaks at ZT15 (Figure 1D), a time that remarkably parallels the minimal transcriptional levels of various clock-controlled genes in the liver (such as dbp,Figure 1C).
To confirm that equal levels of SIRT1 could still generate circadian HDAC function, we complemented cellular extracts with equal amounts of recombinant SIRT1 protein and acetylated p53 peptide as SIRT1 substrate (Luo et al., 2001; Vaziri et al., 2001). This assay likely reflects the intracellular relative concentrations of NAD+ and NAM, or of yet undefined circadian metabolites, whose ratio determines SIRT1 activity (Imai et al., 2000; Luo et al., 2001). Extracts from wild-type (WT) MEFs were prepared every 6 hr post-serum shock (Figure 1E), and liver extracts were prepared at four different ZT from entrained mice (Figure 1F). Also, under these conditions we found that SIRT1 deacetylase activity is rhythmic, peaking 24 hr post-serum shock in MEFs (Figure 1E) and at ZT15 in the liver (Figure 1F). Importantly, the peak of SIRT1 deacetylase activity is consistent with the cyclic acetylation of histone H3 at promoters of clock-controlled genes; at 24 hr, this acetylation is at its lowest levels (see later,Figure 4).

SIRT1 Contributes to the Stringency of Circadian Gene Expression

The finding that SIRT1 activity is regulated in a circadian manner prompted us to investigate its role in clock gene expression and chromatin remodeling. MEFs generated from WT and Sirt1 null mice were serum-shocked, RNA was prepared at various times, and quantitative RNase protection assay was used to monitor dbp circadian gene transcription (Figure 2A). The analysis reveals that genetic ablation of SIRT1 causes changes in circadian gene expression, including an overall increase in the transcription levels and a broadening of the oscillation cycles, with earlier onsets of increasing transcription and later decreases. Importantly, the expression of a nonoscillating gene (clock) is not affected (Figure 2A). These observations are consistent with SIRT1 having a role in controlling the stringency of circadian gene expression and being involved in the oscillatory silencing that periodically follows a transcriptional peak. These results were confirmed by quantitative RT-PCR and reproduced also on the Per2 gene (Figure 2B). Finally, a point-by-point circadian analysis of the differential transcription of both dbp and per2 between WT and Sirt1−/− MEFs confirms that the lack of Sirt1 induces a significantly higher transcriptional efficacy at specific times that normally precede and follow each circadian expression peak (Figure 2C).