SIRT1 Is in a Chromatin Complex with CLOCK:BMAL1 on the Dbp Promoter

Based on the pattern of histone acetylation associated with circadian genes (Figures 3 and and4A),4A), it is conceivable that CLOCK and SIRT1 converge in a coordinate manner to the same regulatory regions. Thus, we decided to test whether SIRT1 is recruited to E-box elements present in the regulatory region of CLOCK: BMAL1-controlled genes. To do so, we performed a dual crosslinking ChIP assay and analyzed two E-box elements within the Dbp gene. As predicted (Ripperger and Schibler, 2006), CLOCK and BMAL1 are recruited to E-box elements in the Dbp gene in a time-dependent manner (Figure 4D). Importantly, we found that SIRT1 is jointly recruited to the same E-box elements in the Dbp gene. Furthermore, the presence of SIRT1 is temporally regulated and parallels the recruitment of the CLOCK:BMAL1 dimer (Figures 4D and 4E). Since SIRT1 is not a DNA-binding protein, we favor a scenario in which SIRT1 recruiting to a circadian promoter is mediated by the CLOCK:BMAL1 dimer. These results indicate that CLOCK:BMAL1 and SIRT1 coexist in a chromatin regulatory complex that operates on circadian promoters.

Direct Interaction of SIRT1 and CLOCK

The coordinated recruiting of the CLOCK:BMAL1 dimer and SIRT1 to circadian gene promoters suggested that these regulators may physically interact. To test this possibility we coexpressed SIRT1 with CLOCK in cultured cells. In coimmunoprecipitation experiments we reveal that SIRT1 interacts with CLOCK but not with PER2 (Figure 5A). Next, we wished to establish whether native, endogenous cellular SIRT1 interacts with CLOCK. Native SIRT1 can be coimmunoprecipitated with both CLOCK and BMAL1 in liver extracts, indicating that it interacts with the CLOCK:BMAL1 complex (Figure 5B). It is unclear why BMAL1 in transfected cells does not seem to coimmunoprecipitate with the SIRT1-CLOCK complex (Figure 5A), but the results on native proteins (Figure 5B) suggest the requirement of some specific physiological conditions.