Fig 2: SIRT1 and MLL1 interact(a) Co-immunoprecipitation experiments from 293 cells transiently transfected with the indicated plasmids (IP, immunoprecipitation; IB, immunoblot). (b) Co-immunoprecipitation experiments in Mll1-/- cells and Mll1-/- stably transfected with Flag–MLL1. (c) Co-immunoprecipitation experiments from nuclear extracts of dexamethasone synchronized MEFs with anti SIRT1 antibody. Western blots were performed from immunoprecipitates (top panel) or nuclear extracts (input) at indicated circadian times (CT, hors post DEX synchronization). (d) Representation of the localization of the MLL1 truncation domains (M1–M6) used for cloning. (e) Co-immunprecipitation experiments using protein extracts from 293 cells transiently expressing the indicated plasmids. (f) Mammalian two–hybrid assays in 293 cells transiently transfected with the CLOCK or CLOCK?19 ‘bait’ plasmids (GAL4–CLOCK and GAL4–CLOCK?19) and the respective ‘prey’ plasmid (VP16 empty control, VP16–M1 to M6). The results are expressed as relative light units (RLU), representing the activation of the (GAL4–UAS)6–luciferase reporter normalized to the internal lacZ control, and units from VP16 empty control were set to 1. Data are presented as the mean±s.e.m. of three independent samples. *P < 0.05, **P < 0.01, ***P < 0.001, two tailed t-test.

Fig 3: SIRT1 and NAD+ levels regulate circadian H3K4 trimethylation(a) ChIP from histone H3K4me3 at specific regions of Dbp gene in mouse embryonic fibroblasts (MEFs) from wild type (WT) or Sirt1-/- mice, and WT MEFs treated with EX527 (50µM during 18 hours). Immunoprecipitated chromatin was quantified by real-time PCR, and data from WT samples at time 0 was set to 1. (b) H3K4me3 ChIP assays at the transcription start site (tss) of circadian genes in livers from wild type mice and Sirt1 KO littermates (Sirt1?Ex4) harvested at the indicated zeitgeber times (ZT, hours after lights on). (c) ChIP from H3K4me3 at HoxA9 tss either in MEFs (top panel) or in livers (bottom panel). (d) Luciferase assay on Dbp-luc from 293 cells transfected with MLL1 and CLOCK–BMAL1 plasmids. (ß–Nicotinamide adenine dinucleotide: NAD+ 2 mM, ß–Nicotinamide mononucleotide: ß–NMN 5 mM, Nicotinic acid: NA 10 mM, Nicotinamide: NAM 10 mM). Light units were normalized to an internal LacZ control, and the relative light units (RLU) from basal expression of Dbp-luc from non-treated cells were set to 1 (means ± s.e.m. of four independent experiments). (e) ChIP from histone H3K4me3 at indicated regions of the Dbp gene from synchronized MEFs either untreated or treated two hours before harvest with 1 mM of NAD+or 1 mM of ß–NMN. For all ChIP experiments, immunoprecipitated chromatin was quantified by real-time PCR, and means ± s.e.m. of three independent replicates analyzed in triplicates are presented. *P < 0.05, **P < 0.01, ***P < 0.001, two tailed t-test.

Fig 4: (a–c) Model depicting the NAD+–dependent molecular interplay between SIRT1 and MLL1 for the control of circadian gene expression and epigenetic states (see discussion).

Fig 5: MLL1 H3K4 methyltransferase activity is modulated by SIRT1(a) Luciferase assays using Dbp-luciferase reporter in 293 cells transiently transfected with the indicated plasmids. Increasing amounts of plasmids encoding SIRT1 and the mutant form SIRT1(H363Y) were assessed. Light units were normalized to an internal LacZ control, and the relative light units (RLU) from basal expression of Dbp-luc plasmid were set to 1 (means ± s.e.m. of three independent experiments). (b) Luciferase assays using Dbp–luciferase (top panel) or Per1–luciferase (bottom panel) reporters. Plasmids encoding MLL1 and CLOCK–BMAL1 were transiently transfected in 293 cells. EX527 treatments were performed 18 hours prior to luciferase assays. Light units were normalized to an internal LacZ control, and the relative light units (RLU) from basal expression of the reporter from non-treated cells were set to 1 (Means ± s.e.m. of four independent experiments). (c) In vitro MLL1 histone lysine methyltransferase assay (HKMT) on recombinant histone H3. The top panel corresponds to a western blot from myc-tagged MLL1 protein used for each assay. CBB, Coomassie Brilliant Blue staining from the gel used to measure radioactive H3. Methyl H3, autoradiography from H3 radioactively labeled with S-adenosyl-L-[methyl-3H]-methionine. Bottom panel, histogram representing quantification of radioactive H3 by liquid scintillation. The counts from the assay without MLL1 protein were set to 1(means ± s.e.m. of three independent measurements). Statistical analyses using two tailed paired t–test are presented; *P < 0.05, **P < 0.01, ***P < 0.001.