NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1

Abstract

The circadian clock controls the transcription of hundreds of genes through specific chromatin-remodeling events. The histone methyltransferase mixed-lineage leukemia 1 (MLL1) coordinates recruitment of CLOCK–BMAL1 activator complexes to chromatin, an event associated with cyclic trimethylation of histone H3 Lys4 (H3K4) at circadian promoters. Remarkably, in mouse liver circadian H3K4 trimethylation is modulated by SIRT1, an NAD+-dependent deacetylase involved in clock control. We show that mammalian MLL1 is acetylated at two conserved residues, K1130 and K1133. Notably, MLL1 acetylation is cyclic, controlled by the clock and by SIRT1, and it affects the methyltransferase activity of MLL1. Moreover, H3K4 methylation at clock-controlled-gene promoters is influenced by pharmacological or genetic inactivation of SIRT1. Finally, levels of MLL1 acetylation and H3K4 trimethylation at circadian gene promoters depend on NAD+ circadian levels. These findings reveal a previously unappreciated regulatory pathway between energy metabolism and histone methylation.

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Figure 1: SIRT1 and NAD+ levels regulate circadian H3K4 trimethylation.
Figure 2: SIRT1 and MLL1 interact.
Figure 3: SIRT1-dependent deacetylation of MLL1 controls its activity.
Figure 4: MLL1 H3K4 methyltransferase activity is modulated by SIRT1.
Figure 5: NAD+-SIRT1 control of cyclic H3K4 trimethylation through circadian deacetylation of MLL1.

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Acknowledgements

We thank all the members of Sassone-Corsi laboratory for helpful discussion and technical support. We also thank C.D. Allis (Rockefeller University), P. Ernst (Geisel School of Medicine at Dartmouth), J. Hess (Indiana University School of Medicine), E. Verdin (University of California, San Francisco), J. Hsieh (Memorial Sloan Kettering), P. Puigserver (Harvard Medical School), M. Oshimura (Tottori University), Y. Murakami (National Institute of Infectious Diseases Japan), J. Hirayama (Tokyo Dental and Medical University), N.J. Zeleznik-Le (Loyola University Chicago), J. Auwerx (École Polytechnique Fédérale de Lausanne) and K. Yagita (Kyoto Prefectural University of Medicine) for sharing reagents and T. Chuang and K. Harvey (Millipore) for their contribution to generate the anti–acetyl-MLL1 antibody. This work was supported in part by European Molecular Biology Organization (EMBO; long-term fellowship ALTF 411-2009 to L.A.-A.), Japan Society for the Promotion of Science (JSPS; Postdoctoral Fellowship for Research Abroad to S.K.), Government of Mexico (postdoctoral fellowship to R.O.-S.), INSERM (France) and the US National Institutes of Health (grants AG041504, GM082634 and DA036408 to P.S.-C.).

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L.A.-A., S.K. and P.S.-C. conceived and designed the project. L.A.-A., S.K. and R.O.-S. contributed newly generated reagents and materials. L.A.-A. designed and performed experiments. L.A.-A. and P.S.-C. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Paolo Sassone-Corsi.

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Integrated supplementary information

Supplementary Figure 1 Specificity of SIRT1-mediated modulation of gene expression and H3K4me3 levels.

(a) RT-PCR analyses of clock genes in mouse embryonic fibroblasts (MEFs) from wild type or Sirt1−/− mice. Additionally, wild type MEFs were treated with the SIRT1 inhibitor EX527 (50μM). Total mRNA was extracted from MEFs after dexamethasone shock and analyzed by quantitative real–time PCR with specific primers. The expression at time point 0 from wild types was set to 1, and gene expression was normalized to actin mRNA levels for each sample. Means ± s.e.m. of three independent samples are presented. (b) Circadian expression of clock genes in livers harvested from entrained wild type or Sirt1ΔEx4 mice. Total mRNA was extracted from livers harvested at the indicated zeitgeber times (ZT). Means ± s.e.m. of three independent samples analyzed per triplicate are presented. (c,d) mRNA expression and ChIP from H3K4me3 at transcription start sites from Tbp, Gapdh, and Emilin1, and mRNA expression of HoxA9 gene from livers (c), or from MEFs (d). Statistical analyses using two tailed paired t–test are presented; *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 2 SIRT1-NAD+ control of epigenetic marks on circadian genes.

(a) ChIP analyses at Dbp transcriptional start site (Dbp tss) or at the first Dbp exon (Dbp E1) or at the Dbp intron 1 E–boxes (Dbp I1) in wild–type MEFs (WT), Sirt1−/–, or wild type treated with the SIRT1 inhibitor EX527. Analyses were done at circadian times (CT) 18 and 30 after dexamethasone shock using specific antibodies against H3K4me2 or H3K4me1. (b) Luciferase assays on Dbp-luc or Per1-luc from 293 cells transfected with MLL1 and CLOCK–BMAL1 plasmids. Transfected cells were treated with increasing doses of β–Nicotinamide adenine dinucleotide (NAD+), β–Nicotinamide mononucleotide (β–NMN), Nicotinic acid (NA) or nicotinamide (NAM). Light units were normalized to an internal LacZ control, and the relative light units (RLU) from basal expression of the luciferase reporter from non-treated cells were set to 1 (means ± s.e.m. of four independent experiments)*P < 0.05, **P < 0.01, ***P < 0.001, two tailed paired t–test. (c) Luciferase assays performed in Sirt1−/− MEFs using the indicated plasmids. Cells were treated with the indicated compounds and data was analyzed as in (b). (d) H3K4me3 ChIP analyses on Dbp intron 1 E-boxes (Dbp I1) in wild-type and Sirt1−/– MEFs either untreated or treated with the indicated compounds. ChIPs were performed at circadian times (CT, hour post synchronization) 18 and 30, and data from non-treated WT MEFs at CT18 were set to 1. (e) ChIP analyses at selected regions on Dbp gene in wild type (WT), Sirt1−/– or wild type treated with the NAMPT inhibitor FK866. Analyses were done at circadian times (CT) 18 and 30 after dexamethasone shock using specific antibodies against H3K4me3 or acetylated H3 (anti H3K9–K14 acetyl). For all ChIP experiments, fragmented chromatin was immunoprecipitated with specific antibodies and quantified by real-time PCR. (Means ± s.e.m. of three independent samples, analyzed per triplicate; *P < 0.05, **P < 0.01, ***P < 0.001, two tailed t–test)

Supplementary Figure 3 MLL1-SIRT1 interaction.

(a) Co-immunoprecipitation experiments from protein extracts of 293 cells transiently transfected with the indicated plasmids using anti Flag antibody followed by western blot with either anti myc or anti Gal4 binding domain (G4BD) (b) Immunoprecipitaion (IP) using anti Flag from total extracts of Mll1−/− and Mll1−/−/Flag–MLL1 and western blot against MLL1, which reveals the expression of MLL1. (c) Rescue of circadian mRNA expression of Dbp gene by stably expressing MLL1 in Mll1−/− MEFs. mRNA was extracted from wild–type, Mll1−/− and Mll1−/−/Flag–MLL1 MEFs after dexamethasone shock, and Dbp expression levels were analyzed by quantitative real–time PCR. Circadian Dbp expression in Mll1−/−/Flag–MLL1 MEFs depicts higher amplitudes upon treatment with the SIRT1 specific inhibitor EX527 (50μM during 18 hr.). Gene expression was normalized to 18S. (Means ± s.e.m. of three independent samples). (d) Co-immunprecipitation experiments using extracts from 293 cells transiently expressing the indicated plasmids. FM1–M6: Flag tagged version of the MLL1 fragments as shown in Fig. 2d. (e) Mammalian two–hybrid assays in 293 cells transfected with the GAL4–SIRT1 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 samples. *P < 0.05, **P < 0.01, ***P < 0.001, two tailed t-test.

Supplementary Figure 4 MLL1 is an acetylated protein and a substrate for SIRT1 deacetylase activity.

(a) Immunoprecipitation of Flag-MLL1-myc with anti Flag antibody in transiently transfected 293 cells. A western blot with anti pan acetyl-lysine reveals acetylated MLL1. (b) MLL1 can be acetylated in transient transfections by CBP, but not by the HATs CLOCK, MOF or HAT1. 293 cells were transiently transfected with the indicated plasmids. MLL1 was immunoprecipitated with anti Flag antibody. A western blot with anti acetyl lysine (anti AcK) reveals MLL1 acetylation. (c) Scheme depicting the in vitro SIRT1 deacetylation assay on MLL1 (see also Methods section). (d) SIRT1, but not SIRT3–SIRT7, can efficiently deacetylate MLL1 in vitro. (SIRT2 is included in Fig. 3f).

Supplementary Figure 5 MLL1 acetylation at K1130 and K1133 is circadian, but total protein does not oscillate.

(a) Sequence alignment of a portion of the MLL1 protein across species. Residues K1130 and K1133 (blue) are highly conserved. The alignment was performed using the ClustalW multiple sequence alignment program. Residues green, yellow, and white represent identity, similarity and no similarity, respectively. (b) Luciferase assays performed on Dbp–Luc reporter using the indicated plasmids, and normalized to lacZ control. Light units from basal Dbp-luc expression were set to 1. Relative light units (RLU) are shown as means ± s.e.m. of four independent samples (*P < 0.05, **P < 0.01, two tailed t–test) (c) Endogenous MLL1 is acetylated at residues K1130 and K1133 in a circadian manner. Livers from WT and Sirt1ΔEx4 entrained mice were harvested at selected times. Nuclear extracts were subjected to western blot using the indicated specific antibodies. P84 and total MLL1 proteins are shown as input control. Oscillation in BMAL1 phosphorylation is shown as a control of the circadian entrainment. Acetyl MLL1 levels were quantified with respect to total MLL1 and p84, and results are presented in the histogram. Relative intensity from WT samples at ZT3 was set to 1. (*P < 0.05, **P < 0.01, ***P < 0.001, two tailed t–test) (d) Western blot from total protein extracts for wild type and Sirt1−/− MEFs synchronized with dexamethasone and harvested at the indicated hours after synchronization. While BMAL1 shows circadian rhythmicity in its phosphorylation levels (middle panel) MLL1 does not oscillate (upper panel). Actin levels are shown as loading control.

Supplementary Figure 6 Luciferase assays assessing MLL1 and SIRT1 transactivation activity on circadian gene promoters.

(ac) Luciferase reporter gene assays using the (a,b) Dbp promoter or (c) Per1 promoter in 293 cells. (a) Plasmids encoding MLL1 full length (MLL1), a truncated version of MLL1 lacking the methyltransferase SET domain (MLL1ΔSET) or a truncated MLL1 lacking the exons 3–5 of the protein (MLL1ΔN) were transiently transfected at increasing doses, with or without CLOCK and BMAL1. Although MLL1 activates Dbp-luc reporter, addition of CLOCK–BMAL1 substantially enhances transactivation indicating a synergistic contribution (b) Plasmids encoding SIRT1 or the catalytic inactive mutant version SIRT1(H363Y) were transiently transfected at increasing doses, with or without CLOCK and BMAL1. SIRT1 represses Dbp-Luc reporter only in the presence of CLOCK–BMAL1, according to a role in modulating the activity of the complex. (c) MLL1 and SIRT1 were transiently transfected with or without CLOCK and BMAL1. SIRT1, but not the catalytic inactive SIRT1(H363Y), showed a decrease in MLL1‐dependent Per1‐luc transactivation. Hence, similar results were obtained with Dbp-Luc and with Per1-Luc reporters (n.s. non-significant). For all experiments, relative light units (RLU) are shown as means ± s.e.m. of four independent samples; *P < 0.05, **P < 0.01, ***P < 0.001, two tailed t‐test.

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Aguilar-Arnal, L., Katada, S., Orozco-Solis, R. et al. NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1. Nat Struct Mol Biol 22, 312–318 (2015). https://doi.org/10.1038/nsmb.2990

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