S-adenosyl-l-homocysteine hydrolase links methionine metabolism to the circadian clock and chromatin remodeling

Abstract

Circadian gene expression driven by transcription activators CLOCK and BMAL1 is intimately associated with dynamic chromatin remodeling. However, how cellular metabolism directs circadian chromatin remodeling is virtually unexplored. We report that the S-adenosylhomocysteine (SAH) hydrolyzing enzyme adenosylhomocysteinase (AHCY) cyclically associates to CLOCK-BMAL1 at chromatin sites and promotes circadian transcriptional activity. SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases, and timely hydrolysis of SAH by AHCY is critical to sustain methylation reactions. We show that AHCY is essential for cyclic H3K4 trimethylation, genome-wide recruitment of BMAL1 to chromatin, and subsequent circadian transcription. Depletion or targeted pharmacological inhibition of AHCY in mammalian cells markedly decreases the amplitude of circadian gene expression. In mice, pharmacological inhibition of AHCY in the hypothalamus alters circadian locomotor activity and rhythmic transcription within the suprachiasmatic nucleus. These results reveal a previously unappreciated connection between cellular metabolism, chromatin dynamics, and circadian regulation.

Fig. 1. AHCY forms a complex with BMAL1.

(A) Table showing the top BMAL1-interacting proteins in the nucleus ranked by log₂ fold change (FC) value at ZT8. (B) Volcano plot of MS analysis of BMAL1-interacting proteins in the nucleus. The x axis indicates log₂ ratio of normalized intensity (iBAQ) of proteins found in BMAL1 to IgG (n = 4). (C) AHCY colocalization with BMAL1 in the nucleus. GFP-AHCY and RFP-BMAL1 were imaged by confocal microscopy in MEF cells. The Pearson’s R coefficient indicates the extent of colocalization of GFP-AHCY and RFP-BMAL1 (n = 53). Scale bar, 10 μm. (D) Intensity (GFP channel) and phasor mapped fluorescence lifetime images (FLIM) of GFP-AHCY and GFP-AHCY/BMAL1-RFP MEF cell nucleus. Two representative cells are shown (n > 30 cells). Scale bar, 1 μm. (E) Histogram of the fractional intensity distributions of the donor (GFP) in the nucleus. The increasing fractional intensity of the shorter lifetime (FRET) component reflects the presence of FRET (red plot). The two average plots are separated by P < 0.001 (Kolmogorov-Smirnov test). (F) Co-IP experiment using 293T cells transiently transfected with the indicated plasmids (IP, immunoprecipitation; IB, immunoblot). (G) Co-IP experiment from nuclear extract of BMAL1 null MEFs with anti-BMAL1 antibody. (H) Co-IP experiment from liver nuclear extracts using anti-AHCY antibody at indicated zeitgeber times (ZT8 and ZT20). (I) Co-IP experiment from liver cytoplasmic, soluble nuclear, and chromatin-bound fractions using anti-AHCY antibody at the indicated zeitgeber time (ZT8). (J) AHCY ChIP at the Dbp, Per2, and Per1 loci in liver at indicated zeitgeber times. The 3′UTR of Dbp was used as a negative control (mean ± SEM, n = 4 per time point). (K) AHCY ChIP at the Dbp, Per2, and Per1 loci in liver at ZT8 and ZT20 (mean ± SEM, n = 4 per time point).

Fig. 2. AHCY is important for amplitude of circadian oscillation.

(A) Circadian bioluminescence traces for Bmal1:luc U2OS cells transfected with either siRNA control (siCtrl) or siRNA targeting Ahcy (siAhcy) (mean values shown, n = 4). (B) Bar graph of relative circadian amplitude of U2OS Bmal1:luc siCtrl and siAhcy cells (mean ± SEM, n = 4; *P ≤ 0.05; unpaired Student’s t test). (C) Heatmaps representing genes cyclic in control MEF cells only (Ctrl; 261 genes) and in both conditions (16 genes) (n = 3 per time point, per group; P < 0.01 in each dataset). Pie chart representing the percentage of genes oscillating in both conditions and in control only. (D) Gene Ontology (GO) term enrichment analysis of genes oscillating in the control group only. (E) Transcription factor binding site (TFBS) analysis of rhythmic transcription factors (TFs) on transcripts rhythmic exclusively in control MEFs. Represented as percentage of TFBS. (F) Bar graph of amplitude analysis of clock genes in control (Ctrl) and AHCY null (KO) MEF cells. (G) Heatmap of hierarchical clustering of genes significantly down-regulated or up-regulated in vehicle-treated MEFs (DMSO) between the two analyzed CT points (CT12 and CT24; n = 3 per time point per group; fold change > 2; FDR < 0.05). Heatmap shows gene expression levels of control (DMSO) and DZnep-treated MEFs. (H) GO term enrichment analysis of genes down-regulated or up-regulated in vehicle-treated MEFs (DMSO) between the two analyzed CT points (CT12 and CT24). (I) Pie chart showing the percentage of time-dependent genes identified in (G) (715 genes) differentially expressed in MEFs treated with DZnep (10 μM). (J) Bar graph displaying log₂ fold change of gene expression of genes related to (G). (K) Bar graph of fold change analysis of clock genes in vehicle (DMSO)– and DZnep-treated MEF cells. ***P ≤ 0.001; unpaired Student’s t test.

Fig. 3. AHCY regulates BMAL1 recruitment to DNA.

(A) Circadian expression of Dbp in control (Ctrl) or AHCY null (KO-2) MEFs, shCntrl and shAhcy MEFs, and MEFs treated with DMSO, 10 μM DZnep, and 100 μM DZ2002 after DEX shock (mean ± SEM, n = 3 per time point, per group; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ANOVA, Holm-Sidak post hoc). (B) Luciferase assay of Dbp-luciferase (Dbp-luc) and Per2-luciferase (Per2-luc) in HEK293 transfected with CLOCK and BMAL1 plasmids and treated with indicated increasing doses of DZnep for 24 hours (means ± SEM, n = 4; *P ≤ 0.05; unpaired Student’s t test). (C) Luciferase assay of Pgc1a-luciferase (Pgc1a -luc) in HEK293 transfected with CLOCK and BMAL1 (left) or TFEB (right) plasmids and treated with indicated doses of DZnep for 24 hours (means ± SEM, n = 4; *P ≤ 0.05; unpaired Student’s t test). (D) BMAL1 ChIP at the Dbp and Per2 loci in control (Ctrl) or AHCY null MEFs (KO). The 3′UTR of Dbp was used as a negative control (mean ± SEM, n = 4 per time point, per group; **P ≤ 0.01; unpaired Student’s t test). (E) BMAL1 ChIP at the Dbp and Per2 loci in MEFs treated with vehicle (DMSO) or DZnep (10 μM) (mean ± SEM, n = 4 per time point, per group; ***P ≤ 0.001; unpaired Student’s t test). (F) Heatmap of BMAL1 ChIP-seq binding profiles in DMSO- and DZnep-treated MEFs at the two analyzed CT points. (G) Venn diagram of BMAL1 binding sites identified at the two analyzed CT points in vehicle (DMSO) and DZnep-treated MEFs. (H) Boxplots of number of BMAL1 tags per peak at CT12 and CT24 shared peaks in DMSO- and DZnep-treated MEFs (unpaired Student’s t test).

Fig. 4. AHCY mediates oscillation of H3K4me3.

(A) Average H3K4me3 coverage around the TSS of expressed transcripts at the two analyzed CTs. (B) Boxplots of number of H3K4me3 tags per peak within promoter regions of expressed genes (Wilcoxon signed-rank test). (C) IGV (Integrative Genomics Viewer) profile of BMAL1- and H3K4me3-enriched regions over the Dbp locus. (D) H3K4me3 ChIP at the Dbp and Per2 loci in MEFs treated with vehicle (DMSO) or DZnep (10 μM). Samples were collected at the indicated CTs, and immunoprecipitated chromatin was quantified by RT-PCR (mean ± SEM, n = 3 per time point, per group; **P ≤ 0.01; ***P ≤ 0.001; ANOVA, Holm-Sidak post hoc). (E) H3K9 and K14-acetyl ChIP at the Dbp and Per2 loci in MEFs treated with vehicle (DMSO) or DZnep (10 μM) (mean ± SEM, n = 3 per time point, per group; ***P ≤ 0.001; ANOVA, Holm-Sidak post hoc). (F) H3K4me3 ChIP at the Dbp and Per2 loci in control (Ctrl) or AHCY null MEFs (KO) (mean ± SEM, n = 4 per time point, per group; **P ≤ 0.01; unpaired Student’s t test).

Fig. 5. In vivo inhibition of AHCY modulates circadian locomotor activity and rhythmic transcription in the SCN.

(A) Schematic of the in vivo experimental protocol. (B) Representative double-plot actograms for diurnal locomotor activity of mice infused with saline or DZnep (100 μM). In each actogram, the first days were recorded under 12-hour light/12-hour dark conditions, after which the light was turned off and recording was continued in constant darkness (DD; black arrow indicates start of DD; red arrow indicates day of surgery and starting point for all analyses). (C) Bar graph of locomotor activity during subjective night and day (mean ± SEM, n = 12 saline and n = 16 DZnep; ***P ≤ 0.001; ANOVA, Holm-Sidak post hoc). (D) Bar graph of amplitude of circadian rhythm represented by fast Fourier transform (FFT) in the circadian range (mean ± SEM, n = 12 saline and n = 16 DZnep; ***P ≤ 0.001; unpaired Student’s t test). (E) Bar graph of period lengths (mean ± SEM, n = 12 saline and n = 16 DZnep; ***P ≤ 0.001; unpaired Student’s t test). (F) Heatmaps of genes cyclic in saline-infused mice only (1513 genes) and in both conditions (saline and DZnep, 664 genes) (n = 4 per time point, per group; P < 0.05 in each dataset). Pie chart representing the percentage of genes oscillating in both conditions and in saline only. (G) Amplitude analysis of genes circadian in both conditions in the SCN. In the graph, the percentages of genes with amplitude higher, lower, or equal to saline condition are reported. (H) GO analysis of genes displaying lower amplitude in DZnep-infused mice compared to saline.