Fig 1: 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.
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 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 5: AHCY expression and activity. (A) Western blot analysis of Tg-hAHCY liver and kidney lysates from zinc-induced animals. Lanes labeled WT are from non-transgenic siblings. Top row shows level of human AHCY as determined by human specific antibody. Middle row shows total AHCY as determined by anti-body that recognizes both species. (B) Western analysis of Tg-mAhcy liver and kidney. Top row is probed with anti-HA antibody that only recognizes mAHCY expressed from the transgene, middle row shows total AHCY. (C) AHCY enzyme activity in liver (n = 7) and kidney (n = 4) from Tg-hAHCY mice and transgene negative controls. (D) AHCY enzyme activity in liver (n = 3) and kidney (n = 4) from Tg-mAhcy mice and transgene negative controls. Error bars show SEM. Asterisk indicates P < 0.05.
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