A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism

Abstract

Disruption of the circadian clock exacerbates metabolic diseases, including obesity and diabetes. We show that histone deacetylase 3 (HDAC3) recruitment to the genome displays a circadian rhythm in mouse liver. Histone acetylation is inversely related to HDAC3 binding, and this rhythm is lost when HDAC3 is absent. Although amounts of HDAC3 are constant, its genomic recruitment in liver corresponds to the expression pattern of the circadian nuclear receptor Rev-erbα. Rev-erbα colocalizes with HDAC3 near genes regulating lipid metabolism, and deletion of HDAC3 or Rev-erbα in mouse liver causes hepatic steatosis. Thus, genomic recruitment of HDAC3 by Rev-erbα directs a circadian rhythm of histone acetylation and gene expression required for normal hepatic lipid homeostasis.

Figure 1. Circadian rhythm of genomic HDAC3 recruitment in mouse liver

A. Heatmap of HDAC3 binding signal at ZT10 (left) and ZT22 (right) from −1kb to +1kb surrounding the center of all the HDAC3 ZT10 binding sites, ordered by strength of HDAC3 binding at ZT10. ChIP-Seq with anti-HDAC3 antibody was performed and data were analyzed as described in Methods. Each line represents a single HDAC3 binding site and the color scale indicates the HDAC3 signal (reads encompassing each locus per million total reads). A read is a unique sequence obtained in ChIP-Seq and then aligned to the mouse genome. ZT, Zeitgeber time (light-on at ZT0, off at ZT12). B. HDAC3 recruitment at 2 selected genomic sites over a 24h cycle by ChIP-PCR. Immunoprecipitated DNA was normalized to input. Values are mean ± s.e.m. (n=4–5). C. HDAC3 diurnal genomic recruitment is maintained in constant darkness. Six HDAC3 binding sites were assessed by ChIP-PCR (n=4–5), and the Bmal1 promoter served as a positive control (23); regions close to the TSS of the Arbp and Ins genes served as negative controls. CT, Circadian time. D. The rhythm of HDAC3 recruitment is reversed by day-time feeding (n=4–5). RF, food was provided only from ZT3 to ZT11 every day for 2 weeks.

Figure 2. Orchestration of genome-wide rhythms of histone acetylation, Pol II recruitment, and gene expression by HDAC3

A. Heatmap of the histone H3 lysine 9 acetylation (H3K9Ac) signal in WT liver at ZT22 (left), ZT10 (middle) and ZT10 in liver depleted of HDAC3 (KO) (right) from −1kb to +1kb surrounding the center of all the HDAC3 ZT10 binding sites, ordered by k means clustering of H3K9Ac signal. Each line represents a single HDAC3 binding site and the color scale indicates the H3K9Ac signal per million total reads. HDAC3 depleted liver was removed from HDAC3^fl/fl mice 1 week after injection of AAV-Cre as in Methods. B. Average H3K9Ac signal from −1kb to +1kb surrounding the center of all the HDAC3 ZT10 binding sites. The Y axis represents the HDAC3 signal per million total reads. C. Heatmap of Pol II signal at ZT10 (left), and ZT22 (right) from −1kb to +1kb surrounding the TSS of genes with HDAC3 recruitment within 10 kb of the TSS, ordered by strength of Pol II binding at ZT22. Each line represents a single HDAC3 bound gene and the color scale indicates the Pol II signal per million total reads. Green marks denote 130 genes under the Gene Ontology term “Lipid Biosynthetic Process”, listed in Supp. Table 1. D. Genes up-regulated in liver depleted of HDAC3 are significantly enriched for HDAC3 binding at ZT10. Expression arrays of WT and HDAC3 KO liver were performed and analyzed as described in Methods, and the percentage of HDAC3 bound genes in each category was calculated. *p~10⁻¹⁵⁶ based on hypergeometic distribution as in Methods.

Figure 3. Recruitment of HDAC3 to the genome by Rev-erbα

A. Immunoblot of HDAC3 and Rev-erbα over a 24h cycle in mouse liver. Hsp90 protein levels are shown as loading control. B. The HDAC3 cistrome at ZT10 largely overlaps with the Rev-erbα cistrome at ZT10. ChIP-Seq with anti-Rev-erbα antibody was performed and analyzed as described in Methods. C. Heatmap of Rev-erbα at ZT10 and ZT22 (left) and of NCoR at ZT10 and ZT22 (right), both at HDAC3 ZT10 sites ordered as in Fig. 1A. Each line represents a single HDAC3 binding site and the color scale indicates the signal per million total reads. D, E. HDAC3 (D) and NCoR (E) recruitment to six binding sites (as in Fig. 1C) were interrogated by ChIP-PCR in liver from mice lacking Rev-erbα. The Bmal1 promoter was used as a positive control (23); regions close to the TSS of the Arbp and Ins genes served as negative controls. Values are mean ± s.e.m. (n=3).

Figure 4. Regulation of hepatic lipid homeostasis by HDAC3

A. Gene Ontology analysis of the HDAC3 and Rev-erbα-bound genes that were upregulated in liver depleted of HDAC3 was performed as described in Methods. B. Oil Red O staining of liver from 12 week old HDAC3^fl/fl mice 2 weeks after tail vein injection of AAV-GFP or AAV-Cre. C. Hepatic triglyceride (TG) levels in mice treated as in “B”. D. Oil Red O staining of livers from 9 week old WT and mice lacking Rev-erbα (Rev-erbα KO). E. Hepatic TG levels in from livers from 9 week old WT and mice lacking Rev-erbα (Rev-erbα KO). Values are mean ± s.e.m. (n=4). *p<0.05 by student’s t-test. F. Hepatic de novo lipogenesis (DNL) in 12 week old HDAC3^fl/fl mice 1 week after infection with AAV-Cre or with AAV-GFP. Hepatic DNL is measured as newly synthesized ²H labeled palmitate. Values are mean ± s.e.m. (n=7–8). *p<0.05 by student’s t-test. G. Model depicting the mechanistic links between the daily cycles of Rev-erbα expression, HDAC3 genomic recruitment, epigenomic status, and hepatic lipogenesis.