Genomic convergence among ERRα, PROX1, and BMAL1 in the control of metabolic clock outputs

Abstract

Metabolic homeostasis and circadian rhythms are closely intertwined biological processes. Nuclear receptors, as sensors of hormonal and nutrient status, are actively implicated in maintaining this physiological relationship. Although the orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) plays a central role in the control of energy metabolism and its expression is known to be cyclic in the liver, its role in temporal control of metabolic networks is unknown. Here we report that ERRα directly regulates all major components of the molecular clock. ERRα-null mice also display deregulated locomotor activity rhythms and circadian period lengths under free-running conditions, as well as altered circulating diurnal bile acid and lipid profiles. In addition, the ERRα-null mice exhibit time-dependent hypoglycemia and hypoinsulinemia, suggesting a role for ERRα in modulating insulin sensitivity and glucose handling during the 24-hour light/dark cycle. We also provide evidence that the newly identified ERRα corepressor PROX1 is implicated in rhythmic control of metabolic outputs. To help uncover the molecular basis of these phenotypes, we performed genome-wide location analyses of binding events by ERRα, PROX1, and BMAL1, an integral component of the molecular clock. These studies revealed the existence of transcriptional regulatory loops among ERRα, PROX1, and BMAL1, as well as extensive overlaps in their target genes, implicating these three factors in the control of clock and metabolic gene networks in the liver. Genomic convergence of ERRα, PROX1, and BMAL1 transcriptional activity thus identified a novel node in the molecular circuitry controlling the daily timing of metabolic processes.

Figure 1. ERRα is involved in diurnal glucose homeostasis.

Blood glucose measurements taken from fed ad libitum (A) and fasted (B) male WT and ERRα-null mice (n = 15 and n = 8 for A and B respectively) at 4 hr intervals over a 24 hr period from ZT 4 to ZT 24. Error bars represent ± SEM. Student's t test, *p<0.05. ZT 0 values are a duplicate of ZT 24 shown for clarity. (C) Blood glucose during a glucose tolerance test in WT and ERRα-null mice (n = 8) at ZT 12 following a 6 hr fast and intraperitoneal glucose administration of 2 mg/g body weight. Error bars represent ± SEM. Student's t test, *p<0.05. (D) Area under the glucose curve at ZT 12. Error bars represent ± SEM. (E) Blood glucose during a glucose tolerance test in WT and ERRα-null mice (n = 6) at ZT 0/24 following a 6 hr fast and intraperitoneal glucose administration of 2 mg/g body weight. Error bars represent ± SEM. Student's t test, *p<0.05. (F) Area under the glucose curve at ZT 0/24. Error bars represent ± SEM. Student's t test, *p<0.05. (G) Diurnal serum insulin levels in fed ad libitum WT and ERRα-null mice. Error bars represent ± SEM. Student's t test, *p<0.05. ZT 0 values are a duplicate of ZT 24 shown for clarity. (H) Blood glucose during a glucose tolerance test in WT and ERRα-null mice (n = 6) at ZT 16 following a 6 hr fast and intraperitoneal glucose administration of 2 mg/g body weight. Error bars represent ± SEM. Inset: area under the glucose curve at ZT 16. Error bars represent ± SEM.

Figure 2. Diurnal variation of serum bile acid, cholesterol, NEFA, and triglyceride levels in WT and ERRα-null mice.

Circulating metabolic parameters were measured in fed ad libitum and fasted WT and ERRα-null mice (n = 4) at 4 hrs intervals over a 24 hr period from ZT 4 to ZT 24. Error bars represent ± SEM. Student's t test, *p<0.05 at individual time points or across a 12 hr light or dark cycle. ZT 0 values are a duplicate of ZT 24 shown for clarity. (A–B) Fed and fasted bile acid, (C–D) fed and fasted total cholesterol, (E–F) fed and fasted NEFA and (G–H) fed and fasted triglyceride levels are shown.

Figure 3. Altered locomotor activity in ERRα-null mice.

(A) Locomotor activity profiles for WT and ERRα-null mice (n = 9–10) maintained in LD conditions in running wheel cages. (B) Representative actograms of one WT and one ERRα-null mouse first entrained in LD conditions and then kept in constant darkness (DD) for 3 weeks in running wheel cages. Grey zones represent darkness. (C) Bar graph representing the endogenous period lengths of WT (n = 9) and ERRα-null mice (n = 10) in constant darkness. Error bars represent ± SEM. Student's t test, *p = 0.008.

Figure 4. ERRα binds to and regulates core clock gene expression.

(A) Binding profiles of ERRα on the extended promoters of core clock genes in mouse liver. Putative ERRα binding sequences (ERREs) are shown on the major binding peaks indicated with an asterisk. (B) Standard ChIP validation reveals that ERRα is enriched at molecular clock target genes using primers amplifying the identified binding peaks in A. (C–D) Male WT and ERRα-null mice (n = 4) kept in LD conditions were sacrificed at 4 hr intervals over a 24 hr period from ZT 4 to ZT 24. Circadian qRT-PCR analysis of Esrra (C) and molecular clock components (D) were performed on RNA isolated from mouse livers and relative expression data normalized to Arbp levels are shown as a function of ZT. Data shown are relative to WT expression levels at ZT 4 arbitrarily set to 1. ZT 0 values are a duplicate of ZT 24 shown for clarity. Error bars represent ± SEM. Student's t test was used to compare WT and ERRα KO liver expression data at the indicated time points, *p<0.05. (E) Western blot analysis of diurnal WT and ERRα-null mouse liver nuclear lysates. Protein extracts from 4 mice at each of the indicated time points were pooled and immunoblotted for ERRα, BMAL1 and CLOCK. (F) Male WT and ERRα-null mice (n = 4) kept in LD conditions were sacrificed at 4 hr intervals over a 24 hr period from ZT 4 to ZT 24 following a 24 hr fast. Circadian qRT-PCR analysis of Esrra and molecular clock components were performed on RNA isolated from mouse livers and relative expression data normalized to Arbp levels are shown as a function of ZT. Data shown are relative to WT expression levels at ZT 4 arbitrarily set to 1. ZT 0 values are a duplicate of ZT 24 shown for clarity. Error bars represent ± SEM. Student's t test was used to compare WT and ERRα KO fasted liver expression data at the indicated time points, *p<0.05.

Figure 5. ERRα is necessary to maintain the rhythmic expression of metabolic genes.

Circadian expression of genes involved in transcriptional regulation (A), glycolysis/gluconeogesesis (B), insulin and AMPK signaling (C) as well as in lipid metabolism (D) are shown. Male WT and ERRα-null mice (n = 4) kept in LD conditions were sacrificed at 4 hr intervals over a 24 hr period from ZT 4 to ZT 24. qRT-PCR analysis was performed on RNA isolated from mouse livers and relative expression data normalized to Arbp levels are shown as a function of ZT. Data shown are relative to WT expression levels at ZT 4 arbitrarily set to 1. ZT 0 values are a duplicate of ZT 24 shown for clarity. Error bars represent ± SEM. Student's t test was used to compare WT and ERRα KO liver expression data at the indicated time points, *p<0.05.

Figure 6. BMAL1/CLOCK regulation of the ERRα promoter.

(A) Pie chart representing the major cellular functions associated with BMAL1 targets enriched in the adult mouse liver using extended promoter arrays. Motif finding algorithm, MDscan, showed that the sequence CACGTG corresponding to the consensus BMAL1/CLOCK E-box binding motif is the most abundant motif present in the first 200 BMAL1 bound segments used in the analysis. (B) Binding profiles of BMAL1 on extended promoters of a subset of core clock gene targets obtained from ChIP-on-chip. Putative BMAL1/CLOCK binding sequences (E-boxes) found, if any, at the binding peaks validated indicated with an asterisk are shown. (C) BMAL1/CLOCK standard ChIP assay in mouse liver on the core clock genes shown in B. (D) Venn Diagram illustrating the overlap in ERRα and BMAL1 direct targets obtained from ChIP-on-chip analyses in mouse liver. (E) Enrichment of a subset of canonical pathways in the ChIP-on-chip target genes determined to be common (yellow) or specific to either ERRα (pink) or BMAL1 (blue). Grey line indicates the p-value threshold cutoff of 0.05. *p<0.05. (F) BMAL1/CLOCK standard ChIP assay in mouse liver on genes associated with transcriptional regulation including ERRα. (G) Schematic representation of a fragment of the mouse, rat and human ERRα promoter showing a conserved BMAL1/CLOCK E-box consensus binding motif, CACGTG, adjacent to the conserved ERRα binding element, TGAAGGTCA. (H) Reporter gene assays in COS-1 cells using a mouse Esrra promoter luciferase reporter construct in the presence of either empty vector, ERRα, CLOCK, BMAL1 or combinations of in the presence or absence of PGC-1α. Luciferase activity was assayed 24 hrs post-transfection and the relative luciferase units (RLU) shown are the mean of duplicate assays.

Figure 7. PROX1 is a regulator of the molecular clock that itself exhibits a rhythmic clock-dependent circadian expression.

(A) Male WT and Clock mutant mice (n = 4) kept in DD conditions were sacrificed every 4 hrs over a 24 hr period from circadian time (CT) 2 to CT 22. qRT-PCR analysis was performed on RNA isolated from mouse livers and relative expression data normalized to Arbp levels are shown as a function of CT. Error bars represent ± SEM. (B) Binding profiles of PROX1 on the extended promoters of core clock genes generated from a recently published ChIP-on-chip experiment in mouse liver. (C) Standard ChIP validation of molecular clock genes identified as direct target genes of PROX1. (D) qRT-PCR was performed on RNA isolated from HepG2 cells treated with control siRNA or an siRNA Dharmacon On-Target Smartpool against PROX1. The expression of molecular clock genes was determined and the data is shown as relative fold expression levels compared to control siRNA and normalized to HPRT1 levels. Data represent mean ± s.d. of triplicate independent experiments. Student's t test, *p<0.05. Western blot analysis on lysates prepared from the HepG2 knockdown samples is shown with the respective antibodies as indicated. (E) Schematic showing the transcriptional crosstalk between ERRα, PROX1 and BMAL1 on molecular clock genes as well as Esrra and Prox1. Genes found to be direct targets of ERRα, PROX1 and/or BMAL1 are indicated by arrows.

Figure 8. ERRα, PROX1, and BMAL1 genomic convergence linking the clock with metabolism.

(A) Venn Diagram illustrating the overlap in ERRα, PROX1 and BMAL1 direct targets obtained from ChIP-on-chip analyses in mouse liver. (B) Comparison between the functionally enriched biological processes associated with BMAL1 targets shared by ERRα and/or Prox1 or bound by BMAL1 only. Target genes specific for BMAL1 association with ERRα and/or Prox1 were enriched for metabolic processes where targets specific to BMAL1 alone were enriched for processes related to chromatin structure and cell cycle. *p<0.01. (C) Schematic representing target genes specific to BMAL1 (blue) or those shared by BMAL1/ERRα (pink), BMAL1/PROX1 (purple) or BMAL1/ERRα/PROX1 (green) associated with diverse metabolic processes.