TCDD abolished daily oscillations in relative liver weight
The effects of TCDD on body and liver weight were examined following oral gavage with sesame oil vehicle or 30 µg/kg TCDD every 4d for 28d. At euthanasia, the average body weight of treated mice was ~15% less than controls (Supplementary Fig. S2). JTK_CYCLE analysis determined that the RLW of controls oscillated in a diurnal manner, peaking at ZT0 (Fig. 1). This is consistent with previous mouse and rat studies in which both RLW and hepatocyte size were found to exhibit circadian oscillations over a 24 h period, reaching maximal size and weight at the end of the feeding phase (ZT0)52,53. TCDD increased RLW at each time point with a maximum increase of 1.9-fold at ZT12, comparable to RLW increases reported in previous studies31,38. Notably, the daily rhythmicity in RLW was lost following TCDD treatment (Fig. 1). TCDD had no effect on daily food consumption over the 28d treatment regimen (Supplementary Fig. S3), consistent with previous studies54. Therefore, TCDD-elicited alterations in body weight, RLW, and hepatic rhythmicity are not driven by changes in overall food intake.
Loss of hepatic rhythmic gene expression following AhR activation
Using RNA-Seq, TCDD-elicited hepatic transcriptomic changes were assessed at 3 h intervals over a 24 h period. JTK_CYCLE analysis of the RNA-Seq data detected 5,636 hepatic genes exhibiting diurnal rhythmic expression (BH q-value ≤ 0.1; period = 21–24 h) in control mice (Fig. 2A), equivalent to 25.7% of the 21,896 genes expressed in the liver. This is higher than the ~11 to 16% of hepatic genes reported to exhibit circadian oscillations in other studies10,11, but less than a recent 37% estimate12. These discrepancies are likely due to differences in the BH q-value cut-off, transcriptomic platforms, sampling intervals, and statistical power. Specifically, 15 core hepatic clock regulators exhibited oscillating expression including (i) the E-box binding transcription factors Arntl (aka Bmal1), Clock, and Npas2, (ii) the PER/CRY genes Per1, Per2, Per3, Cry1, and Cry2, (iii) the RORE-binding transcription factors Nr1d1 (encodes REV-ERBα), Nr1d2 (encodes REV-ERBβ), and Rorc (encodes RORϒ), and (iv) the D-box binding transcription factors Dbp, Tef, Hlf, and Nfil3. Hepatic expression of the RORE-binding transcription factor Rora did not exhibit rhythmicity, consistent with previous reports that it lacks circadian oscillation in peripheral tissues55. Collectively, these results confirmed that our study was appropriately designed to examine the effects of persistent AhR activation on the hepatic circadian clock.
The classical AhR target genes Cyp1a1, Cyp1a2, and Cyp1b1 did not exhibit rhythmicity in vehicle mice according to JTK_CYCLE analysis. In TCDD-treated mice, these cytochrome P450s were induced 542.9-, 19.5-, and 562.7-fold, respectively, confirming hepatic AhR activation. Overall, 8,970 arrhythmic genes were differentially expressed by TCDD at one or more time points. According to JTK_CYCLE analysis, TCDD abolished the rhythmicity of 5,613 hepatic genes, equivalent to 99.6% of the genes which exhibited circadian regulation in controls. The coefficient of variation (COV = standard deviation / average) of the normalized log2-transformed RNA-Seq read counts (n = 3) for each circadian-regulated gene was calculated at each ZT, averaged across the 8 timepoints, and compared between vehicle and TCDD-treated mice (Supplementary Fig. S4). Linear regression analysis revealed a slope of 0.83, suggesting variation in circadian-regulated gene expression was roughly equivalent between control and treated mice.
Following TCDD treatment, only 23 circadian-regulated genes retained rhythmicity, while 5 acquired rhythmic expression (Sdhaf2, Nsrp1, Marveld3, Folr1, and Gm17068) (Fig. 2A). Linear regression analysis of the time at which each gene expression cycle peaks/crests (acrophase) between vehicle- and TCDD-treated animals revealed a slope of 0.95, suggesting TCDD had little effect on the acrophase of most genes. For example, of the 23 genes which maintained rhythmicity, 15 exhibited similar acrophases (±1.5 h) between control and TCDD-treated animals (Fig. 2B; Supplementary Table S3). TCDD increased the acrophase of Rorc, Tef, Leprotl1, Rtel1, and Ddo ≥ 3 h (+ve phase shift), and decreased the acrophase of Kpna2, Max, and Polr1b ≥ 4.5 h (−ve phase shift) (Fig. 2C,D). In contrast to limited effects on acrophase, TCDD altered the amplitude of most rhythmic genes. Of the 23 genes identified as rhythmic in both controls and treated mice, 15 exhibited a ≥ 1.3-fold reduction in amplitude, while only 3 genes increased in amplitude (Rtel1, Bclaf1, Slc39a10; 1.4-, 1.6-, 1.7-fold) (Fig. 2E). Overall, TCDD abolished the rhythmicity of the vast majority of clock-controlled hepatic genes, while those that continued oscillating following treatment exhibited reduced amplitudes (in most cases). Beyond the collapse of the hepatic circadian clock, previous studies have shown TCDD also causes the loss of liver-specific and sexually dimorphic gene expression56. Comparison of these gene sets revealed 47% of repressed male-specific genes and 39% of repressed liver-specific genes exhibited rhythmicity in vehicle- but not TCDD-treated mice (Fig. 2F).
TCDD dampened rhythmicity of hepatic core clock regulators
The striking global loss in hepatic gene expression rhythmicity is likely due to the dampened expression of core circadian clock regulators. Notably, TCDD repressed hepatic expression of all 16 core clock regulators at one or more time points (Fig. 3; Table 1). Furthermore, the rhythmic expression of all core clock regulators was diminished by TCDD, involving either a ≥ 3.3-fold reduction in amplitude (Arntl, Npas2, Nr1d1, Nr1d2, Rorc, Per2, Per3, Cry1, Nfil3, Dbp, and Tef) or a complete loss of oscillation (Clock, Per1, Cry2, and Hlf). For example, the amplitude of Dbp, Rorc, and Per3 was repressed 27.3-, 9.1-, and 7.2-fold by TCDD (Fig. 3; Table 1). qRT-PCR analysis confirmed that TCDD dose-dependently repressed select hepatic core clock genes (Fig. 4).
Capillary electrophoresis (WES ProteinSimple System) was used to evaluate the effect of TCDD on hepatic protein levels of select clock regulators. JTK_CYCLE analysis (BH q-value ≤ 0.1; period = 21–24 h) confirmed ARNTL, REV-ERBα, and NFIL3 protein levels exhibited rhythmic oscillations in control samples. TCDD reduced hepatic ARNTL and NFIL3 protein levels 14.4- and 80.6-fold, respectively, while REV-ERBα protein was undetected in TCDD-treated mouse liver samples at every time point (Fig. 5). Furthermore, TCDD abolished the diurnal rhythmicity of these three proteins, consistent with reduced amplitudes in gene expression.
ChIP-PCR was used to evaluate hepatic ARNTL genomic enrichment following persistent AhR activation over 28d. In control livers, oscillating genomic ARNTL binding within previously identified target genes was confirmed, with peak binding detected at ZT9. TCDD reduced ARNTL binding within Per1, Dbp, and Nr1d1 3.4-, 4.0-, and 3.7-fold, respectively. Moreover, ARNTL binding rhythmicity was abolished within Per1 and Dbp, while the amplitude of rhythmicity at Nr1d1 was reduced 5.9-fold (Fig. 6A–C). The specificity of the ARNTL immunoprecipitation was confirmed using a negative control region on chromosome 6 (Fig. 6D). These results demonstrate that TCDD dampened Arntl rhythmic mRNA expression, reduced ARNTL protein levels, and impaired ARNTL genomic binding within target genes.
Although rhythmic expression of most hepatic genes is regulated by the local molecular clock, some genes retain rhythmicity in the absence of a functional hepatic oscillator57,58. These “system-driven genes” are regulated by systemic oscillating cues including feeding/fasting cycles, body temperature fluctuations, and diurnal hormones (e.g. glucocorticoids). JTK_CYCLE analysis classified these system-driven genes as arrhythmic following TCDD treatment, with the exception of Per2. However, visual assessment suggests some of these genes still exhibit a diurnal oscillating trend (e.g. Hsph1, Hspa1b. Hspa8, Hsp90aa1, Chordc1, Stip1, Fus), albeit with reduced amplitude (Supplementary Figs S5 and S6). Despite discrepancies between visual assessment and JTK_CYCLE analysis, the oscillating expression of system-driven genes was altered by TCDD. In many cases, hepatic ChIP-Seq analysis identified increased AhR genomic binding in these genes31, implying disruption of their rhythmic expression may be a direct consequence of TCDD.
Increased genomic AhR binding within core clock genes
A previously published hepatic RNA-Seq time-course analysis revealed differential expression of several E-box-containing core clock genes as early as 4 h after TCDD treatment, prior to any phenotypic effects, suggesting direct AhR-dependent regulation of the hepatic clock. Specifically, TCDD repressed Per1 (2.0-fold; not-significant), Per3 (3.0-fold), Dbp (3.3-fold), and Tef (1.8-fold) at 4 h, while Per2 was induced 3.0-fold59. At least one pDRE (MSS ≥ 0.856) is present within the regulatory region (10 kb upstream of TSS to TES) of each mouse core clock gene with the exception of Nr1d246. Furthermore, AhR ChIP-Seq analysis of male livers 2 h following TCDD treatment showed increased AhR genomic binding (fold change ≥ 1.9 vs. IgG) within the loci of all core clock genes except Per331, providing strong evidence that hepatic core clock genes are direct AhR targets (Table 1). Among these 15 core clock genes, only 7 exhibited AhR enrichment at a site containing a pDRE, while the other 8 genes exhibited AhR enrichment within regions lacking a pDRE (Table 1). Therefore, TCDD-elicited disruption of hepatic rhythmicity likely involves both DRE-dependent and DRE-independent AhR signaling.
In vitro studies using Hepa1c1c7 cells treated with β-naphthoflavone (βNF) for 1.5 h report AhR interacts with ARNTL and binds at E-box response elements within the Per1 promoter. This decreases ARNTL/CLOCK heterodimerization at E-boxes within Per1 and represses its expression25. To further investigate DRE-independent mechanisms involved in circadian dysregulation in vivo, ChIP-PCR was used to compare AhR, ARNTL, and CLOCK genomic binding in liver samples of male mice orally gavaged with TCDD for 2 h. The primers described by Xu et al. (2010) were used to assess binding within the Per1 promoter at a site containing a canonical E-box but no pDRE (promoter site 1)25. In contrast to in vitro βNF treatment, in vivo TCDD treatment had no effect on AhR, CLOCK, or ARNTL enrichment within this region (Fig. 7A). A 3.4-fold increase in AhR binding was detected further upstream of the Per1 transcription start site (promoter site 2), near an E-box and pDRE. However, neither CLOCK nor ARNTL binding were affected by TCDD (Fig. 7B). Similarly, 2.1-fold enrichment in AhR binding was detected in a region containing a non-canonical E-box (5′-CACGTT-3′60) within the Per2 promoter, despite a lack of pDREs. Again, AhR binding did not interfere with CLOCK or ARNTL binding at this site (Fig. 7C). AhR binding was also enriched 21.8-fold at a pDRE within a Per2 intron. Despite a lack of canonical E-boxes, CLOCK and ARNTL binding were detected but unaffected by TCDD (Fig. 7D). AhR enrichment was also increased 4.3- and 1.8-fold within a Dbp intron and the Nr1d1 promoter, respectively, with no effect on CLOCK or ARNTL binding (Fig. 7E,F). A 43.8-fold increase in AhR binding at a pDRE within the Cyp1a1 promoter served as a positive control (Fig. 7G), while no enrichment was detected in the negative control region on chromosome 6 (Fig. 7H). Overall, TCDD-activated AhR did not interfere with ARNTL or CLOCK binding at the regions examined in this study.
TCDD disrupts circadian regulation of hepatic metabolism
To identify circadian-controlled biological processes and metabolic pathways affected by TCDD, a functional enrichment analysis was performed on 2,804 hepatic genes that: (i) exhibited rhythmicity in controls but not TCDD-treated mice, and (ii) were differentially expressed (|fold change| ≥ 1.5; P1(t) ≥ 0.8) by TCDD at three or more time points. DAVID identified 19 enriched functional clusters (ES ≥ 1.3) including metabolism of lipids (e.g. fatty acids, phospholipids, cholesterol/sterols, and sphingolipids), glycogen, and heme, as well as oxidation-reduction reactions and DNA repair (Fig. 8A). Indeed, the hepatic peripheral clock is known to regulate nutrient metabolism, oxidative defense, and DNA repair, facilitating synchronization with feeding/fasting cycles and ultraviolet (UV) radiation exposure during daylight3.
The effect of TCDD on circadian-controlled hepatic metabolism was further evaluated through an untargeted metabolomics analysis of the liver. Negative mode electrospray ionization of polar hepatic extracts detected 6,569 metabolite peaks, of which 1,638 (24.9%) were classified as rhythmic by JTK_CYCLE (BH q-value ≤ 0.1; period = 21–24 h). Similarly, the positive mode analysis detected 5,055 metabolite peaks, where 900 (17.8%) were rhythmic in controls (Fig. 8B). This fraction of rhythmic hepatic metabolites is comparable to the percentage of oscillating hepatic genes detected (25.7%), but lower than the ~50% reported in a targeted metabolomics study12. Following TCDD treatment, 1,637 of the 1,638 oscillating peaks detected in negative mode lost rhythmicity (99.9%), while 100% of the positive mode peaks were arrhythmic (Fig. 8B). Using the mummichog algorithm within MetaboAnalyst51, 11 enriched KEGG pathways were identified among the peaks which lost rhythmicity (7 in negative mode, 4 in positive mode), including several biological processes also enriched at the gene expression level (Fig. 8C). Glucose and glycogen metabolism, heme biosynthesis, bile acid homeostasis (Supplementary Information), and redox homeostasis (Supplementary Information) were examined further through the integration of transcriptomic, metabolomic, and enzymatic analyses.
Glucose and glycogen metabolism
During the active phase, glucose is primarily obtained through the consumption of dietary polysaccharides, while the breakdown of glycogen (glycogenolysis) and de novo glucose biosynthesis (gluconeogenesis) provide glucose during the fasting phase. Several key gluconeogenesis genes including phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) are directly regulated by core clock transcription factors (e.g. REV-ERBα), as well as the circadian-regulated transcriptional activators Krüppel-like factor 15 (KLF15) and cAMP responsive element binding protein 3-like 3 (CREB3L3)61,62,63. Both Klf15 and Creb3l3 lost rhythmicity and were repressed (3.1- and 3.0-fold, respectively) by TCDD. Accordingly, Pck1 and G6pc were repressed 17.7- and 6.9-fold, respectively, consistent with results reported in KLF15 and CREB3L3 knockout models61,63. Pck1 rhythmic oscillation was concurrently abolished, while G6pc was classified as arrhythmic in both control and treated animals (Fig. 9). KLF15 also controls the availability of gluconeogenic precursors (e.g. pyruvate) by regulating amino acid catabolism and nitrogenous waste excretion (i.e. urea cycle). Several other KLF15 target genes were also repressed including glutamic pyruvic transaminase (Gpt; 12.3-fold), 4-hydroxyphenylpyruvic acid dioxygenase (Hpd; 3.7-fold), proline dehydrogenase (Prodh; 5.4-fold), tryptophan 2,3-dioxygenase (Tdo2; 2.0-fold), and ornithine transcarbamylase (Otc; 57.9-fold), impairing amino acid catabolism and gluconeogenic precursor availability. Consistent with this repression of key gluconeogenesis enzymes and regulators, hepatic glucose levels were reduced (up to 5.8-fold) at each time point (Fig. 9).
Excess glucose can be stored as glycogen and called upon as an energy source during fasting. As such, hepatic glycogen levels oscillate in a circadian manner peaking at the end of the active phase (ZT0) (Fig. 9). TCDD not only decreased glycogen levels (up to 31.4-fold), but also abolished the diurnal rhythmic pattern. This is consistent with the 88.7-fold repression and loss of rhythmicity in glycogen synthase 2 (Gys2) (Fig. 9), the rate-limiting step of hepatic glycogenesis which is directly regulated by CLOCK64. Loss of circadian regulation of hepatic Gys2 expression and glycogen content was also reported in CLOCK mutant mice64. Paradoxically, glycogenin (Gyg), the core protein required for the initiation of glycogenesis, was induced 4.1-fold while losing its oscillating pattern. Additionally, 3.1-fold induction of UDP-glucose pyrophosphorylase 2 (Ugp2) was consistent with the 26.7-fold increase in hepatic levels of UDP-glucose, the activated monomer required for glycogen synthesis (Fig. 9). Continuous induction of Gyg and Ugp2 may be an attempt to restore depleted glycogen levels. The inability to store glucose during the feeding phase would compromise energy availability during fasting and limit optimal nutrient utilization. This, combined with impaired gluconeogenesis, suggests TCDD disrupted circadian regulation of carbohydrate metabolism, consistent with reports of lower circulating glucose levels and altered glucose tolerance in TCDD-treated mice31,65.
Heme regulates circadian cycling by serving as a cofactor for REV-ERBα/β, NPAS2, CLOCK, and PER234,35,36,37, and in turn, several key clock components regulate heme biosynthesis. Specifically, NPAS2, ARNTL, PER1, and PER2 regulate the rhythmic expression of aminolevulinic acid synthase 1 (Alas1), the rate-limiting step in hepatic heme biosynthesis66. In accordance with the diminished rhythmicity of Npas2, Arntl, Per1, and Per2, TCDD abolished the diurnal oscillation of Alas1 and induced its expression 8.2-fold. As a result, hepatic protoporphyrinogen IX and heme levels were arrhythmic and increased 88.3- and 176.8-fold, respectively, following treatment (Fig. 10). Alas1 is also controlled by PPAR coactivator 1α (PPARGC1A; aka PGC-1α), which is transcriptionally regulated by nutrient availability67. Consequently, heme serves as a signal of nutritional status, allowing the hepatic clock to respond to changes in nutrient availability. Interestingly, Ppargc1a was repressed 4.0-fold by TCDD, indicating the link between Ppargc1a and Alas1 expression was disrupted. Therefore, heme levels no longer reflect nutrient availability, rendering the clock less responsive to nutritional status. Four of the seven genes downstream of Alas1 in the heme biosynthesis pathway (Uros, Cpox, Ppox, and Fech) also lost their rhythmicity following TCDD treatment (Fig. 10). Overall, the effects of TCDD on heme biosynthesis would not only disrupt diurnal rhythmicity of the hepatic clock, but also impair entrainment with nutrient availability.