Disruption of CLOCK-BMAL1 transcriptional activity is responsible for aryl hydrocarbon receptor-mediated regulation of Period1 gene

Abstract

The aryl hydrocarbon receptor (AhR) is a period-aryl hydrocarbon receptor nuclear transporter-simple minded domain transcription factor that shares structural similarity with circadian clock genes and readily interacts with components of the molecular clock. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters behavioral circadian rhythms and represses the Period1 (Per1) gene in murine hematopoietic stem and progenitor cells. Per1 expression is driven by circadian locomotor activity cycles kaput-brain muscle ARNT-like (CLOCK-BMAL1)-dependent activation of Eboxes in the Per1 promoter. We hypothesized that the effects of AhR activation on the circadian clock are mediated by disruption of CLOCK-BMAL1 function and subsequent Per1 gene suppression. Effects of AhR activation on rhythmic Per1 transcripts were examined in livers of mice after treatment with the AhR agonist, TCDD; the molecular mechanisms of Per1 repression by AhR were determined in hepatoma cells using TCDD and beta-napthoflavone as AhR activators. This study reports, for the first time, that AhR activation by TCDD alters the Per1 rhythm in the mouse liver and that Per1 gene suppression depends upon the presence of AhR. Furthermore, AhR interaction with BMAL1 attenuates CLOCK-BMAL1 activity and decreases CLOCK binding at Ebox1 and Ebox3 in the Per1 promoter. Taken together, these data suggest that AhR activation represses Per1 through disrupting CLOCK-BMAL1 activity, producing dysregulation of rhythmic Per1 gene expression. These data define alteration of the Per1 rhythm as novel signaling events downstream of AhR activation. Downregulation of Per1 could contribute to metabolic disease, cancer, and other detrimental effects resulting from exposure to certain environmental pollutants.

FIG. 1.

Circadian profiles of Per1 transcripts in the mouse liver during the light-dark cycle. After 2 weeks of entrainment in 12:12-h ligh-dark, mice were orally dosed with 1 μg/kg body weight of vehicle (corn oil) or TCDD during their lights-on period. Five days later, animals were placed in constant darkness to allow expression of their endogenous circadian rhythm. After 48 h in constant darkness, vehicle- and TCDD-treated mice were sacrificed and livers were collected every 4 h. Quantitative PCR was used to measure Per1 (A) and Cyp1a1 (B) mRNA. Results are shown as mean fold change relative to levels at CT0 ± SEM. n = 4–6 per group, *p < 0.05 TCDD-treated peak versus vehicle-treated peak; **p < 0.001 peak versus vehicle control.

FIG. 2.

Activation of AhR inhibits Per1 gene expression. (A) Proteins from Hepa-1c1c7 and c12 cell lines were prepared and 30 μg of total cell lysates for each cell line was subjected to Western blot for AhR using α-tubulin as a loading control. (B) One hour after the addition of vehicle (DMSO, 0.01%), increasing concentrations (0.1, 1, and 10μM) of β-NF, or 10nM of TCDD, total RNA was harvested in Hepa-1c1c7 and c12 cells. Quantitative PCR was used to measure Per1 mRNA. (C) RNA was harvested at the various times (0, 0.5, 1, 2, and 4 h) after the addition of DMSO vehicle media or 10μM β-NF in Hepa-1c1c7 and c12 cells. (D) c12 cells were transfected by FLAG-pcDNA3 control vector or pcDNA3-FLAG-AhR vector and incubated for 24 h, and total protein and RNA was extracted. Quantitative PCR was used to measure Per1 and Cyp1a1 mRNA. Western blot was used to detect transfected FLAG-tagged protein. (E) Hepa-1c1c7 and c12 cells were treated with 10μM of β-NF for 4 h. Cell lysates were collected and Per1 protein was analyzed by Western blot using 60 μg of total cell lysates. Quantitative PCR and Western blot was used to measure Per1 mRNA and protein levels, respectively. Results are shown as mean fold change relative to the vehicle or 0-h level ± SEM. n = 3–4 per group, *p < 0.05 and **p < 0.01 versus the vehicle or control. Results are a representative of three to four independent experiments.

FIG. 2.

Activation of AhR inhibits Per1 gene expression. (A) Proteins from Hepa-1c1c7 and c12 cell lines were prepared and 30 μg of total cell lysates for each cell line was subjected to Western blot for AhR using α-tubulin as a loading control. (B) One hour after the addition of vehicle (DMSO, 0.01%), increasing concentrations (0.1, 1, and 10μM) of β-NF, or 10nM of TCDD, total RNA was harvested in Hepa-1c1c7 and c12 cells. Quantitative PCR was used to measure Per1 mRNA. (C) RNA was harvested at the various times (0, 0.5, 1, 2, and 4 h) after the addition of DMSO vehicle media or 10μM β-NF in Hepa-1c1c7 and c12 cells. (D) c12 cells were transfected by FLAG-pcDNA3 control vector or pcDNA3-FLAG-AhR vector and incubated for 24 h, and total protein and RNA was extracted. Quantitative PCR was used to measure Per1 and Cyp1a1 mRNA. Western blot was used to detect transfected FLAG-tagged protein. (E) Hepa-1c1c7 and c12 cells were treated with 10μM of β-NF for 4 h. Cell lysates were collected and Per1 protein was analyzed by Western blot using 60 μg of total cell lysates. Quantitative PCR and Western blot was used to measure Per1 mRNA and protein levels, respectively. Results are shown as mean fold change relative to the vehicle or 0-h level ± SEM. n = 3–4 per group, *p < 0.05 and **p < 0.01 versus the vehicle or control. Results are a representative of three to four independent experiments.

FIG. 3.

β-NF induces AhR translocation and degradation. (A) Hepa-1c1c7 cells were treated with vehicle (DMSO, 0.01%) or increasing concentrations (0.1, 1, and 10μM) of β-NF for 4 h. (B) and (C) Hepa-1c1c7 cells were treated with 10μM of β-NF for various times (0, 0.5, 1, 2, and 4 h). Total cell lysates and nuclear lysates were prepared as detailed in the “Materials and Methods” section. Western blot was performed using 20 μg lysates of each sample to measure AhR protein. All blots were stained with α-tubulin or β-actin (nuclear lysates) antibody as a loading control. Results are a representative of three independent experiments.

FIG. 4.

β-NF induces AhR-BMAL1 and decreases CLOCK-BMAL1 associations. Hepa-1c1c7 cells were treated with vehicle (DMSO, 0.01%) or 10μM of β-NF for 1.5 h. (A) A total of 500 μg of vehicle or β-NF-treated cell lysates was used for co-IP. Two micrograms of AhR (goat) antibody or BMAL1 (rabbit) was used for precipitation of protein. IgG was a negative control. Protein complexes were collected using protein A/G subjected to Western blot. Lysates in the top two blots were precipitated with anti-AhR (goat) primary antibody and then probed with anti-BMAL1 primary antibody (rabbit, 1:200, top blot) or with anti-AhR (rabbit, 1 μg/ml, second blot) primary antibody, respectively. Lysates shown in the bottom two blots were precipitated with anti-BMAL1 (rabbit) primary antibody and probed with anti-CLOCK (goat, 1:500, third blot from top) primary antibody, or with anti-BMAL1 (rabbit, 2 μg/ml, fourth blot from top) primary antibody, respectively. (B) Cell lysates of vehicle or β-NF treated for 1.5 h were subjected to Western blot analysis for CLOCK, BMAL1, and α-tubulin (loading control). Results are representative of three independent experiments.

FIG. 5.

Activation of AhR represses CLOCK-BMAL1–induced transactivation. c12 cells were transfected with a reporter construct mPer1-luc (10 ng), CLOCK-BMAL1 (250 ng each), AhR construct (250 ng), CLOCK (250 ng), BMAL1 (250 ng), β-gal (10 ng), or the empty vectors. After 24 h, cells were harvested for luciferase assay. The relative firefly luciferase of mPer1-luc from analysis for individual groups was normalized against β-gal luciferase. Results are shown as mean fold change relative to the untransfected CLOCK-BMAL1 and AhR relative firefly luciferase level ± SEM. n = 3–4 per group, *p < 0.05 versus cotransfected CLOCK and BMAL1 vector group; **p < 0.001 versus cotransfected empty vector group.

FIG. 6.

Recruitment of AhR to the Per1 promoter results in the inhibition of Per1. Hepa-1c1c7 cells were treated with either vehicle (DMSO, 0.01%) or 10μM β-NF for 1.5 h. Cells were harvested and native chromatin from nuclear lysates was prepared. IPs were carried out using AhR and CLOCK antibodies. A control (no antibody) was included to confirm the specificity of the precipitation. Purified genomic DNA was subjected to PCR amplifications with gene-specific primers. Input lane represents promoter-specific amplification of 0.2% of the total chromatin sample. (A) Products were separated and analyzed using a 2% agarose gel. (B) Densitometric analysis of AhR and CLOCK. Results are shown as mean fold change relative to vehicle level ± SEM. n = 3–4 group, *p < 0.05 and **p < 0.001 versus vehicle. Results are a representative of three independent experiments.