Computational identification of dioxin response elements in human, mouse, and rat genomes

Dioxin response elements were computationally identified and matrix similarity scores estimated as previously described (PMIDs: 15328365, 21762485, and 26582802

AHR ChIP-Seq of male mouse liver following 2 hour TCDD exposure

Aryl hydrocarbon receptor ChIP-Seq performed in livers of male mice gavaged with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 2hr

Genome-Wide ChIPseq Analysis of AhR, COUP-TF, and HNF4 Enrichment in TCDD-Treated Mouse Liver

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators that interact with AhR have been identified. Enrichment analysis of motifs in AhR-bound genomic regions implicated co-operation with COUP transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF4). The present study investigated AhR, HNF4α and COUP-TFII genomic binding and effects on gene expression associated with liver-specific function and cell differentiation in response to TCDD. Hepatic ChIPseq data from male C57BL/6 mice at 2 h after oral gavage with 30 µg/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2–72 h) and dose–response (0.01–30 µg/kg) datasets to assess putative AhR, HNF4α and COUP-TFII interactions associated with differential gene expression. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4α to regions within liver-specific genes, suggesting intersections associated with the loss of liver-specific functions and hepatocyte differentiation. Analysis found that the repression of liver-specific, HNF4α target and hepatocyte differentiation genes, involved increased AhR and HNF4α binding with decreased COUP-TFII binding. Collectively, these results suggested TCDD-elicited loss of liver-specific functions and markers of hepatocyte differentiation involved interactions between AhR, COUP-TFII and HNF4α

Loss of liver-specific and sexually dimorphic gene expression by aryl hydrocarbon receptor activation in C57BL/6 mice

<div><p>The aryl hydrocarbon receptor (AhR) is a highly conserved transcription factor that mediates a broad spectrum of species-, strain-, sex-, age-, tissue-, and cell-specific responses elicited by structurally diverse ligands including 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD). Dose-dependent effects on liver-specific and sexually dimorphic gene expression were examined in male and female mice gavaged with TCDD every 4 days for 28 or 92 days. RNA-seq data revealed the coordinated repression of 181 genes predominately expressed in the liver including albumin (3.7-fold), α-fibrinogen (14.5-fold), and β-fibrinogen (17.4-fold) in males with corresponding AhR enrichment at 2 hr. Liver-specific genes exhibiting sexually dimorphic expression also demonstrated diminished divergence between sexes. For example, male-biased <i>Gstp1</i> was repressed 3.0-fold in males and induced 4.5-fold in females, which were confirmed at the protein level. Disrupted regulation is consistent with impaired GHR-JAK2-STAT5 signaling and inhibition of female specific CUX2-mediated transcription as well as the repression of other key transcriptional regulators including <i>Ghr</i>, <i>Stat5b</i>, <i>Bcl6</i>, <i>Hnf4a</i>, <i>Hnf6</i>, <i>Foxa1/2/3</i>, <i>and Zhx2</i>. Attenuated liver-specific and sexually dimorphic gene expression was concurrent with the induction of fetal genes such as alpha-fetoprotein. The results suggest AhR activation causes the loss of liver-specific and sexually dimorphic gene expression producing a functionally “de-differentiated” hepatic phenotype.</p></div

2,3,7,8-Tetrachlorodibenzo-p-dioxin abolishes circadian regulation of hepatic metabolic activity in mice

Abstract Aryl hydrocarbon receptor (AhR) activation is reported to alter the hepatic expression of circadian clock regulators, however the impact on clock-controlled metabolism has not been thoroughly investigated. This study examines the effects of AhR activation on hepatic transcriptome and metabolome rhythmicity in male C57BL/6 mice orally gavaged with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days. TCDD diminished the rhythmicity of several core clock regulators (e.g. Arntl, Clock, Nr1d1, Per1, Cry1, Nfil3) in a dose-dependent manner, involving either a ≥ 3.3-fold suppression in amplitude or complete loss of oscillation. Accordingly, protein levels (ARNTL, REV-ERBα, NFIL3) and genomic binding (ARNTL) of select regulators were reduced and arrhythmic following treatment. As a result, the oscillating expression of 99.6% of 5,636 clock-controlled hepatic genes was abolished including genes associated with the metabolism of lipids, glucose/glycogen, and heme. For example, TCDD flattened expression of the rate-limiting enzymes in both gluconeogenesis (Pck1) and glycogenesis (Gys2), consistent with the depletion and loss of rhythmicity in hepatic glycogen levels. Examination of polar hepatic extracts by untargeted mass spectrometry revealed that virtually all oscillating metabolites lost rhythmicity following treatment. Collectively, these results suggest TCDD disrupted circadian regulation of hepatic metabolism, altering metabolic efficiency and energy storage

Gene expression changes of sexually dimorphic genes.

<p>Gene set enrichment analysis (GSEA) of sexually dimorphic genes in (A-B) male and (B-C) female mice gavaged with TCDD every 4 days for 28 days. GSEA was performed using male-enriched and female-enriched gene sets determined as described in materials and methods. Gene expression changes were ranked from largest positive to largest negative. The top panel (green line) represents a ‘running-sum statistic’ which increases when a gene is in a gene set (denoted by black bars), and decreasing when it is not. TCDD repressed the majority of male-specific dimorphic genes (A) while inducing many female-specific genes (B) in male mice. Conversely, TCDD induced many male-specific genes (C) while repressing a majority of the female-specific genes (D) in female mice. (E) Heat map demonstrates the gene expression changes in male-specific and female-specific genes at 28 days.</p

Albuminoid genomic region.

<p>Albuminoid genomic region including <i>Alb</i>, <i>Afp</i>, and <i>Afm</i>. UCSC genome browser tracks show (1) the scale, (2) male AhR ChIP-seq peaks at 2 h, (3) male AhR enriched sites (FDR ≤ 0.05), (4) female AhR ChIP-seq peaks, (5) female AhR enriched sites (FDR ≤ 0.05), (6) location of pDREs (diagonal line indicates pDREs with a matrix similarity score ≥0.85), and (7) location of <i>Alb</i>, <i>Afp</i> and <i>Afm</i> genes within the albuminoid genomic region. <i>Gc</i>, the fourth albuminoid, is located 1 Mb upstream of <i>Alb</i> (not shown). Tracks are available for visualization at <a href="http://dbzach.fst.msu.edu/index.php/publications/supplementary-data/" target="_blank">http://dbzach.fst.msu.edu/index.php/publications/supplementary-data/</a>.</p

Hepatic protein levels changes by TCDD.

<p>Hepatic protein levels in male and female mice gavaged with sesame oil vehicle or 30 μg/kg TCDD every 4 days for 28 days were measured using the ProteinSimple Wes system. Bars represent mean log₂fold-change + SEM for at least 4 animals (N = 4–5). Asterisks (*) indicate a significant difference (<i>P</i> ≤ 0.05) compared to vehicle control determined by Mann-Whitney U-test.</p

AhR-mediated changes in sexually dimorphic regulator expression.

<p>(A) GHR-JAK2-STAT5 signaling cascade interactions with liver-specific gene expression regulators [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref016" target="_blank">16</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref018" target="_blank">18</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref019" target="_blank">19</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184842#pone.0184842.ref043" target="_blank">43</a>]. GH-mediated activation of GHR induces STAT5 phosphorylation promoting male-specific gene expression, and induction of female-specific CUX2 (red outline) which regulates female-specific gene expression. CUX2 and STAT5 compete for binding sites resulting in sexually dimorphic gene expression. BCL6, HNF4α, HNF6, and FOXA also regulate sexually dimorphic and liver-specific gene regulation. Blue identifies repressed genes while white represents genes unaffected by TCDD. (B) Heatmap of sexually dimorphic and liver-specific gene expression regulators (A) in males (28d) and females (28 and 92d). Genes in blue were repressed while red indicates induction. STAT5 protein levels were determined in (C) female and (D) male mice gavaged with 30 μg/kg TCDD every 4 days for 28 days. Bars represent mean ± SEM for at least 3 animals (N = 3–4). Asterisks (*) indicate a significant difference compared to vehicle control as determined by Mann-Whitney U-test.</p