An integrated genomic analysis of aryl hydrocarbon receptor-mediated inhibition of B-cell differentiation

Abstract

The aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters differentiation of B cells and suppresses antibody production. A combination of whole-genome, microarray-based chromatin immunoprecipitation (ChIP-on-chip), and time course gene expression microarray analysis was performed on the mouse B-cell line CH12.LX following exposure to lipopolysaccharide (LPS) or LPS and TCDD to identify the primary and downstream transcriptional elements of B-cell differentiation that are altered by the AHR. ChIP-on-chip analysis identified 1893 regions with a significant increase in AHR binding with TCDD treatment. Transcription factor binding site analysis on the ChIP-on-chip data showed enrichment in AHR response elements. Other transcription factors showed significant coenrichment with AHR response elements. When ChIP-on-chip regions were compared with gene expression changes at the early time points, 78 genes were identified as potential direct targets of the AHR. AHR binding and expression changes were confirmed for a subset of genes in primary mouse B cells. Network analysis examining connections between the 78 potential AHR target genes and three transcription factors known to regulate B-cell differentiation indicated multiple paths for potential regulation by the AHR. Enrichment analysis on the differentially expressed genes at each time point evaluated the downstream impact of AHR-regulated gene expression changes on B-cell-related processes. AHR-mediated impairment of B-cell differentiation occurred at multiple nodes of the B-cell differentiation network and potentially through multiple mechanisms including direct cis-acting effects on key regulators of B-cell differentiation, indirect regulation of B-cell differentiation-related pathways, and transcriptional coregulation of target genes by AHR and other transcription factors.

FIG. 1.

Flow chart depicting the integration of the ChIP-on-chip and time course gene expression microarray analysis. The integrated analysis was used to identify candidate genes and cellular processes for direct and indirect regulation by the AHR in mouse B cells.

FIG. 2.

Comparison of genes showing significant alterations in expression at the early time points (8 or 12 h) and increased AHR binding. (A) Venn diagram comparing genes with increased expression with genes showing increased AHR binding. (B) Venn diagram comparing genes with decreased expression with genes showing increased AHR binding.

FIG. 3.

Correlation between increased AHR binding and altered gene expression. (A) Scatter plot of all 78 genes comparing log₂ fold-change (FC) AHR binding by ChIP-on-chip versus log₂ fold-change of gene expression changes at 8 h. (B) Scatter plot of all 78 genes comparing log₂ fold-change AHR binding by ChIP-on-chip versus log₂ fold-change of gene expression changes at 12 h. (C) Scatter plot of gene subset showing increased AHR promoter binding comparing log₂ fold-change AHR binding by ChIP-on-chip versus log₂ fold-change of gene expression changes at 8 h. (D) Scatter plot of gene subset showing increased AHR promoter binding comparing log₂ fold-change AHR binding by ChIP-on-chip versus log₂ fold-change of gene expression changes at 12 h. The correlation coefficient (r) and associated p value are shown for each plot. The CYP1A1 gene was removed from all graphs for better visualization of the data points.

FIG. 4.

Network analysis of the 78 direct AHR target genes and Prdm1. Molecules with red circles denote directly regulated genes that were upregulated, whereas molecules with blue circles denote genes that were downregulated. Green arrows denote positive regulation, red arrows denote negative regulation, and gray arrows are not defined as to the direction of regulation.

FIG. 5.

Network analysis of the 78 direct AHR target genes and Pax5. Molecules with red circles denote directly regulated genes that were upregulated, whereas molecules with blue circles denote genes that were downregulated. Green arrows denote positive regulation, red arrows denote negative regulation, and gray arrows are not defined as to the direction of regulation.

FIG. 6.

Network analysis of the 78 direct AHR target genes and Bcl-6. Molecules with red circles denote direct AHR target genes that were upregulated, whereas molecules with blue circles denote genes that were downregulated. Green arrows denote positive regulation, red arrows denote negative regulation, and gray arrows are not defined as to the direction of regulation.

FIG. 7.

Time course hierarchical clustering of all genes showing significant changes in expression. Mouse CH12.LX cells were treated with LPS (10 μg/ml) + DMSO (vehicle) or LPS (10 μg/ml) + TCDD (10nM) and harvested at 8, 12, 24, 36, and 48 h postexposure. At each time point, cells treated with TCDD were compared with the DMSO vehicle control. Hierarchical clustering was performed on the log2 fold-change for all five time points. The maximum fold-change (FC) for each gene by ChIP-on-chip is shown in the right-hand column for comparison. The locations of typical AHR responsive genes are shown to the right of the figure.

FIG. 8.

Bar chart of statistically enriched GeneGo process networks at each time point in the gene expression microarray analysis. Time points are color coded as follows: yellow, 8 h; blue, 12 h; red, 24 h; green, 36 h; and brown, 48 h. Vertical gray line depicts an FDR-adjusted p value of 0.01.

FIG. 9.

Confirmation of AHR binding and expression changes for a subset of genes in primary mouse B cells. (A) Primary mouse B cells were activated with 10 μg/ml LPS and treated with 30nM TCDD or 0.01% DMSO for 1 h. ChIP-qPCR was performed on the same Bach2, Pdgfrb, Blk, and Il5ra genomic regions identified in the ChIP-on-chip experiment. A control untranscribed region (Untr6) was included as a negative control. The data are presented as binding events per 1000 cells and represent mean ± SE of triplicate measurements in B cells isolated from separate animals. *p < 0.05; ^†p = 0.058. (B) Primary mouse B cells were activated with 10 μg/ml LPS and treated with 30nM TCDD or 0.01% DMSO for 12 h. qRT-PCR was performed on Bach2, Pdgfrb, Blk, and Il5ra. Cyp1a1 was included as a positive control. The data represent mean ± SE of quadruplicate measurements in B cells isolated from separate animals. *p < 0.05, **p < 0.01, ***p < 0.001.