The Aryl Hydrocarbon Receptor Displaces p300 from E2F-dependent Promoters and Represses S Phase-specific Gene Expression

Abstract

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wide range of toxic, teratogenic, and carcinogenic effects. TCDD is a ligand for the aromatic hydrocarbon receptor (AHR), a ligand-activated transcription factor believed to be the primary mediator of these effects. Activation of the AHR by TCDD also elicits a variety of effects on cell cycle progression, ranging from proliferation to arrest. In this report, we have characterized further the role of the activated AHR in cell cycle regulation. In human mammary carcinoma MCF-7 and mouse hepatoma Hepa-1 cells, TCDD treatment decreased the number of cells in S phase and caused the accumulation of cells in G 1 . In Hepa-1 cells, this effect correlated with the transcriptional repression of several E2F-regulated genes required for S phase progression. AHR-mediated gene repression was dependent on its interaction with retinoblastoma protein but was independent of its transactivation function because AHR mutants lacking DNA binding or transactivation domains repressed E2F-dependent expression as effectively as wild type AHR. Overexpression of p300 suppressed retinoblastoma protein-dependent gene repression, and this effect was reversed by TCDD. Chromatin immunoprecipitation assays showed that TCDD treatment caused the recruitment of AHR to E2F-dependent promoters and the concurrent displacement of p300. These results delineate a novel mechanism whereby the AHR, a known transcriptional activator, also mediates gene repression by pathways involving combinatorial interactions at E2F-responsive promoters, leading to the repression of E2F-dependent, S phase-specific genes. The AHR seems to act as an environmental checkpoint that senses exposure to environmental toxicants and responds by signaling cell cycle inhibition.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) 1 is the prototypical compound of a class of environmental contaminants that includes many halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons and is a model for the outcome of exposure to these compounds as well as coplanar polychlorinated biphenyls. Exposure to TCDD results in a plethora of toxic and carcinogenic responses in animals, including liver toxicity (1), immunosuppression (2, 3), reproductive and developmental dysfunction (4 -6), endometriosis (7,8), and tumor promotion (9). The most immediate exposure outcome in humans is chloracne (10). Long term effects in humans range from immunological and reproductive perturbations (11) to cardiovascular disease (12, 13) and cancer (14 -18). TCDD itself is poorly metabolized, which leads to biological accumulation and production of sustained effects (19).Although the in vivo effects of TCDD are wide ranging, the in vitro effects are just as varied and often contradictory, affecting cell proliferation, apoptosis, and differentiation, depending on the cell type examined. TCDD acts as an endocrine disruptor in cell cultures, inhibiting several estrogen-induced responses such as growth of human mammary and endometrial canc...

The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

“…The values shown are the mean Ϯ SD of three determinations relative to ␤-actin. As shown previously Marlowe et al, 2004) no ligand is necessary to activate the AHR in these cells. (C) AHR and RB inhibit apoptosis induction by E2F1.…”

“…The plasmid pcDNAI/B6AHR, used to express the high-affinity murine AHR, and its truncation mutant derivatives, have been described previously (Marlowe et al, 2004). Expression of the peptides encoded by each AHR truncation mutant presented in Supplemental Figure S2 was confirmed by Western blot (data not shown).…”

“…Most evidence shows that AHR activation delays cell cycle progression and G 1 to S phase transition, although this effect seems to be cellular context specific, because in rat oval progenitor cells the AHR promotes rather than delays cell cycle progression (Weiss et al, 2008). A canonical RB-binding cyclin D-like motif in the AHR protein sequence mediates a direct interaction between AHR and RB, and experimental work focused on the characterization of this interaction as the mediator of AHR-dependent cell cycle delay (Ge and Elferink, 1998;Puga et al, 2000;Marlowe et al, 2004;Huang and Elferink, 2005) has shown that the activated AHR cooperates with RB in its ability to repress E2F-dependent transcription and delay cell cycle progression Strobeck et al, 2000).…”

“…Most evidence shows that AHR activation delays cell cycle progression and G 1 to S phase transition, although this effect seems to be cellular context specific, because in rat oval progenitor cells the AHR promotes rather than delays cell cycle progression (Weiss et al, 2008). A canonical RB-binding cyclin D-like motif in the AHR protein sequence mediates a direct interaction between AHR and RB, and experimental work focused on the characterization of this interaction as the mediator of AHR-dependent cell cycle delay (Ge and Elferink, 1998;Puga et al, 2000;Marlowe et al, 2004;Huang and Elferink, 2005) has shown that the activated AHR cooperates with RB in its ability to repress E2F-dependent transcription and delay cell cycle progression Strobeck et al, 2000).Paradoxically, embryo fibroblasts from Ahr gene-knockout mice also show a relative delay in cell cycle progression, which has been primarily associated with posttranscriptional stabilization of Tgfb1 mRNA (Chang et al, 2007). In these cells, several E2F transcriptional targets with AHR and E2F binding motifs in their promoters, conserved in both the human and mouse sequences (Supplemental Tables S1 and S2), were also found to be AHR targets, being repressed by both E2f1 and Ahr ablation (Supplemental Figure S1).…”

Repression of Ah receptor and induction of transforming growth factor-β genes in DEN-induced mouse liver tumors

“…The AHR has also been recognized as a cell cycle regulator (Ma and Whitlock, 1996;Weiss et al, 1996;Ge and Elferink, 1998;Kolluri et al, 1999;Puga et al, 2000;Strobeck et al, 2000;Marlowe et al, 2004) although the precise molecular mechanisms responsible for this role have not been fully elucidated. In the absence of an exogenous ligand, or after deletion of the ligand-binding PAS-B domain, the AHR has been shown to promote cell cycle progression (Ma and Whitlock, 1996;Elizondo et al, 2000;Chang et al, 2007), whereas exposure to its prototypical ligand, TCDD, inhibits cell cycle proliferation in an AHR-dependent manner (Bauman et al, 1995;Hushka and Greenlee, 1995;Levine-Fridman, Chen, and Elferink, 2004;Marlowe et al, 2004).…”