Differential proteomics analysis reveals a role for E2F2 in the regulation of the Ahr pathway in T lymphocytes

Abstract

E2F transcription factors (E2F1-8) are best known for their role in cell proliferation, although it is clear that they regulate many other biological processes through the transcriptional modulation of distinct target genes. However, the specific set of genes regulated by each E2F remains to be characterized. To gain insight into the molecular pathways regulated by E2F2, we have analyzed the proteome of antigen receptor-activated T cells lacking E2F2. We report that loss of E2F2 results in a deregulated Aryl-hydrocarbon-receptor pathway. Proliferating E2F2(-/-) T lymphocytes expressed significantly higher levels of Aip, Ahr, and Arnt relative to wild-type (WT)(1) controls. The mechanism for increased levels of Aip appears straightforward, involving direct regulation of the Aip gene promoter by E2F2. Although the Ahr and Arnt promoters also bind E2F2, their regulation appears to be more complex. Nevertheless, exposure to the environmental xenobiotic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known exogenous ligand of the Ahr pathway, led to overexpression of the Ahr target gene Cyp1a1, and to increased sensitivity to TCDD-triggered apoptosis in E2F2(-/-) T cells compared with WT controls. These results suggest that E2F2 modulates cellular sensitivity to xenobiotic signals through the negative regulation of the Ahr pathway.

Fig. 1.

Experimental design and workflow summary. Two independent experiments were performed, and three biological replicates were used per genotype and experiment. Differentially expressed proteins with statistical significance (t test p < 0.05) were identified by LC-MS/MS, resulting two proteins commonly deregulated in both experiments: Aip and Crkl.

Fig. 2.

Validation of deregulated expression of Aip and Crkl expression in activated E2F2^−/− T lymphocytes. (A, C) Western analysis of Aip and Crkl expression values showing normalized ratios. The ratios Aip/β-actin and Crkl/β-actin in WT were considered as a unit. The experiment shown is representative of three independent experiments (n = 8 WT; n = 8 E2F2^−/−). (B, D) Quantitative RT-PCR analysis of Aip and Crkl mRNA expression. Values were normalized against the mRNA expression levels of an irrelevant gene (eEf1a1) and given as relative values (n = 6 WT; n = 6 E2F2^−/−; two independent experiments, each in triplicate).

Fig. 3.

E2F2 binds to the promoters of the Ahr pathway genes in quiescence or after activation. (A) Schematic representation of Aip, Ahr, Arnt, Ahrr and Chk1 promoters, indicating the position of the E2F-binding sites and the primers used for ChIP analysis. (B, C) Results of the ChIP analysis. Cell lysates from quiescent or anti-CD3 stimulated T lymphocytes were used for ChIP analysis with anti-E2F2 antibodies. Immunoprecipitated DNA was analyzed by Q-PCR using primers flanking the E2F sites of each promoter. Antibodies to large T-antigen were used as irrelevant antibody control (mock). The promoter of β-actin, a gene that has no E2F sites but is highly expressed in T lymphocytes, was used as a negative control. (B) Specificity of anti-E2F2 antibodies used in ChIP assays. Control ChIP assay of Chk1 promoter using anti-E2F2 antibodies in WT or E2F2^−/− T lymphocytes. Each data point depicts the amount (ng) of immunoprecipitated DNA ± S.D. (average of three independent ChIP experiments, each analyzed in triplicate, n = 6 WT; n = 6 E2F2^−/−). (C) ChIP assays of cell lysates from quiescent or proliferating T lymphocytes derived from WT mice, using antibodies anti-E2F2. Each data point depicts the amount (ng) of immunoprecipitated DNA ± S.D. (average of three independent ChIP experiments, each analyzed in triplicate, n = 9 WT). PCR amplification values for Ahr pathway promoters were compared with those of β–actin promoter by a one-way analysis of variance (p < 0.05).

Fig. 4.

Deregulation of Ahr pathway genes in activated E2F2^−/− T lymphocytes. (A, C) Western analysis of Ahr and Arnt expression. Values were normalized against β-actin. The ratios Ahr/β-actin and Arnt/β-actin in WT were considered as a unit. The experiment shown is representative of three independent experiments (n = 8 WT; n = 8 E2F2^−/−). (B, D, E) Quantitative RT-PCR analysis of Ahr, Arnt and Ahrr. mRNA expression values were normalized against the mRNA expression levels of an irrelevant gene (eEf1a1) and given as relative values (n = 6 WT; n = 6 E2F2^−/−; two independent experiments, each in triplicate).

Fig. 5.

(A) Western blot analysis of Cyp1a1 and Gsta1 expression demonstrating comparable protein levels in WT and E2F2^−/− T lymphocytes activated through their T-cell receptor. Expression values were normalized against β-actin. The ratios Cyp1a1/β-actin and Gsta1/β-actin in WT were considered as a unit. (B) Western blot analyzes of quiescent (0h) or proliferating (36h) T lymphocytes showing differential subcellular localization of Ahr pathway proteins upon E2F2 loss. Accumulation of Ahr pathway proteins was observed in the cytosolic fraction of E2F2 deficient T lymphocytes. LamB1 was used as nuclear marker and β-actin as loading control. The experiment shown is representative of two independent experiments (n = 6 WT; n = 6 E2F2^−/−).

Fig. 6.

TCDD-mediated activation of Ahr pathway in proliferating T lymphocytes leads to an increased apoptosis in E2F2^−/− T cells. T cells were activated using anti-CD3 antibody for 36 hours and subsequently treated with TCDD for the referred times. (A) Apoptosis ratio in the presence of TCDD and vehicle toluene (TOL) of activated WT and E2F2^−/− T lymphocytes. (B–D) RT-PCR analysis of Cyp1a1, Bax and FasL in TCDD treated activated T cells derived from WT and E2F2^−/− mice (B) Quantitative RT-PCR analysis of Cyp1a1 mRNA expression. Values were normalized against the mRNA expression levels of an irrelevant gene (eEf1a1) and given as relative values. (C, D) Quantitative RT-PCR analysis of the proapoptotic genes FasL and Bax. Values were normalized against the mRNA expression levels of an irrelevant gene (eEf1a1), and relative values obtained in wild-type samples were considered as a unit in each experimental time point (n = 9 WT; n = 9 E2F2^−/−; three independent experiments).

Fig. 7.

Model of E2F2-mediated regulation of the Ahr pathway. E2F2 represses the expression of Ahr pathway components Ahr, Aip and Arnt. In the absence of E2F2, higher levels of Ahr pathway gene products accumulate in the cytosol. In response to xenobiotics, E2F2-deficient cells exhibit greater Ahr-dependent transcriptional activation, resulting in increased apoptosis.