Chromium cross-links histone deacetylase 1-DNA methyltransferase 1 complexes to chromatin, inhibiting histone-remodeling marks critical for transcriptional activation

Abstract

Transcriptional regulation of gene expression requires posttranslational modification of histone proteins, which, in concert with chromatin-remodeling factors, modulate chromatin structure. Exposure to environmental agents may interfere with specific histone modifications and derail normal patterns of gene expression. To test this hypothesis, we coexposed cells to binary mixtures of benzo[a]pyrene (B[a]P), an environmental procarcinogen that activates Cyp1a1 transcriptional responses mediated by the aryl hydrocarbon receptor (AHR), and chromium, a carcinogenic heavy metal that represses B[a]P-inducible AHR-mediated gene expression. We show that chromium cross-links histone deacetylase 1-DNA methyltransferase 1 (HDAC1-DNMT1) complexes to Cyp1a1 promoter chromatin and inhibits histone marks induced by AHR-mediated gene transactivation, including phosphorylation of histone H3 Ser-10, trimethylation of H3 Lys-4, and various acetylation marks in histones H3 and H4. These changes inhibit RNA polymerase II recruitment without affecting the kinetics of AHR DNA binding. HDAC1 and DNMT1 inhibitors or depletion of HDAC1 or DNMT1 with siRNAs blocks chromium-induced transcriptional repression by decreasing the interaction of these proteins with the Cyp1a1 promoter and allowing histone acetylation to proceed. By inhibiting Cyp1a1 expression, chromium stimulates the formation of B[a]P DNA adducts. Epigenetic modification of gene expression patterns may be a key element of the developmental and carcinogenic outcomes of exposure to chromium and to other environmental agents.

FIG. 1.

Chromium cross-links HDAC1 to the Cyp1a1 promoter and inhibits its B[a]P-induced release. Hepa-1 cells were treated for 2 h with DMSO vehicle (D) or 50 μM chromium (Cr and C), for 1.5 h with 5 μM B[a]P (B), for 2 h with chromium with 5 μM B[a]P for the last 1.5 h (Cr+B[a]P and CB), or for 1.5 h with 5 μM B[a]P with chromium added for the last hour (B[a]P+Cr). (A) ChIP assays were performed as described with specific modifications. For the cross-linking step, cells were incubated with or without formaldehyde, and all subsequent steps were performed the in presence or in the absence of EDTA. HDAC1-immunoprecipitated DNA was amplified by real-time PCR using specific primers spanning the Cyp1a1 proximal promoter region. Quantification is expressed as percentage of total input, and results are the means (± standard deviations) from three independent experiments. (B) Hepa-1 cells were grown in medium with or without 2 μM Aza for 72 h prior to treatment. Re-ChIP assays were performed by sequential IP with the indicated antibodies. Immunoprecipitated DNA was amplified by real-time PCR using specific primers spanning the Cyp1a1 proximal promoter region. (C) ReChIP assays were performed on chromatin from cells treated as indicated by sequential IP with antibodies against AHR and ARNT. The DNA was amplified by PCR using specific primers spanning the Cyp1a1 enhancer region, and PCR products were visualized by ethidium bromide staining after gel electrophoresis. (D) Quantification of the data in panel B is expressed as percentage of total input, and results are the means (± standard deviations) from three independent experiments. All results shown in gels are representative of three independent experiments.

FIG. 2.

Chromium inhibits B[a]P-inducible gene expression without affecting AHR binding. (A) Hepa-1 cells were treated for 9 h with DMSO (D), 50 μM chromium (C), 50 μM chromium for 1 h followed by 5 μM B[a]P for 8 h (CB), or 5 μM B[a]P for 8 h with 50 μM chromium added 2 h after B[a]P (BC). Total RNA was prepared, and Cyp1a1, Aldh3a1, and Nqo1 mRNA expression levels were evaluated by real-time PCR after reverse transcription. Relative mRNA expression is expressed as the induction (n-fold) calculated from the ratio of the target signal to β-actin relative to the same ratio in control cells. The data are the means (± standard deviations) from three independent experiments. (B) ChIP assays were performed with anti-AHR antibodies after treatment for 2 h with DMSO (D), 50 μM chromium (C), 5 μM B[a]P (B) for 1.5 h, or 50 μM chromium for 0.5 h before (CB) or 1 h after (BC) treatment with 5 μM B[a]P for 1.5 h. Immunoprecipitated DNA was amplified and quantified by real-time PCR using specific primers for the Cyp1a1, Aldh3a1, and Nqo1 promoter domains containing AHR binding sites. DNA enrichment was normalized to inputs, and data shown are the means ± standard deviations from three independent experiments.

FIG. 3.

Chromium blocks B[a]P-mediated RNA polymerase II recruitment without affecting the kinetics of AHR binding to the enhancer. Cells were pretreated with 50 μM chromium or with vehicle for 0.5 h, induced with 5 μM B[a]P, and subjected to ChIP every 20 min for 3 h with anti-AHR and anti-RNA polymerase II antibodies. (A and C) PCR products resulting from the amplification of the enhancer (kbp −0.8) and the proximal promoter (kbp −0.1) regions (see Fig. 4A for their locations in the Cyp1a1 promoter), respectively. (B and D) Quantification by real-time PCR, normalized to input DNA, for the AHR binding kinetics to the enhancer (B) and for the binding kinetics of AHR (D, top) and RNA polymerase II (D, bottom) to the proximal promoter. All results are representative of three independent experiments, and quantification data are the means ± standard deviations from three independent experiments.

FIG. 4.

Chromium inhibits B[a]P-induced modification of specific histone amino acids in Cyp1a1 promoter chromatin. (A) Schematic representation of the mouse Cyp1a1 promoter from kbp −4.0 to +1.0 with the PCR primer positions used to map the promoter in ChIP experiments. Positions of AhRE motifs and primers (purple arrows) relative to the transcriptional start site (blue arrow, numbered as +1) are indicated. A nucleosome (green dashed circle), probably positioned over the promoter, is localized on the scheme. (B and C) Cells were treated for 2 h with DMSO (D) or 50 μM chromium (Cr or C), for 1.5 h with 5 μM B[a]P (B), for 2 h with chromium with 5 μM B[a]P for the last 1.5 h, (Cr+B[a]P or CB), or for 1.5 h with 5 μM B[a]P with chromium added for the last hour (B[a]P+Cr or BC). (B) ChIP used the indicated antibodies specific for posttranslationally modified histone amino acids. Real-time PCR products amplified with primers for the distal (kbp −3.2), enhancer (kbp −0.8), and proximal promoter (kbp −0.1) domains were resolved by electrophoresis. (C) Representative results of two or three independent real-time PCR amplification experiments are expressed as percentage of total input.

FIG. 5.

HDAC1 siRNA, NaB, and Aza block chromium-induced transcriptional repression of B[a]P-inducible Cyp1a1 expression. Cells were transfected with GAPDH siRNA, scrambled siRNA, DNMT1 siRNA, HDAC1 siRNA, or no siRNA. (A to C) At 48 h posttransfection, the effect of the siRNAs on target gene expression was evaluated for mRNA (A) and protein levels (B), or the cells were treated with DMSO, 50 μM chromium for 9 h, 5 μM B[a]P for 8 h, or 50 μM chromium for 1 h followed by 5 μM B[a]P for an additional 8 h. Cyp1a1 mRNA expression was determined in these samples by real-time PCR (C). Relative mRNA expression is expressed as induction (n-fold) calculated as the ratio of target signal to β-actin relative to the same ratio in the control cells. (D) Hepa-1 cells were maintained in a medium with or without 2 μM Aza for 72 h, 2 mM NaB for 16 h, or both and treated with chromium or B[a]P as for panel C. Total RNA was extracted, and Cyp1a1, Aldh3a1, and Nqo1 mRNA expression levels were determined by real-time PCR after reverse transcription. Relative mRNA expression is expressed as the induction (n-fold) calculated from the ratio of the target signal to β-actin relative to the same ratio in control cells. The data are the means (± standard deviations) from three independent experiments.

FIG. 6.

NaB and Aza partially reduce occupancy of the Cyp1a1 promoter by HDAC1 and DNMT1 but do not recruit AHR and p300. Hepa-1 cells were maintained in medium with or without 2 μM Aza for 72 h, 2 mM NaB for 16 h, or a combination of both agents. Cells were treated for 2 h with DMSO, 50 μM chromium, 5 μM B[a]P, or 50 μM chromium for 0.5 h followed by 5 μM B[a]P for an additional 1.5 h. ChIP assays used HDAC1, DNMT1, AHR, and p300 antibodies. DNA enrichment was quantified by real-time PCR and expressed as percentage of total input. Data are the means (± standard deviations) from three independent experiments. ND, not done.

FIG. 7.

NaB and Aza reverse the inhibition of histone modifications induced by chromium. Hepa-1 cells were maintained in medium with or without 2 μM Aza for 72 h, 2 mM NaB for 16 h, or a combination of both agents and were treated for 2 h with DMSO, 50 μM chromium, 5 μM B[a]P for 1.5 h, or 50 μM chromium for 0.5 h followed by 5 μM B[a]P for an additional 1.5 h. ChIP assays were performed with the indicated antibodies raised against the specific posttranslational histone modifications indicated in the graphs. Immunoprecipitated DNA was amplified by real-time PCR using primers specific for the indicated enhancer and proximal promoter regions. DNA enrichment is expressed as percentage of the total input. Results are the means ± standard deviations from two independent experiments.

FIG. 8.

Formation of BPDE-DNA adducts is dose dependent and is potentiated by chromium pretreatment. Hepa-1 cells were pretreated with the indicated concentration of sodium chromate for 30 min before addition of 1 μM B[a]P to the culture medium. Cells were harvested for adduct determination after 2 h and 24 h of B[a]P treatment. The total adduct level is shown as the number of adducts per 10⁹ nucleotides. Each bar represents the mean ± standard error from two independent experiments. The asterisks denote statistical significance (P < 0.05) for the comparison of chromium pretreatment to DMSO treatment for the same length of B[a]P treatment.

FIG. 9.

Schematic model of chromium-induced transcriptional repression. This model summarizes results reported here and in previous reports (25, 50). In the inactive state, Cyp1a1 is silent due to the presence of the complexes formed by DNMT1 and HDAC1, the hypoacetylation of histone tails, and a high level of dimethylated Lys4-H3. AHR activation by a ligand, such as B[a]P, causes ligand-activated AHR-ARNT complexes (represented here by a single heterodimer) to bind to the cognate AhREs in the enhancer region. Binding of the AHR-ARNT complex promotes an active state characterized by the phosphorylation of Ser10-H3 in the enhancer region, trimethylation of Lys4-H3 in the proximal promoter, and hyperacetylation of histone tails across the promoter. Here shown as a chromatin-bending loop, but possibly also the result of sliding, the enhancer complex makes contact with the proximal promoter; releases the repressive HDAC1-DNMT1 complexes; allows recruitment of coactivators, general transcription factors, and the RNA polymerase II complex; and initiates Cyp1a1 trans-activation. Preexposure to Cr(VI), rapidly reduced to Cr(III), causes cross-linking (coordination) of HDAC1-DNMT1 complexes to the promoter, maintaining a chronic state of histone deacetylation that inhibits recruitment of p300 and the AHR complex to the proximal promoter and establishing a chromium-repressed state.