Abstract
Co-contamination with complex mixtures of carcinogenic metals, such as chromium, and polycyclic aromatic hydrocarbons is a common environmental problem with multiple biological consequences. Chromium exposure alters inducible gene expression, forms chromium-DNA adducts and chromium-DNA cross-links, and disrupts transcriptional activator-co-activator complexes. We have shown previously that exposure of mouse hepatoma Hepa-1 cells to chromate inhibits the induction of the Cyp1a1 and Nqo1 genes by dioxin. Here we have tested the hypothesis that chromium blocks gene expression by interfering with the assembly of productive transcriptional complexes at the promoter of inducible genes. To this end, we have studied the effects of chromium on the expression of genes induced by benzo[a]pyrene (B[a]P), another aryl hydrocarbon receptor agonist, and characterized the disruption of Cyp1a1 transcriptional induction by chromium. Gene expression profiling by using high density microarray analysis revealed that the inhibitory effect of chromium on B[a]P-dependent gene induction was generalized, affecting the induction of over 50 different genes involved in a variety of signaling transduction pathways. The inhibitory effect of chromium on Cyp1a1 transcription was found to depend on the presence of promoter-proximal sequences and not on the cis-acting enhancer sequences that bind the aryl hydrocarbon receptor-aryl hydrocarbon receptor nuclear translocator complex. By using transient reporter assays and chromatin immunoprecipitation analyses, we found that chromium prevented the B[a]P-dependent release of HDAC-1 from Cyp1a1 chromatin and blocked p300 recruitment. These results provide a mechanistic explanation for the observation that chromium inhibits inducible but not constitutive gene expression.
Highlights
Co-contamination with complex mixtures of carcinogenic metals, such as chromium, and polycyclic aromatic hydrocarbons is a common environmental problem with multiple biological consequences
The list of B[a]P-inducible genes inhibited by chromium includes genes involved in a variety of signaling transduction pathways, such as calcium-dependent regulation, receptor-associated kinases, transcription factors, cell cycle regulation, differentiation, and apoptosis in addition to genes involved in drug metabolism
The results presented in this article show that inhibition of Ah receptor-dependent expression by chromium is a generalized phenomenon that extends to at least 50 B[a]P-inducible genes involved in a variety of cellular processes (Table I)
Summary
B[a]P, benzo[a]pyrene; AHR, aryl hydrocarbon receptor; AH, aryl hydrocarbon; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; ARNT, Ah receptor nuclear translocator; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; ChIP, chromatin immunoprecipitation; HAT, histone acetyltransferase; HDAC, histone deacetylase; HDAC, ICP-MS, inductively coupled plasma mass spectrometry; SRC, steroid receptor co-activator; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Cy3, cytidine-3; Cy5, cytidine-5; HSV-1, herpes simplex virus, type 1; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. The molecular mechanism responsible for this effect is likely to involve the reduction process in the persistent stimulation of regulatory pathways affecting interactions of transcription factors with transcriptional co-regulators and chromatin remodeling factors, more so than the binding of the factors themselves to their cognate recognition sites Earlier studies from this laboratory have shown that exposure of mouse hepatoma Hepa-1 cells to chromate inhibits the induction of the Cyp1a1 and Nqo genes by dioxin, an AHR ligand [17]. The AHR is a ligand-activated basic region helixloop-helix/Per-AHR-ARNT-Sim transcription factor that forms heterodimers with ARNT and binds to cis-acting AHR-response enhancer elements in the regulatory domains of target genes, such as Cyp1a1 and Nqo, leading to changes in chromatin structure and activation of gene transcription [18] These changes include the recruitment to the transcription machinery of associated co-regulator proteins. We find that chromium prevents the B[a]P-dependent release of HDAC-1 from chromatin, preventing the association of p300 with the Cyp1a1 transcriptional complex and blocking gene transcription
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