No abstract

Cytochrome P450 (CYP) 1A1 and CYP1B1 are inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) in the human breast cancer cell line, MCF-7. Since CYP1A1 was inducible to a much greater degree than CYP1B1, we hypothesized that there may be differences in coactivator recruitment to the promoter and/or enhancer regions of these genes. Dioxin treatment leads to recruitment of the aryl hydrocarbon receptor to the enhancer regions but not to the proximal promoter regions of both the CYP1A1 and CYP1B1 genes. On the other hand, dioxin treatment facilitated recruitment of RNA polymerase II to the promoters but not the enhancer regions. Dioxin treatment also elicited recruitment of the transcriptional coactivators, steroid receptor coactivator 1 (SRC-1) and steroid receptor coactivator 2 (SRC-2) and p300, which possess intrinsic histone acetyltranferase activities, to both genes, whereas Brahma (BRM)/Switch 2-related gene 1 (BRG-1), a subunit of nucleosomal remodeling factors, was recruited more robustly to CYP1A1 relative to CYP1B1. Small inhibitory RNA-mediated knockdown of p300 and SRC-2 adversely affected dioxin induction of both genes, whereas knockdown of BRM/BRG-1 reduced CYP1A1 induction but had little, if any, effect on CYP1B1 induction. These results suggest that nucleosomal remodeling is less significant for dioxin-mediated induction of CYP1B1 than that of CYP1A1 and may be related to the more modest inducibility of the former. Interestingly, simultaneous knockdown of SRC-2 and BRM/BRG-1 had no greater effect on CYP1A1 induction than knockdown of each coactivator individually, while simultaneous knockdown of p300 and BRM/BRG-1 had a much greater effect than knockdown of each individual gene, suggesting that the recruitment of SRC-2 to CYP1A1 depends upon BRM/BRG-1, while the recruitments of p300 and BRM/BRG-1 are independent of each other. These observations provide novel insights into the functional roles of the endogenous coactivators in dioxin induction of the human CYP1A1 and CYP1B1 genes in their natural chromosomal configurations.

BackgroundHypoxia-inducible expression of the erythropoietin (EPO) gene is mediated principally by hypoxia-inducible factor 2α (HIF-2α) in Hep3B cells under physiologic conditions. How/whether p300/CBP and the members of p160 coactivator family potentiate hypoxic induction of endogenous EPO and other HIF-2α and hypoxia-inducible factor 1α (HIF-1α) target genes remains unclear.Methodology/Principal FindingsWe demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that the histone acetyl transferase (HAT) coactivators p300, SRC-1 and SRC-3 are recruited to the 3′ enhancer of the EPO gene upon hypoxic stimulation, and that each associates with the enhancer in a periodic fashion. Hypoxia induced acetylation of the EPO gene 5′ promoter at histone 4 and lysine 23 of histone 3. Knocking down SRC-3, but not SRC-1 or SRC-2, using short interfering RNAs (siRNAs), reduced EPO transcriptional activity. Knocking down p300 resulted in dramatic down-regulation of hypoxic stimulation of EPO gene transcription, negated recruitment of RNA polymerase II to the gene's promoter, and eliminated hypoxia-stimulated acetylation at the promoter and recruitments of SRC-1 and SRC-3 to the enhancer. The inhibitory effects of knocking down p300 and the chromatin remodeling coactivator, Brm/Brg-1, on EPO transcription were additive, suggesting that p300 and Brm/Brg-1 act independently. p300 was also required for hypoxia induced transcription of the HIF-1α target gene, VEGF, but was dispensable for induction of two other HIF-1α target genes, PGK and LDHA. Knocking down CBP, a homolog of p300, augmented hypoxic induction of VEGF, LDHA and PGK. Different HIF target genes also exhibited different requirements for members of the p160 coactivator family.Conclusions/Significancep300 plays a central coactivator role in hypoxic induction of EPO. The coactivators exhibit different specificities for different HIF target genes and each can behave differently in transcriptional regulation of different target genes mediated by the same transcription factor.

Background: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) activates transcription of CYP1A1. Results: Ectopic expression of SIN3A reverses the inhibitory effect of SIN3A siRNAs on CYP1A1 induction. TCDD stimulates SIN3A binding to the CYP1A1 enhancer and promoter. Conclusion: SIN3A coactivates TCDD induction of CYP1A1 transcription. Significance: The anomalous stimulatory effect of SIN3A on transcription is extended to a gene that is rapidly and dramatically induced.

Hypoxia increased the ability of two human cancer cell lines, PC-3M and T24, to invade through Matrigel, while sodium nitroprusside (SNP), a nitric oxide (NO) donor, strongly inhibited this invasion, along with down-regulating HIF-1alpha. SNP also inhibited the function of mitochondria in PC-3M cells, and mitochondrion-specific inhibitors reduced the invasion of these cells. Furthermore, knocking down either Rieske iron-sulfur protein (Fe-S) of mitochondrial complex III or HIF-1beta in these cells decreased their invasive potential. Our findings suggest that NO inhibits invasion of cancer cells via both inhibition of HIF-1, and impairment of mitochondria.

Abstract:The aromatic hydrocarbon receptor (AhR) mediates biological responses to certain exogenous ligands, such as the environmental contaminant 2, 3,7,, and has also been demonstrated to modulate the cell cycle and differentiated state of several cell lines independently of exogenous ligands. In this study, we used DNA micorarray analysis to elucidate the profi le of genes responsive to the expression of unliganded AhR by re-introducing AhR into an AhR-defi cient mouse derivative (c19) of the mouse hepatoma cell line Hepa1c1c7. 22 gene products were up-regulated and 8 were down-regulated two-fold or more in c19 cells infected with a retroviral vector expressing mouse AhR. Surprisingly, expression of genes involved in cell proliferation or differentiation were not affected by introduction of AhR. AhR also did not restore expression of the albumin gene in c19 cells. Introduction of AhR into c12, a similar AhRdefective mouse hepatoma cell line, also did not restore albumin expression, and furthermore, did not lead to changes in cellular morphology or cell cycle parameters. These observations fail to support the notion that unliganded AhR regulates proliferation and differentiation of liver-derived cells.

No abstract