BBB Seminar: Jason Matthews
New mechanisms of AHR function using zinc finger nuclease-mediated gene knockout and identification of TiPARP (ARTD14) as a repressor of AHR transactivation
Department of Pharmacology and Toxicology, University of Toronto, ON, Canada
The aryl hydrocarbon receptor (AHR) was initially identified as a receptor that mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other environmental toxicants; however, there is an increasing amount of data suggesting that AHR is an important therapeutic target for a number of human diseases including inflammatory disease and breast cancer. We used zinc finger nucleases (ZFNs) targeting AHR to create estrogen receptor (ER) positive (MCF-7 AHR-/-) and ER negative (MDA-MB-231 AHR-/-) AHR knockout in human breast cancer cell lines. These data show that AHR is critical for the constitutive expression levels of cytochrome P450 1B1 (CYP1B1), an enzyme that is over-expressed in many different cancers. Loss of AHR reduced the proliferation of both MCF-7 and MDA-MB-231 cells. These findings show the key role AHR plays in the regulation of CYP1B1 and in the proliferation of MCF-7 and MDA-MB-231 breast cancer cells, supporting AHR as a therapeutic target for breast cancer. We have also became very interested in the role of TCDD-inducible poly(ADP-ribose) polymerase (TiPARP, ARTD14), an AHR target gene that is regulated in almost all cell and animal models examined, in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T-47D breast cancer and HuH-7 hepatoma cells increased TCDD-dependent CYP1A1 and CYP1B1 mRNA expression and recruitment of AHR to both genes. Over-expression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by over-expression of AHR, but not ARNT (AHR nuclear translocator). Deletion and mutagenesis studies showed that TiPARP-mediated inhibition of AHR required the zinc finger and catalytic domains. TiPARP and AHR co-localized in the nucleus, directly interacted and both were recruited to CYP1A1 in response to TCDD. Over-expression of TiPARP enhanced whereas RNAi-mediated knockdown of TiPARP reduced TCDD-dependent AHR proteolytic degradation. TCDD-dependent induction of AHR target genes was also enhanced in Tiparp-/- mouse embryonic fibroblasts compared to wildtype controls. Our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop in AHR signalling.
Host: Marit Bakke, Department of Biomedicine