2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP/ARTD14) is a member of the ARTD family and is regulated by the aryl hydrocarbon receptor (AHR); however, little is known about TiPARP function. In this study we examined the catalytic function of TiPARP and determined its role in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T47D breast cancer and HuH7 hepatoma cells increased TCDD-dependent cytochrome P450 1A1 (CYP1A1) and CYP1B1 mRNA expression and recruitment of AHR to both genes. Overexpression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by overexpression of AHR, but not ARNT. 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. Overexpression 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. Moreover, livers excised from TCDD-treated Tiparp-/- mice displayed significantly greater AHR target gene expression compared with wildtype or heterozygous mice. Comparison of TiPARP to known negative regulator, AHR repressor (AHRR) revealed TiPARP and AHRR some notable similarities and differences between their mechanisms of repression. Similar to TiPARP, the AHRR was recruited to AHR target regulatory regions in response to TCDD and its overexpression repressed reporter gene activity. However unlike TiPARP, knockdown of the AHRR did not affect AHR transactivation or its proteasomal degradation. Despite some mechanistic similarities, our data suggest that TiPARP and AHRR independently repress AHR transactivation. Overall, our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop controlling AHR transcriptional regulation.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/65686 |
| Date | 22 July 2014 |
| Creators | MacPherson, Laura |
| Contributors | Matthews, Jason |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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