The aryl hydrocarbon receptor (AHR) and estrogen receptor alpha (ERα) are ligand-activated transcription factors. Reciprocal crosstalk between these two receptor systems has been previously established but the exact molecular mechanisms of their interactions remain incompletely understood. Using chromatin immunoprecipitation followed by DNA microarrays (ChIP-chip), I assessed the role of ERα in AHR signalling after dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) treatment in the T-47D human breast cancer cell line. I determined that ERα is recruited to a subset of AHR target genes suggesting that it is a gene-specific modulator of AHR activity. Transcription factor binding site analysis of our data set also revealed that forkhead motifs were over-represented, implying that they may be important in AHR signalling. To address this, I focused on the regulation of cyclin G2 (CCNG2) to determine the importance of FOXA1 (forkhead box A1) in AHR signalling. CCNG2 is a negative regulator of cell cycle and known to be repressed by ERα. Using ChIP, Co-IP, CCNG2 reporter gene constructs and RNA interference targeting FOXA1, I demonstrated that FOXA1 was important for the AHR-mediated and TCDD-dependent induction of CCNG2. Another finding from the ChIP-chip study was that AHR was recruited to estrogen target genes. To determine the importance of this I used zinc-finger nuclease mediated knockout of AHR and studied ERα signalling as well as the role of AHR in the cell cycle using breast cancer cell lines. Focusing on the regulatory regions of trefoil factor 1 (TFF-1) and gene upregulated in breast cancer 1 (GREB1) I determined that AHR had an inhibitory effect. Cell cycle analysis indicated that AHR facilitated cell cycle progression with cells accumulating in both the G¬1 and G2/M phases in the absence of AHR. My novel findings demonstrated the complexity of AHR-ERα crosstalk, its importance in the cell cycle, and the need for further study.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/33900 |
| Date | 06 December 2012 |
| Creators | Ahmed, Shaimaa |
| Contributors | Matthews, Jason |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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