Novel Gene Regulation Mechanisms of Aryl Hydrocarbon Receptor

Novel Gene Regulation Mechanisms of Aryl Hydrocarbon Receptor: A ChIP-seq and RNA-seq Study of Ahrdbd/dbd Mice Expressing an Aryl Hydrocarbon Receptor with Mutated DNA-binding Domain

Peng Shao

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Pharmacology and Toxicology University of Toronto

© Copyright by Peng Shao (2019) Novel Gene Regulation Mechanisms of Aryl Hydrocarbon Receptor: A ChIP-seq and RNA-seq Study of Ahrdbd/dbd Mice Expressing an Aryl Hydrocarbon Receptor with Mutated DNA-binding Domain

Peng Shao

Master of Science

Department of Pharmacology and Toxicology University of Toronto

2019

Abstract

Aryl hydrocarbon receptor (AHR) is a transcription factor known for mediating the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and regulating many physiological and pathological processes. In its canonical signalling pathway, ligand-activated AHR translocates into nucleus, associates with its heterodimerization partner, and binds the cognate aryl hydrocarbon response element (AHRE) to regulate transcription. Based on recent evidence, we hypothesize that

AHR may mediate gene regulation through unrevealed mechanisms, namely indirect DNA binding via tethering to other transcription factors, or direct DNA binding to regions not containing any

AHRE. To explore this, Ahrdbd/dbd mice whose AHR is incapable of binding AHRE were used.

ChIP-seq showed Ahrdbd/dbd mice indeed had an increased number of AHR-bound regions across the genome upon activation by TCDD. However, RNA-seq found almost no alteration in gene expression. These results support the presence but refute the physiological significance of AHR tethering to other transcription factors or non-AHRE binding to DNA.

Acknowledgments

I would like to express my sincere gratitude to my supervisor Dr. Denis Grant and my co- supervisor Dr. Jason Matthews for their immense support throughout my study. It has been an enjoyable and invaluable experience to learn under their guidance.

My sincere appreciation goes to Dr. David Hutin for his extensive assistance, advice, and encouragement. I also appreciate the kindness of all other members of our laboratory: Dr. Daniel

Hanna, Dr. Alexandra Long, and Dr. Kim Sugamori.

I thank Dr. David Riddick for being my supportive advisor, as well as Dr. Ali Salahpour and Dr.

Leonardo Salmena for being members of my examination committee.

I would like to acknowledge the Canadian Institutes of Health Research as the funding agency for our laboratory and this project. I also appreciate the financial support for my study from the Ontario

Graduate Scholarship and the University of Toronto.

I am eternally grateful to my parents for their boundless love. My wish is that my grandparents would be proud of me, though they are no longer here to see it.

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Table of Contents

Abstract ...... ii Acknowledgments...... iii Table of Contents ...... iv List of Figures ...... vii List of Tables ...... viii List of Abbreviations ...... ix Chapter 1 Introduction ...... 1 1.1 The AHR ...... 1 1.1.1 History of Identification ...... 1 1.1.2 Functional Domains of AHR ...... 2 1.1.3 Canonical Signalling Pathway of AHR ...... 4 1.1.4 Non-canonical Signalling Pathway ...... 7 1.2 Role of AHR in Toxicology ...... 7 1.2.1 Synthetic Compounds ...... 8 1.2.2 History of TCDD ...... 10 1.2.3 AHR Mediated Toxicity ...... 11 1.3 Role of AHR in Physiology ...... 14 1.3.1 Natural AHR Ligands ...... 14 1.3.2 Role of AHR in Detoxification ...... 17 1.3.3 Role of AHR in Development...... 17 1.3.4 Other Cellular Roles of AHR ...... 19 1.4 Evidence for AHR Transcriptional Regulation via Tethering ...... 19 1.4.1 Non-canonical Functions of AHR ...... 20 1.4.2 Transcriptional Regulation via Tethering ...... 21 1.4.3 AHR and ER ...... 21 1.4.4 AHR and Immune Regulators (NFκB and AP-1) ...... 23 1.4.5 AHR and Other Candidate Partners ...... 24 1.5 Ahrdbd/dbd Mice ...... 26 1.5.1 Generation and Characterization of Ahrdbd/dbd Mice ...... 27 1.5.2 Ahrb1 and Ahrd alleles...... 28 1.6 Rationale and Objective ...... 29 Chapter 2 Materials and Methods ...... 32 2.1 Materials ...... 32

2.1.1 Chemical and Biological Reagents ...... 32 2.1.2 Plasticware and Other Consumables ...... 36 2.1.3 Instruments ...... 37 2.2 Methods...... 40 2.2.1 Animal Facility and Colony Maintenance ...... 40 2.2.2 Mouse Genotyping ...... 41 2.2.3 Ahr Gene Cloning and Sequencing ...... 41 2.2.4 Luciferase Reporter Assay ...... 43 2.2.5 Animal Treatment and Tissue Collection ...... 45 2.2.6 RNA Extraction, cDNA synthesis, and RT-qPCR ...... 45 2.2.7 Western Blot ...... 46 2.2.8 ChIP-seq and Data Analysis ...... 48 2.2.9 RNA-seq and Data Analysis ...... 53 2.2.10 Statistical Analysis ...... 54 Chapter 3 Results ...... 55 3.1 Characterization of Ahrdbd/dbd Mice ...... 55 3.1.1 The Mouse Colony ...... 55 3.1.2 Gene Sequencing of Ahrwt and Ahrdbd ...... 59 3.1.3 mRNA and Protein Expression of AHR in Mouse Liver ...... 62 3.1.4 Comparison of AHRwt and AHRdbd for Activation of AHRE-driven Reporter ...... 65 3.1.5 mRNA Expression of Cyp1a1 and Cyp1b1 in Mouse Liver ...... 68 3.2 ChIP-seq ...... 71 3.2.1 ChIP-qPCR ...... 71 3.2.2 AHR-bound Regions ...... 77 3.2.3 De novo and Enriched Transcription Factor Binding Site Analysis ...... 80 3.3 RNA-seq ...... 85 3.3.1 Genes Revealed by RNA-seq ...... 85 3.3.2 Validation by RT-qPCR ...... 91 Chapter 4 Discussion ...... 96 4.1 The Impact of AHR on Reproduction, Fertility and Sex Ratio...... 96 4.2 The Genotype and Functionality of AHRdbd ...... 98 4.3 AHR Expression and Conjectural Self-regulation ...... 100 4.4 AHR-bound Regions Revealed by ChIP-seq ...... 102 4.5 Transcription Factor Binding Sites Enriched in AHR-bound Regions ...... 104

4.6 Alteration in Gene Expression Revealed by RNA-seq ...... 108 4.7 Limitations ...... 110 4.8 Future Directions ...... 112 4.9 Conclusion ...... 113 References ...... 115

List of Figures

Figure 1.1 Domain architecture of AHR and ARNT...... 4 Figure 1.2 AHR canonical signalling pathway...... 6 Figure 1.3 Synthetic ligands of AHR – HAHs and PAHs...... 10 Figure 1.4 Selected natural ligands of AHR...... 16 Figure 2.1 Schematic representation of the ChIP-seq pipeline from ENCODE...... 53 Figure 3.1 The sex ratio of the offspring of Ahrwt/dbd breeders...... 57 Figure 3.2 The genotypic ratio of the offspring of Ahrwt/dbd breeders...... 58 Figure 3.3 Amino acid sequence alignment of the cloned AHRwt and AHRdbd proteins...... 61 Figure 3.4 Hepatic Ahr mRNA levels in Ahrwt/wt and Ahrdbd/dbd mice after treatment with DMSO or 100 µg/kg TCDD for 2, 6, and 24 hours...... 63 Figure 3.5 Hepatic AHR protein levels in Ahrwt/wt and Ahrdbd/dbd mice after treatment with DMSO or 100 µg/kg TCDD for 2, 6, and 24 hours...... 64 Figure 3.6 Relative Cyp1a1-regulated luciferase activity of COS-1 cells transfected with empty pcDNA3.1, pcDNA3.1-Ahrwt, and pcDNA3.1-Ahrdbd plasmids...... 66 Figure 3.7 Relative AHR protein expression of COS-1 cells transfected with empty pcDNA3.1, pcDNA3.1-Ahrwt, and pcDNA3.1-Ahrdbd plasmids...... 67 Figure 3.8 Hepatic Cyp1a1 mRNA expression levels in Ahrwt/wt and Ahrdbd/dbd mice after treatment with DMSO or 100 µg/kg TCDD for 2, 6, or 24 hours...... 69 Figure 3.9 Hepatic Cyp1b1 mRNA expression levels in Ahrwt/wt and Ahrdbd/dbd mice after treatment with DMSO or 100 µg/kg TCDD for 2, 6, or 24 hours...... 70 Figure 3.10 qPCR analysis of ChIP samples using Cyp1a1 and Cyp1b1 promoters, presented as percent input...... 73 Figure 3.11 qPCR analysis of ChIP samples using Cyp1a1 and Cyp1b1 promoters, presented as fold enrichment...... 74 Figure 3.12 qPCR analysis of the amplified libraries using Cyp1a1 promoter, presented as fold enrichment...... 76 Figure 3.13 Venn diagram comparing AHR-bound regions determined by ChIP-seq among all groups .. 78 Figure 3.14 UpSet plot comparing AHR-bound regions determined by ChIP-seq among all groups...... 79 Figure 3.15 Overlap of genes differently expressed in Ahrwt/wt mice versus Ahrdbd/dbd mice...... 86 Figure 3.16 Overlap of genes differently expressed with TCDD versus with DMSO...... 87 Figure 3.17 mRNA expression confirmed by RT-qPCR of genes selected from the RNA-seq result – Part one...... 94 Figure 3.18 mRNA expression confirmed by RT-qPCR of genes selected from the RNA-seq result – Part two...... 95

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List of Tables

Table 2.1 Primers for Ahrdbd genotyping ...... 41 Table 2.2 Primers for Ahr gene cloning and sequencing ...... 43 Table 2.3 Primers for gene expression RT-qPCR ...... 46 Table 2.4 Primers for ChIP-qPCR...... 50 Table 3.1 Top 6 de novo motifs discovered in AHR-bound regions of TCDD-treated Ahrwt/wt mice and TCDD-treated Ahrdbd/dbd mice...... 81 Table 3.2 Top 10 enriched transcription factor binding sites in AHR-bound regions of DMSO- treated and TCDD-treated Ahrwt/wt mice...... 83 Table 3.3 Top 10 enriched transcription factor binding sites within 10 to 50 bp from AHR binding motifs in TCDD-treated Ahrwt/wt mice...... 83 Table 3.4 Top 10 enriched transcription factor binding sites in AHR-bound regions of DMSO- treated and TCDD-treated Ahrdbd/dbd mice...... 84 Table 3.5 Top 25 upregulated genes and top 25 downregulated genes in DMSO-treated Ahrwt/wt mice relative to DMSO-treated Ahrdbd/dbd mice ...... 88 Table 3.6 Top 25 upregulated genes and top 25 downregulated genes in TCDD-treated Ahrwt/wt mice relative to TCDD-treated Ahrdbd/dbd mice ...... 89 Table 3.7 Top 25 upregulated genes and the only 5 downregulated genes in TCDD-treated Ahrwt/wt mice relative to DMSO-treated Ahrwt/wt Mice ...... 90 Table 3.8 The only gene differently expressed in TCDD-treated Ahrdbd/dbd mice relative to DMSO-treated Ahrdbd/dbd Mice ...... 90

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List of Abbreviations

3-MC 3-methylcholanthrene AHH Aryl hydrocarbon hydroxylase AHR Aryl hydrocarbon receptor AHRE Aryl hydrocarbon response element AHRR Aryl hydrocarbon receptor repressor ALDH Aldehyde dehydrogenase AP-1 Activator protein 1 ARNT Aryl hydrocarbon receptor nuclear translocator B[a]P Benzo[a]pyrene bHLH-PAS Basic Helix-Loop-Helix – Period/ARNT/Single minded ChIP-chip Chromatin immunoprecipitation followed by DNA microarray ChIP-seq Chromatin immunoprecipitation-sequencing COX2 Cyclooxygenase 2 Cspg4 Chondroitin sulfate proteoglycan 4 c-Src Cellular Src kinase Cux2 Cut like homeobox 2 CYP Cytochrome P450 DIM 3,3'-diindolylmethane DMEM Dulbecco’s modified Eagle’s medium DRE Dioxin response element DSS Dextran sodium sulfate DTT Dithiothreitol DV Ductous venosus E2F E2 factor Ecel1 Endothelin converting enzyme like 1 EDTA Ethylenediaminetetraacetic acid Egfr Epidermal growth factor receptor EGR Early growth response EMSA Electrophoretic mobility shift assays ER Estrogen receptor ERE Estrogen response element Esrrg Estrogen related receptor gamma FICZ 6-formylindolo[3,2-b]carbazole Fmr1 Fragile X-mental retardation 1 GR Glucocorticoid receptor GST Glutathione S-transferase HAH Halogenated aromatic hydrocarbon HIF Hypoxia inducible factor HSP90 90-kDa heat shock protein I3C Indole-3-carbinol ICZ Indolo[3,2-b]carbazole IL-6 LPS-induced interleukin 6 KA Kynurenic acid LPS Lipopolysaccharide

MAPK Mitogen-activated protein kinase Moxd1 Monooxygenase DBH like 1 NCOA1 Nuclear receptor coactivator 1 NES Nuclear export signal NF1 Nuclear factor 1 NFκB Nuclear factor kappa B NLS Nuclear localization signal NQO1 NAD(P)H quinone oxidoreductase 1 NRF1 Nuclear respiratory factor 1 NRF2 Nuclear respiratory factor 2 NR2F Nuclear receptor subfamily 2 NRIP1 Nuclear receptor-interacting protein 1 ONPG o-Nitrophenyl-β-D-Galactopyranoside p23 23-kDa co-chaperone PAH Polycyclic aromatic hydrocarbon PBS Dulbecco’s phosphate buffered saline PCB Polychlorinated biphenyl PCDD Polychlorinated dibenzodioxin PCDF Polychlorinated dibenzofuran PIC Protease inhibitor cocktail PKA Protein kinase A PKCα Protein kinase Cα PLA2 Phospholipase A2 PPAR Peroxisome proliferator-activated receptor RNA-seq RNA-sequencing RXR Retinoid X receptor SDS Sodium dodecyl sulfate Serpine1 Serpin family E member 1 SP1 Specificity protein 1 Tbp TATA-box binding protein TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TEF Toxicity equivalency factor TGF-β Transforming growth factor β Th17 T helper type 17 cells TIPARP TCDD-inducible poly-ADP-ribose polymerase Tpo Thyroid peroxidase TR Thyroid hormone receptor Treg Regulatory T cells Tris Tris(hydroxymethyl)aminomethane TSE Tris/Saline/EDTA UGT UDP-glucuronosyltransferase XAP2 Hepatitis B Virus X-associated protein 2 XRE Xenobiotic response element

Chapter 1 Introduction

The aryl hydrocarbon receptor (AHR) is a transcription factor traditionally known for regulating gene expression by directly binding to its cognate DNA motif, aryl hydrocarbon response element (AHRE). In contrast, recent evidence suggests a possibility for AHR to indirectly bind DNA via tethering to other transcription factors, or to directly bind DNA regions that do not contain an AHRE. This thesis performed Chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) on Ahrdbd/dbd mice expressing AHR with a mutated DNA-binding domain incapable of binding AHRE, in order to explore tethering and non-AHRE binding of AHR.

1.1 The AHR

The AHR is a ligand-activated transcription factor and a member of the basic Helix-Loop-

Helix – Period/ARNT/Single minded (bHLH-PAS) family (Larigot et al. 2018). In the classical or canonical mechanism of AHR action, ligand binding causes it to translocate into the nucleus where it associates with AHR nuclear translocator (ARNT) (Denison et al. 2011). The AHR-ARNT heterodimer then binds a DNA motif, the AHRE, and regulates the transcription of target genes

(Denison et al. 1988). These genes include many drug metabolizing enzymes that could metabolize the AHR ligands (Nebert et al. 1990).

1.1.1 History of Identification

In the late 1940s and early 1950s, James and Elizabeth Miller observed the metabolism of xenobiotics mediated by microsomal enzymes into reactive derivatives (Mueller and Miller 1948).

Shortly after, Allan Conney found that microsomal enzymes can be induced by benzo[a]pyrene

(B[a]P) (Conney et al. 1959). In the 1970s, it was observed that exposure of mice to B[a]P

1 upregulated aryl hydrocarbon hydroxylase (AHH) enzyme activity only in certain strains (Gielen et al. 1972). Crossing and backcrossing of different mouse strains enabled the identification of the

Ah (aryl hydrocarbon) locus, which controls the expression of cytochrome P450 (CYP) enzymes that have AHH activity (Gielen et al. 1972). The B[a]P-responsive allele found in the C57BL/6J mouse strain was named Ahb, while the nonresponsive allele found in the DBA/2J strain was named

Ahd. Nonresponsive DBA mice were later found to respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD), a more potent AHH inducer (Poland et al. 1974). This suggested that the Ah locus encodes a receptor protein and that the reduced response of DBA mice is due to a mutation in this receptor that reduces its affinity for ligands (Poland et al. 1974). A landmark study by Alan Poland demonstrated that a receptor binding TCDD found in the hepatic cytosol is responsible for inducing hepatic AHH activity (Poland et al. 1976).

A photoaffinity ligand that covalently binds to AHR under ultraviolet light, 2-azido-3-iodo-

7,8-dibromodibenzo-p-dioxin, aided in its further characterization (Poland et al. 1986). Four polymorphic alleles of the Ahr gene, Ahrb1, Ahrb2, Ahrb3, and Ahrd, were eventually identified

(Poland et al. 1987). The purification and N-terminal sequencing of the AHR protein led to the eventual cloning of the Ahr gene (Perdew and Poland 1988; Bradfield et al. 1991), and allowed for the identification of distinct structural and functional domains of AHR. The Ahr cDNA was cloned in 1992 (Burbach et al. 1992; Ema et al. 1992), and the Ahr gene was cloned shortly after (Schmidt et al. 1993; Mimura et al. 1994).

1.1.2 Functional Domains of AHR

As a member of the bHLH-PAS family, the three major functional regions of AHR are the bHLH domain, the PAS domain, and the C-terminal domain (Figure 1.1). The bHLH domain is a

2 structural motif commonly found in dimerizing transcription factors (Jones 2004). It is located at the N-terminal end of AHR. As its name indicates, it consists of a region with basic residues followed by two α-helices that are interconnected by a short loop. The basic amino acid residues are responsible for DNA binding (Fukunaga et al. 1995; Jones 2004). The HLH region participates in protein-protein interactions, including the heterodimerization with ARNT and the association with the 90-kDa heat shock protein (HSP90) chaperone (Whitelaw et al. 1995; Jones 2004). The nuclear localization signal (NLS) and nuclear export signal (NES) are also found within the N- terminus (Eguchi et al. 1997; Ikuta et al. 1998).

The PAS domain was named after the three proteins in which it was first discovered – period circadian protein, ARNT, and single-minded protein (Huang et al. 1993). PAS contains a five-stranded antiparallel β-sheet and several α-helices, and it typically functions as a molecular velcro, allowing the binding of small molecules and other proteins (Möglich et al. 2009). In AHR, the two structural repeats, PAS-A and PAS-B, are adjacent to the bHLH (Fukunaga et al. 1995).

Both PAS-A and PAS-B participate in the heterodimerization of AHR with ARNT, while only

PAS-B comprises the ligand binding pocket (Whitelaw et al. 1995; Fukunaga et al. 1995).

The C-terminal domain of AHR is where coactivators and corepressors interact, and it is therefore also called the transactivation domain (Jain et al. 1994; Ko et al. 1997). It is further divided into three subdomains: an acidic region enriched with glutamate and aspartate, a Q-rich region enriched with glutamine, and a P/S/T region enriched with serine, threonine, and proline

(Ridolfi et al. 2014; Larigot et al. 2018). Because each subdomain is able to recruit different co- regulators, this structural complexity allows for flexibility of transactivation.

ARNT, the heterodimerization partner of AHR, shares structural similarities with AHR

(Figure 1.1). The bHLH and PAS-A domains mediate dimerization with AHR, and the C-terminal

3 domain allows for interactions with coactivators and corepressors (Larigot et al. 2018). However, in contrast to AHR, the PAS-B region of ARNT is not able to bind ligands and thus remains constitutively active (Andersson et al. 2002).

Figure 1.1 Domain architecture of AHR and ARNT. AHR is composed of the bHLH domain, the PAS domain, and the transactivation domain. The basic region allows DNA binding; the HLH, PAS-A, and PAS-B regions allows heterodimerization; the PAS-B region alone is responsible for ligand binding. ARNT is structurally similar to AHR, except that its PAS-B domain does not bind ligands (Schulte et al. 2017).

1.1.3 Canonical Signalling Pathway of AHR

In the latent state, AHR exists as a complex in the cytoplasm with chaperone proteins, including two HSP90, one 23-kDa co-chaperone (p23), and one hepatitis B Virus X-associated protein 2 (XAP2) (Perdew 1988; Denis et al. 1988; Nair et al. 1996; Ma and Whitlock 1997; Carver and Bradfield 1997). These chaperones maintain proper protein folding and ligand recognition capacity, maintaining the transcriptional function of AHR in a latent state. The association with

HSP90 is important for increasing the ligand binding capacity of AHR (Carver et al. 1994;

Whitelaw et al. 1995). Meanwhile, HSP90 attaches to AHR at the HLH and PAS domains, which are exactly the ARNT binding region; in other words, it blocks AHR-ARNT heterodimerization in the absence of ligands (Antonsson et al. 1995; Fukunaga et al. 1995; Perdew and Bradfield

1996). Similarly, p23 also prevents unliganded AHR from dimerizing with ARNT (Kazlauskas et al. 1999); its expression was found to increase the DNA binding and transcriptional regulation of

AHR-ARNT (Cox and Miller 2002; Shetty et al. 2003). XAP2 enhances AHR transcriptional activity by increasing the cellular level of AHR (Meyer and Perdew 1999; Meyer et al. 2000).

Due to their lipophilicity, AHR ligands enter cells by passive diffusion through the plasma membrane (Denison et al. 2011). Upon ligand binding to the pocket at the PAS-B site, AHR changes its conformation and exposes its NLS at the N-terminus, allowing the AHR complex to translocate into the nucleus (Denison et al. 2011). In the nucleus, as AHR dimerizes with ARNT,

HSP90 and other chaperons dissociate (Soshilov and Denison 2008; Denison et al. 2011). The

AHR-ARNT complex then recognizes and binds to DNA motifs called AHREs that are located in regulatory regions of AHR target genes. AHREs, also known as dioxin response elements (DRE) and xenobiotic response elements (XRE), have been determined to contain 5’-TnGCGTG-3’, with the minimal core sequence of 5’-GCGTG-3’ (Denison et al. 1988). The DNA-bound AHR-ARNT complex then recruits a number of coactivators, including p300, nuclear receptor coactivator 1

(NCOA1), and nuclear receptor-interacting protein 1 (NRIP1) (Kobayashi et al. 1997; Kumar and

Perdew 1999; Kumar et al. 1999). These are responsible for regulating transcriptional machinery and altering chromatin structure (Carlson and Perdew 2002).

Figure 1.2 AHR canonical signalling pathway. Unliganded AHR is sequestered in the cytoplasm by chaperone proteins. AHR ligands enter the cytoplasm via simple diffusion. Ligand binding changes AHR conformation and exposes its NLS for nuclear translocation. In the nucleus, ARNT displaces the chaperones, forming AHR-ARNT heterodimer. By recognizing AHREs on DNA, the heterodimer regulates transcription with the aid of coactivators and corepressors.

The most well-known AHR induced genes encode drug metabolizing enzymes of the oxidation (also known as Phase I) and conjugation (Phase II) classes, respectively (Nebert et al.

1990). These include the cytochrome P450 isoforms CYP1A1, CYP1A2 and CYP1B1, aldehyde dehydrogenase 3A1 (ALDH3A1), glutathione S-transferase A1 (GSTA1), NAD(P)H quinone oxidoreductase 1 (NQO1), and UDP-glucuronosyltransferase 1A6 (UGT1A6) (Nebert et al. 2004;

Aleksunes and Klaassen 2012; Becker et al. 2016). These enzymes can exert an adaptive role by metabolizing the xenobiotics that activated AHR (Becker et al. 2016). However, the metabolites formed can also be reactive species with electrophilic groups that can covalently bind to DNA and protein molecules, leading to cytotoxicity and mutagenesis (Becker et al. 2016).

Due to the presence of NES at its N-terminal end, AHR in the nucleus is constantly exported to the cytoplasm, where it is ubiquitinated and then degraded by the proteasome (Pollenz

2002). Some AHR induced genes are negative regulators of AHR, forming a negative feedback loop to control its activity. AHR repressor (AHRR) structurally resembles AHR except for the absence of the PAS-B domain; therefore, AHRR is unable to bind ligands, but is able to associate with ARNT (MacPherson et al. 2014). It was originally thought AHRR reduces the availability of active AHR-ARNT complex by sequestering ARNT (Mimura et al. 1999). However, one report showed that increasing ARNT expression did not rescue AHR function, and proposed that AHRR action is through transrepression, or protein-protein interaction with promoter-bound AHR-ARNT

(Evans et al. 2007). Similarly, TCDD-inducible poly-ADP-ribose polymerase (TIPARP) is an

AHR-induced mono-ADP-ribosylase that promotes AHR proteolytic degradation and represses

AHR transactivation (MacPherson et al. 2014).

1.1.4 Non-canonical Signalling Pathway

Aside from the well-established canonical signalling pathway through direct DNA-binding, research in recent years has provided evidence that AHR can interact with other proteins to alter transcription in a fashion that is independent of DNA binding (Beischlag et al. 2008). This topic is a central focus of the present thesis, and will be discussed in detail in Section 1.4.

1.2 Role of AHR in Toxicology

As a promiscuous receptor, AHR binds to different classes of molecules. It appears to mediate several important physiological functions when activated by naturally occurring compounds, but causes serious toxicological responses when activated by certain synthetic

7 compounds. Halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons

(PAHs) are the two major groups of anthropogenic molecules implicated in AHR-mediated toxicology (Denison et al. 2011). Many of these compounds, especially TCDD, are environmental pollutants of public health concern (Lindén et al. 2010; Sorg 2014). This section will introduce these synthetic compounds, and then focus on the prototypical toxicant ligand TCDD to describe

AHR-mediated toxicity.

1.2.1 Synthetic Compounds

The first discovered and the best described AHR ligands are anthropogenic molecules that exist as environmental pollutants. They are considered as the classic high affinity ligands of AHR

(Denison et al. 2011). In fact, the discovery and early characterization of AHR resulted from attempts to explain the toxicology of these molecules (Gielen et al. 1972; Poland et al. 1974). They all exhibit aromatic, planar, and hydrophobic structures, giving them high stability to accumulate and persist in organisms and in the environment. They are subdivided into HAHs and PAHs depending on whether halogen atoms, usually chlorine, are attached to the molecule (Denison et al. 2011).

Examples of HAHs include polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) (Sorg 2014). TCDD, the most potent and toxic AHR agonist known, is one of the PCDDs (Lindén et al. 2010). TCDD is commonly referred to simply as dioxin, while other HAHs are loosely referred to as dioxin-like compounds (Lindén et al. 2010). A toxicity equivalency factor (TEF) is used to compare the potency of all PCDDs, PCDFs, and PCBs (Sorg 2014), where TCDD is given a value of 1 and all other compounds are lower than 1 (Van den Berg et al. 2006). HAHs are typically by-products of

8 organic synthesis and fuel combustion in industrial processes and waste incineration (Safe 1990).

PCBs were widely used as dielectric and coolant fluids in electrical apparatus, such as transformers, capacitors, and motors (Safe 1990). As persistent organic pollutants, HAHs remain a major concern of public health and environmental toxicity (Lindén et al. 2010).

In contrast, PAHs are structures of multiple rings containing only carbon and hydrogen

(Abdel-Shafy and Mansour 2016). B[a]P and 3-methylcholanthrene (3-MC) are two classic examples (Denison et al. 2011). They are typically produced by thermal decomposition of organic matter (Abdel-Shafy and Mansour 2016). Cigarette smoke, charbroiled meat, and exhaust emission are all sources of exposure (Abdel-Shafy and Mansour 2016). The AHR binding affinity of PAHs is in the nM to µM range, lower than the pM to nM affinity of HAHs (Denison et al. 2011). The potency of PAHs can be dependent on the duration of exposure (Riddick et al. 1994). For example,

3-MC is 10 times less potent than TCDD after 4 hrs of treatment, but it becomes 1000 times lower after 14 hours (Riddick et al. 1994). This time dependency is at least partially attributed to the fact that PAHs are more readily metabolized than the stable TCDD (Riddick et al. 1994).

Halogenated aromatic hydrocarbons (HAHs)

Polychlorinated dibenzodioxins Polychlorinated dibenzofurans Polychlorinated biphenyls (PCDDs) (PCDFs) (PCBs)

Polycyclic aromatic hydrocarbons (PAHs)

Benzo[a]pyrene 3-methylcholanthrene (B[a]P) (3-MC)

Figure 1.3 Synthetic ligands of AHR – HAHs and PAHs. HAHs include PCDDs, PCDFs, and PCBs. Each R-group can be a chlorine atom or a hydrogen atom. TCDD is one of the PCDDs, with R2, R3, R6, and R7 being chlorine atoms, and R1, R4, R5, and R8 being hydrogen atoms. The most well-known examples of PAHs are B[a]P and 3-MC.

1.2.2 History of TCDD

Much of our knowledge of AHR mediated toxicity came from historical instances of TCDD exposure. Sandermann first reported the synthesis of TCDD in 1957 (Sandermann et al. 1957).

Unfortunately, an assistant in the laboratory developed chloracne, an acne-like skin condition, after accidental exposure to TCDD (Beischlag et al. 2008). Chloracne is now known as the most sensitive marker of possible toxicity to dioxin-like compounds in humans (Caputo et al. 1988;

Saurat et al. 2012). During the Vietnam War in the 1960s, the US Army used Agent Orange to defoliate forests, and TCDD was released as a trace by-product contained in this herbicide (Steele et al. 1990). This exposure has been associated with skin conditions, teratogenic effects, and

10 cancers among Vietnamese civilians and American veterans (No authors listed 1987; Ngo et al.

2006). In 1976, up to 1 kilogram of TCDD and dioxin-like compounds were dispersed into the environment following an industrial accident in Seveso, Italy, causing serious health issues in the local population and killing thousands of animals (Caputo et al. 1988; Ngo et al. 2006). In 2004,

Ukrainian President Viktor Yushchenko was poisoned with a large quantity of pure TCDD. Close monitoring of his case contributed to our knowledge about TCDD toxicity in humans (Sorg et al.

2009; Saurat et al. 2012).

1.2.3 AHR Mediated Toxicity

TCDD toxicity is primarily mediated through AHR, as Ahr-deficient (Ahr-/-) mice are resistant (Fernandez-Salguero et al. 1996). Accordingly, AHR-mediated toxicology has been largely revealed by studies using TCDD. This section will therefore describe studies of AHR mediated toxicity mostly using TCDD. Among the many aspects of TCDD toxicity that have been observed, the major ones include a generalized wasting syndrome, skin disorders, carcinogenesis, developmental abnormalities, and immunosuppression (Bock and Köhle 2006; Lindén et al. 2010;

Sorg 2014).

Laboratory animals treated even with high doses of TCDD do not die immediately. Instead, they undergo dramatic weight loss, which can be over 50% of their body weight, and eventually die after several days or a few weeks (Seefeld et al. 1984; Pohjanvirta and Tuomisto 1994). This wasting syndrome is an uncommon phenomenon associated with chemical toxicity (Lindén et al.

2010). Exposed animals do not appear to have either malabsorption or increased metabolism, and this weight loss has been attributed primarily to reduced food intake, or hypophagia (Seefeld and

Peterson 1984; Kelling et al. 1985; Potter et al. 1986). Energy storage depletion caused by

11 hypophagia has been found to cause their eventual death. Compared with TCDD-treated rats, pair- fed controls had an essentially identical weight loss time course and survival curve (Christian et al. 1986). However, other mechanisms leading to death must exist, since force-feeding prevented the weight loss but did not postpone the death of rats treated with TCDD (Tuomisto et al. 1999).

One hypothesis to explain the wasting syndrome is that TCDD lowers the body weight set-point in animals; hypophagia is simply a secondary effect of TCDD due to this lowered set-point (Lindén et al. 2010). The biochemical basis for the wasting syndrome is still not well understood.

The clinical hallmark and the most sensitive marker of TCDD exposure in humans is chloracne (Caputo et al. 1988; Saurat et al. 2012). This disorder manifests as comedones that become deep cysts on the face. With increased toxicity, it may spread to the posterior neck, trunk, and extremities. One mechanistic theory suggests that AHR is important in the differentiation of keratinocytes, so that excess AHR activation causes chloracne (Bock and Köhle 2006). For example, the keratin-binding protein filaggrin is a target of AHR regulation. Viktor Yushchenko, whose TCDD level in blood was 50,000 times greater than the general population, had dermal hamartomas with disappearance of sebaceous glands (Sorg et al. 2009). One hypothesis is that

TCDD turns off the cutaneous stem cells that normally generate new sebocytes (Sorg 2014).

As mentioned in Section 1.1.3, AHR upregulates enzymes that can metabolize the ligands that activated it. This ideally serves as a compensatory feedback mechanism to decrease the levels of these ligands and thus prevent excessive or prolonged activation of AHR. However, these drug metabolizing enzymes can also turn many PAHs into reactive metabolites that can bind covalently to DNA, causing mutations and leading to cancer initiation or progression. In addition, by regulating genes involved in cancer, activated AHR can also act as a non-genotoxic promoter of cancer by selecting and expanding preneoplastic cells (Bock and Köhle 2006). For example, it has

12 been observed that TCDD causes cell cycle arrest and apoptosis in normal hepatocytes, but inhibits apoptosis and enhances cell division in preneoplastic hepatocytes (Bock and Köhle 2006). This protection may take place through suppressing cell-cell contact inhibition, oxidative stress, and p53-mediated apoptosis (Bock and Köhle 2006). Based on mechanistic considerations, the World

Health Organization has classified TCDD as a Group 1 established human carcinogen (Baan et al.

2009; Boffetta et al. 2011). Surprisingly, however, the carcinogenicity of TCDD in humans is still a matter of debate (Sorg 2014). One major reason is that sensitivity to the toxic effects of TCDD varies hugely between species and even between strains within a single species, so that animal studies may be limited in predicting human response (Peterson et al. 1993; Sorg 2014). Also, because the half-life of TCDD in humans is around 7 years due to both its accumulation in fat stores and its very limited metabolism, the formation rate of reactive metabolites such as epoxides from TCDD may have limited biological significance (Sorg 2014).

TCDD affects reproduction and in utero development (Peterson et al. 1993). Retrospective studies following the Seveso accident found that the semen quality of young men was decreased, and more female than male children were born to couples after TCDD exposure (Mocarelli et al.

2000, 2008). TCDD acts a teratogen, producing various birth defects such as cleft palate in laboratory animals (Abbott et al. 1992). The effect of TCDD seems to depend on the developmental period during which the fetus is exposed (Matthews and Ahmed 2013). Various pieces of evidence support the role of TCDD as an endocrine disruptor, affecting estrogen, testosterone, and thyroid hormone signalling that are all important to normal development (Safe

1998; Beischlag et al. 2008). AHR has tightly regulated crosstalk with estrogen receptor (ER)

(Beischlag et al. 2008; Matthews and Ahmed 2013). TCDD treatment caused fertilized oocyte loss

13 in mice and inhibition of follicular development in zebrafish (Kitajima et al. 2004; King Heiden et al. 2006).

TCDD also disrupts the immune system. The size of thymus is greatly decreased in rodents treated with TCDD (Poland and Knutson 1982). Similarly, mice expressing constitutively active

AHR had fewer thymocytes (Nohara et al. 2005). Both B-cell and T-cell mediated immunities are impaired, leading to susceptibility to bacterial and viral infections (Matthews and Ahmed 2013).

1.3 Role of AHR in Physiology

The physiological role of AHR has been traditionally thought to be (1) metabolizing or detoxifying xenobiotics, and (2) regulating normal development (Nebert et al. 1990; Schmidt et al.

1996). More recent research has now found many additional regulatory functions of AHR, including roles in cell proliferation and in the immune system (Matthews and Ahmed 2013). AHR mediates its normal physiological functions via activation by natural ligands (Denison et al. 2011).

1.3.1 Natural AHR Ligands

After the characterization of AHR activation by synthetic ligands, researchers looked for natural ligands that may be involved in the physiological functions of AHR (Nguyen and Bradfield

2008). The promiscuity of AHR allows structurally diverse molecules to be ligands (Denison et al.

2011). In contrast to synthetic ligands such as HAHs and PAHs, which are strong agonists, natural ligands are usually weak agonists or even antagonists (Denison et al. 2011). Natural ligands of

AHR are often categorized as either exogenous or endogenous.

Ingestion of vegetables is the major source of exogenous natural ligands of AHR (Jeuken et al. 2003). Indole-3-carbinol (I3C) and flavonoids are two examples. I3C is well-known to be

14 found in cruciferous vegetables, such as broccoli and cauliflower (Bjeldanes et al. 1991). Dietary

I3C supplement has been found to supress various cancers and inflammatory diseases, such as colitis and multiple sclerosis (Huang et al. 2013; Rouse et al. 2013). Under acidic conditions in the gastrointestinal tract, I3C is condensed into various products, including indolo[3,2-b]carbazole

(ICZ) and 3,3'-diindolylmethane (DIM), which are also typical AHR ligands (Matthews and

Ahmed 2013). The AHR affinity of ICZ is one of the highest found among all natural ligands, about 100,000 times stronger than I3C, and only about 30 times weaker than TCDD (Bjeldanes et al. 1991). Interestingly, ICZ does not produce TCDD-like toxic responses, despite their comparable affinities (Pohjanvirta et al. 2002). As another example, a number of flavonoids comprise the largest group of exogenous natural AHR ligands (Ashida et al. 2008). The majority of molecules in this group are AHR antagonists, while some are agonists (Denison et al. 2011).

Humans may ingest around 1 g of flavonoids per day from fruits and vegetables (Scalbert and

Williamson 2000). The anti-cancer roles of flavonoids have been investigated (Moon et al. 2006).

The generation of Ahr-/- mice has allowed researchers to observe physiological functions of AHR in the absence of exogenous ligands, including both synthetic and dietary ones (Schmidt et al. 1996). The fact that Ahr-/- mice suffer from a wide range of phenotypic abnormalities supports the existence and importance of endogenous AHR ligands (Schmidt et al. 1996). Tryptophan derivatives, heme-related tetrapyroles (e.g. bilirubin), and arachidonic acid metabolites (e.g. lipoxin A4) have been identified as endogenous ligands (Denison et al. 2011). The division between “exogenous” and “endogenous” may sometimes be ambiguous, as in the case of tryptophan derivatives. Tryptophan is an essential amino acid that must be obtained from the diet.

After ingestion, it is metabolized by the intestinal microbiota and the host into various molecules that have AHR agonist activities, such as kynurenic acid (KA) (Hubbard et al. 2015). At

15 physiologically relevant levels, KA has been found to induce AHR target genes (DiNatale et al.

2010). In addition, 6-formylindolo[3,2-b]carbazole (FICZ) is formed from tryptophan upon exposure to ultraviolet and visible light (Rannug et al. 1987, 1995). As the endogenous ligand with the highest AHR affinity and potency to date, FICZ is thought to have many physiological effects, including the differentiation of immune cells (Jurado-Manzano et al. 2017).

Natural Exogenous Ligands

Indole-3-carbinol Flavone (I3C)

Endogenous Ligands

Kynurenic acid 6-formylindolo[3,2-b]carbazole (KA) (FICZ) Figure 1.4 Selected natural ligands of AHR. Examples of exogenous ligands include I3C and flavonoids. Flavone is the simplest flavonoid. A major group of endogenous ligands is tryptophan derivatives, which include KA and FICZ.

1.3.2 Role of AHR in Detoxification

As the first known and the best characterized physiological function of AHR, AHR upregulates the expression of many drug metabolizing enzymes upon ligand activation, and this serves as a protective mechanism to reduce the presence of ligands (Nebert et al. 1990). This negative regulation loop prevents toxic effects of exogenous and endogenous AHR ligands

(Becker et al. 2016). It is noteworthy that not all AHR ligands are readily metabolized by these upregulated drug metabolizing enzymes, and TCDD is a typical example that has a half-life of over 7 years in humans (Pirkle et al. 1989). Other synthetic compounds, such as 3-MC, can be more susceptible to biotransformation (Riddick et al. 1994). Highlighting the “double-edge sword” aspect of biotransformation, the metabolism of PAHs, such as B[a]P, often leads to their bioactivation to carcinogens (Conney 1982). This concept has been previously mentioned as one mechanism of AHR toxicity in Section 1.2.3. However, B[a]P-treated Cyp1a1+/+ mice survive, while Cyp1a1-/- mice die within 30 days (Uno 2004). Therefore, despite the potential to generate carcinogenic metabolites, this biotransformation process seems to be essential for survival after exposure to excess PAHs. In addition to xenobiotics, endogenous ligands also need this pathway to be regulated. For example, FICZ is a substrate of CYP enzymes, and this negative feedback loop ensures maintenance of physiological levels of FICZ and the normal function of AHR (Wei et al. 2000). Some endogenous signalling molecules, such as certain steroid hormones and neurotransmitters, are also regulated by these biotransformation enzymes (Bock and Köhle 2006).

1.3.3 Role of AHR in Development

AHR is an ancient gene found in all studied vertebrates, and its homologues are also found in many invertebrates (Bock and Köhle 2006). Interestingly, primitive forms of AHR are not able

17 to bind synthetic and natural ligands (Bock and Köhle 2006). Thus, the detoxification of xenobiotics is a role that seems to have been obtained late in evolution (Oesch-Bartlomowicz et al.

2005). The role of AHR in development has been observed in invertebrates. For example, it is involved in the development of neurons in Caenorhabditis elegans, and in the development of antenna in Drosophila melanogaster (Duncan et al. 1998; Huang 2004).

The critical role of AHR in development is revealed by studies using Ahr-/- mice (Schmidt et al. 1996). Ahr-/- mice have a high neonatal mortality (Fernandez-Salguero et al. 1995; Abbott et al. 1999), and surviving mice have decreased liver size, increased heart size, and increased spleen size (Fernandez-Salguero et al. 1995; Schmidt et al. 1996). The cause of the liver atrophy is the continued presence of ductous venosus (DV), which is the abnormal phenotype that has drawn the most attention (Walisser et al. 2004). DV is a shunt that directs blood flow from the umbilical vein to the inferior vena cava, allowing a portion of oxygenated blood to bypass the liver. DV normally closes several days after birth, shifting blood flow to the liver. Failure of DV closure leads to decreased blood supply to the liver, and thus decreased liver size (Walisser et al. 2004; Bock and

Köhle 2006). TCDD treatment was shown to restore DV closure in Ahr hypomorphic mice, confirming the requirement of AHR signalling in this process (Walisser et al. 2004). In addition to liver atrophy, Ahr-/- mice also exhibit portal fibrosis, which could be explained by elevated transforming growth factor β (TGF-β) (Zaher et al. 1998). Other vascular abnormalities outside of the liver are also present, such as the failure of hyaloid artery closure in the eyes after birth (Larigot et al. 2018). Interestingly, Ahr-/- rats do not exhibit liver atrophy or abnormalities in DV or hyaloid artery; instead, Ahr-/- rats have renal and ureter dilation, which are not observed in Ahr-/- mice

(Harrill et al. 2013).

1.3.4 Other Cellular Roles of AHR

Among many other functions of AHR that have received increased attention, one is cell cycle control. AHR acts as a promoter of cell proliferation in some experiments, but as an inhibitor in others. Embryonic fibroblasts from Ahr-/- mice show decreased growth and G2/M phase accumulation (Elizondo et al. 2000). AHR-directed siRNAs delay G1/S phase transition in HepG2 cells (Abdelrahim 2003). In contrast, many other studies have found that ligand-activated AHR causes G1 phase arrest (Marlowe and Puga 2005). AHR effects on cell proliferation are probably cell type dependent (Matthews and Ahmed 2013; Larigot et al. 2018). This may be related to the carcinogenenic consequences of AHR-mediated toxicity.

The role of AHR in immunity is demonstrated by the fact that TCDD suppresses B-cell and T-cell function, as previously mentioned in Section 1.2.3. In addition, AHR is involved in the differentiation of regulatory T (Treg) cells and T helper type 17 (Th17) cells from the common precursor, naive CD4+ T cells. Th17 cells are pro-inflammatory, causing autoimmunity and inflammation, while Treg cells are immunosuppressive, preventing immunity from overactivation

(Lee 2018). Th17/Treg cell balance is therefore critical in maintaining immune homeostasis.

Different studies have found that TCDD promotes Treg cell differentiation, whereas FICZ promotes

Th17 cell differentiation (Quintana et al. 2008; Veldhoen et al. 2008). This is likely relevant to the fact that Ahr-/- mice are more susceptible to colitis, a form of inflammatory bowel disease

(Arsenescu et al. 2011).

1.4 Evidence for AHR Transcriptional Regulation via Tethering

Besides its canonical role as a direct DNA-binding transcription factor, non-canonical functions of AHR are emerging as a new direction of research (Beischlag et al. 2008). Evidence

19 suggests that AHR may tether to other transcription factors and thereby indirectly regulate their target genes (Dere et al. 2011). This section will first introduce two non-canonical functions of

AHR, and then focus on the concept that AHR may mediate indirect gene regulation via tethering.

1.4.1 Non-canonical Functions of AHR

Recent studies have provided evidence that AHR can function independent of its classical role as a transcription factor (Matsumura 2009; Larigot et al. 2018). Specifically, ligand-activated

AHR participates in protein kinase networks. The proto-oncogene cellular Src kinase (c-Src) exists as a component of the quiescent AHR complex along with other chaperone proteins in the cytoplasm. Upon ligand binding, AHR releases c-Src, which then activates mitogen-activated protein kinases (MAPKs) (Park et al. 2005). MAPKs eventually upregulate the transcription of cyclooxygenase 2 (COX2), an enzyme that produces prostaglandin from arachidonic acid

(Matsumura 2009). Simultaneously, ligand-activated AHR leads to rapid influx of Ca2+ into the cytoplasm from both the extracellular space and the endoplasmic reticulum, activating protein kinase Cα (PKCα) (Dong and Matsumura 2008). PKCα phosphorylates phospholipase A2 (PLA2), and PLA2 then releases arachidonic acid from plasma membrane, providing an additional source of prostaglandin which leads to inflammation (Matsumura 2009).

AHR may also be activated by phosphorylation and act without ligand binding. This may be related to the fact (mentioned in Section 1.3.3) that AHR homologues in invertebrates do not bind to ligands, but have important functions in development (Bock and Köhle 2006). The protein kinase A (PKA) activated by cAMP was shown to phosphorylate and activate AHR (Oesch-

Bartlomowicz et al. 2005). The binding sites of AHR activated by TCDD versus cAMP were overlapping but different, and cAMP-activated AHR did not heterodimerize with ARNT (Oesch-

Bartlomowicz et al. 2005). These findings suggest that AHR activated by cAMP-mediated phosphorylation may interact with different partners for transcriptional regulation (Bock and

Köhle 2006). These are two examples showing that the current picture of AHR function is probably still incomplete, and AHR may have many as yet unknown interactions with other proteins.

1.4.2 Transcriptional Regulation via Tethering

An emerging feature of many transcription factors is that they are able to regulate gene expression by tethering to other transcription factors (Beischlag et al. 2008). Historically, this tethering mechanism was first revealed by studies on the glucocorticoid receptor (GR). Mice with

GR defective in DNA-binding and dimerization, but not GR-null mice, are able to survive

(Reichardt et al. 1998). This indicates that repression and coactivation capacities may be equally as important as the direct DNA-binding of transcription factors. GR was found to repress target genes of activator protein 1 (AP-1) and nuclear factor kappa B (NFκB) through protein-protein interaction, and this phenomenon was called “transrepression” (Jonat et al. 1990; Ray and

Prefontaine 1994). Thyroid hormone receptor (TR) and estrogen receptor (ER) were also later found to transrepress AP-1 and NFκB target genes (Lee et al. 2000; Jakacka et al. 2001). Whether

AHR is able to coactivate or transrepress target genes of other transcription factors via tethering to them is still in the early exploratory phase. This is certainly one important direction to complete the full picture of AHR function.

1.4.3 AHR and ER

In the field of AHR crosstalk with other transcription factors, crosstalk with ER has been the most well studied. The anti-estrogenic effect of AHR has been well-known for a long time, as

21 reports in the 1980s revealed that TCDD decreases estrogen function in the uterus and liver of rats

(Astroff and Safe 1988). Subsequent studies showed that AHR ligands reduce the estrogen-induced upregulation of numerous ER-targeted genes at both the mRNA and protein levels (Safe and

Wormke 2003). Several different mechanisms for AHR-mediated ER inhibition have been proposed and supported (Matthews and Ahmed 2013). First, AHR increases the metabolism of ER ligands by upregulating biotransformation enzymes (Spink et al. 1994). Second, AHR induces other inhibitory proteins of ER, such as proteasomes (Wormke et al. 2003). Third, ER-responsive genes can simply have AHRE in their regulatory region to directly recruit AHR (Krishnan et al.

1995). Fourth, AHR competes with ER for common co-activators (Matthews and Ahmed 2013).

On the other hand, different studies found that transfected ER enhances or represses the transcriptional regulation of AHR (Matthews et al. 2005; Beischlag and Perdew 2005). Glutathione

S-transferase (GST) pull-down assays revealed that ERα associates directly with AHR as well as with ARNT, and chromatin immunoprecipitation experiments found that ERα and AHR appear at the enhancer of CYP1A1 at the same time (Matthews et al. 2005; Beischlag and Perdew 2005).

These results support the concept that ER transrepresses AHR via tethering. ER-ARNT tethering may be more important than ER-AHR tethering in this transrepression. Ligand-activated ER was still able to cause transrepression when the transactivation domain of AHR for ER binding was mutated (Beischlag and Perdew 2005). ER also repressed the transcriptional regulation of

Gal4/ARNT chimera (Beischlag and Perdew 2005).

Similarly, coming back to AHR-mediated ER inhibition, a relatively recent hypothesis is that AHR tethers to ER, resulting in the transrepression of ER-responsive genes (Ohtake et al.

2003). Subdomains of the AHR transactivation domain expressed from transfected constructs inhibited estrogen response element (ERE)-driven luciferase reporter assay signals (Reen et al.

2002). Direct interactions between AHR and ER as well as between ARNT and ER have been verified by coimmunoprecipitation and GST pull-down experiments (Ohtake et al. 2003;

Brunnberg et al. 2003). However, despite these pieces of evidence, more research needs to be done to fully understand the mechanisms and complexities of AHR and ER crosstalk.

1.4.4 AHR and Immune Regulators (NFκB and AP-1)

NFκB is a key transcription factor that regulates inflammatory responses. There are five members in the NFκB family: RelA, RelB, c-Rel, p50, and p52 (Karin et al. 2004). The canonical

NFκB pathway is mediated by the heterodimer formed by RelA and p50 (Lawrence 2009). Upon infection, bacterial products, such as lipopolysaccharide (LPS), bind to cell surface receptors and trigger a signalling cascade that results in the release of RelA/p50 heterodimer and its movement from the cytoplasm into the nucleus (Lawrence 2009). RelA/p50 heterodimer then binds to its cognate response element, recruits coactivators, and upregulates the expression of anti-bacterial genes, especially cytokines (Lawrence 2009).

AP-1 is another transcription factor family mediating inflammatory responses (Karin et al.

1997). Members of the AP-1 family recognize core elements as either homodimers or heterodimers

(Karin et al. 1997). The promoters of many inflammatory genes contain AP-1 binding sites in addition to NFκB binding sites.

Transcriptional activation of NFκB and AP-1 has been shown to be impaired by the tethering of other transcription factors. This often leads to transrepression, which is thought to be a mechanism to attenuate inflammation during chronic and acute challenges. For example, GR,

ER, and TR are all able to transpress both NFκB and AP-1 (Jonat et al. 1990; Ray and Prefontaine

1994; Lee et al. 2000; Jakacka et al. 2001).

AHR activity has been shown to decrease NFκB-mediated cytokine production in response to inflammatory stimuli. For example, in bone marrow stromal cells AHR agonists decrease the

DNA binding of NFκB and suppress LPS-induced interleukin 6 (IL-6) mRNA expression (Dalvie et al. 2003). Another study identified the direct physical interaction between AHR and RelA using coimmunoprecipitation, and used electrophoretic mobility shift assays (EMSA) and luciferase reporter assays to show that AHR decreased RelA/p50 DNA-binding and transcriptional activity; conversely, RelA/p50 also repressed AHR transcriptional activity (Tian et al. 1999). Interestingly,

AHR and RelB/p52 were found to enhance each other’s transcriptional activities, and a direct physical interaction between AHR and RelB was also observed (Vogel et al. 2007). The conflicting results for whether the association of AHR and NFκB leads to inhibition or activation is thought to be due to differences in cell types, culture conditions, and culture serum batches (Vogel et al.

2007). Since NFκB is a major candidate partner for AHR to physically interact with, inflammation is a field in which the non-canonical tethering activity of AHR is suspected to have a key influence.

Similarly, AP-1 is another candidate for AHR tethering. TCDD reduced the DNA-binding and transcriptional activity of AP-1 in the presence of LPS (Suh et al. 2002). Although a direct physical association between AHR and AP-1 has not been identified, AP-1 can tether to ER, and hence may at least indirectly associate with AHR (Beischlag and Perdew 2005; Beischlag et al.

2008).

1.4.5 AHR and Other Candidate Partners

In contrast to traditional methods for studying associations between AHR and other transcription on a gene-by-gene basis, the Matthews laboratory performed ChIP followed by DNA microarray (ChIP-chip) as well as ChIP-seq to explore this issue on a genome-wide scale. ChIP-

24 chip analysis of mouse liver collected 2 hours and 24 hours after TCDD exposure revealed that

56% (at 2 hours) and 48% (at 24 hours) of the AHR binding sites did not contain any AHRE core motif (Dere et al. 2011). When these binding sites were mapped to genes, 37% (at 2 hours) and

55% (at 24 hours) of AHR-targeted genes did not contain an AHRE (Dere et al. 2011). In addition to the expected AHRE, AHR-enriched regions exhibited an over-representation of the binding sites of many other transcription factors, including specificity protein 1 (SP1), hypoxia inducible factor

(HIF), E2 factor (E2F), nuclear respiratory factor 1 (NRF1), nuclear receptor subfamily 2 (NR2F), nuclear factor 1 (NF1), early growth response (EGR), peroxisome proliferator-activated receptor

(PPAR), and retinoid X receptor (RXR) (Dere et al. 2011). Similar results were found from ChIP- seq experiments using MCF-7 cells treated with TCDD. About 50% (1,283/2,594) of AHR- enriched regions did not contain an AHRE (Lo and Matthews 2012). Aside from AHR, the most over-represented binding motifs were those of activator protein 2 (AP-2), AP-1, EGR, SP1, ER, and HIF (Lo and Matthews 2012). These results strongly support the notion that tethering to other transcription factors to regulate their targeted genes is an important mode of AHR function in addition to direct binding to AHREs, and suggest that the abovementioned transcription factors are all direct or indirect partners for AHR tethering.

As stated in Sections 1.4.3 and 1.4.4, ER and AP-1 have been proposed to be AHR tethering targets. Like AHR, HIF is another member of the bHLH/PAS transcription factor family, and it also dimerizes with ARNT in order to function (Nie et al. 2001). Both AP-2 and EGR are known to physically interact with a number of transcription factors and other proteins, and one study found that AHR, AP-2, and EGR-1 formed a single complex under conditions of high glucose (Tremblay and Drouin 1999; Eckert et al. 2005; Dabir et al. 2008). AHR and E2F1 were found to coimmunoprecipitate, and this AHR binding inhibited E2F1-induced apoptosis (Marlowe et al.

2008). PPAR, which functions as a heterodimer with RXR, is also known to physically interact with many other transcription factors. PPARα was found to negatively interfere with the DNA binding of both NFκB and AP-1, and PPARβ/δ was found to coimmunoprecipitate with NFκB

(Delerive et al. 1999; Planavila et al. 2005). ERα and ERβ are both partners that PPARγ physically interacts with (Bonofiglio 2005; Xiang et al. 2010). Thus, in addition to possible direct tethering to AHR, PPAR may also tether to AHR indirectly through NFκB and/or AP-1. SP1 has been known to physically bind AHR, ER, and NFκB, modulating their transcriptional activities (Perkins et al. 1993; Kobayashi et al. 1996; Porter et al. 1997). NR2F is able to form heterodimers with

RXR, but it is better known as a repressor for many other transcription factors, such as PPAR and

ER (Giguere 1999). Formation of inactive complexes has been proposed as one possible mechanism (Giguere 1999). Direct association between AHR and NR2F has been reported (Klinge et al. 2000).

1.5 Ahrdbd/dbd Mice

As an approach to study the non-canonical role of AHR independent of its binding to the

AHRE, the group of Dr. Christopher Bradfield generated Ahrdbd/dbd mice, which express an AHR protein with mutated DNA-binding domain (Bunger et al. 2008). Since these mice demonstrated developmental defects and TCDD resistance like Ahr-/- mice, these authors proposed that AHRE binding is required for the developmental and toxicological functions of AHR (Bunger et al. 2008).

The Ahrdbd/dbd mouse line was subsequently used by other groups to show that AHRE binding of

AHR is necessary for mediating TCDD-induced mammary gland developmental defects during pregnancy, neutrophil recruitment and inducible nitric oxide synthase expression in response to infection, and regulation of the expression of miR196a in apoptosis (Lew et al. 2011; Wheeler et

26 al. 2013; Hecht et al. 2014). On other hand, one report showed that AHR destabilization of cyclooxygenase-2 (COX-2) mRNA and suppression of its protein expression is independent of binding to the AHRE (Zago et al. 2013).

1.5.1 Generation and Characterization of Ahrdbd/dbd Mice

Information in this section was extracted from the original paper describing the generation of Ahrdbd/dbd mice (Bunger et al. 2008). Ahrdbd/dbd mice were generated by introducing an I25G mutation and a GGATTC insertion into exon 2 of the Ahr gene isolated from 129/SvJ mice via megaprimer-based site-directed PCR mutagenesis. The final construct was introduced into GS1 embryonic stem cells by electroporation. After screening, correct clones were injected into

C57BL/6J blastocysts. The offspring of the chimeras and normal C57BL/6J mice were genotyped for the GGATTC insertion, and then bred with CMV-Cre/tg mice to remove the neomycin cassette.

The resulting mice were then backcrossed to C57BL/6J mice for three generations.

The six-nucleotide GGATTC insertion in exon 2, which corresponds to the bHLH DNA- binding region, places a glycine-serine between arginine-39 (R39) and aspartate-40 (D40). R39 is the last residue of the basic domain, whereas D40 is the first residue of the bHLH domain. Two- hybrid assays demonstrated that AHRdbd and ARNT still interact, while EMSA and luciferase reporter assays showed that the heterodimer of AHR and ARNT binds AHRE and regulates gene expression in the presence of the AHR ligand BNF, but that of AHRdbd and ARNT does not.

Coimmunoprecipitation experiments showed that AHRdbd also retains its association with the chaperone proteins HSP90 and XAP-2. In contrast to the wildtype, the ethoxyresorufin-O- deethylase assay demonstrated that microsomes isolated from Ahrdbd/dbd mice had extremely low

CYP1A1 activity both in the absence and presence of TCDD. However, the DNA-binding domain mutations caused AHRdbd protein to be constitutively localized in the nuclear compartment.

Ahrdbd/dbd mice exhibit the same developmental defects as Ahr-/- mice. Compared to wildtype littermates, Ahrdbd/dbd mice have smaller livers, larger hearts and larger spleens. They have patent DV throughout life, just as Ahr-/- mice. Ahrdbd/dbd and Ahr-/- mice also have the same resistance to TCDD. The weights of liver and thymus of Ahrdbd/dbd mice did not change with 6-day treatment of 100 µg/kg TCDD, whereas Ahrwt/wt mice had a 23% increase in liver and 59% decrease in thymus. TCDD-treated Ahrdbd/dbd and Ahr-/- mice did not have lipid accumulation in their liver, in contrast to Ahrwt/wt mice. Ahrdbd/dbd embryos were also entirely resistant to TCDD-induced cleft palate and hydronephrosis.

1.5.2 Ahrb1 and Ahrd alleles

As mentioned above, the Ahrdbd mutation was made on the Ahr allele from 129/SvJ mice, but the genetic background of Ahrdbd/dbd mice is C57BL/6J. Historically, gene targeting was performed using embryonic stem cells from 129 mouse strains, which are the most permissive to manipulation (Nagy and Vintersten 2006). Thus, the 129/SvJ Ahr allele was required to allow the homologous recombination with the gene in the GS1 embryonic stem cell from the 129/SvJ strain.

Since 129 stains do not breed well and have abnormal anatomy and behavior, it has been a common practice to backcross them to C57BL/6J mice after the mutation is made (Rivera and Tessarollo

2008). This was how the Ahrdbd/dbd line was made. However, C57BL/6J mice and 129/SvJ mice possess different versions of Ahr, the Ahrb1 allele and Ahrd allele, respectively (Thomas et al. 2002).

The difference between the two alleles can be a complicating factor that needs to be taken into consideration for the Ahrdbd/dbd mouse line.

Existing mouse strains have any of four Ahr alleles: Ahrd, Ahrb1, Ahrb2, and Ahrb3 (Thomas et al. 2002). It was discovered in the 1970s that some strains express a more ligand-responsive version of AHR than that of other strains (Gielen et al. 1972; Poland et al. 1976). The more responsive allele was named Ahrb after the C57BL/6 strain, while the less responsive allele was named Ahrd for the DBA/2 strain (Poland and Glover 1990). Additional variants were later identified within Ahrb, giving rise to Ahrb1, Ahrb2, and Ahrb3 (Poland and Glover 1990). Generally speaking, AHRb1, AHRb2, and AHRb3 have 10-fold higher affinity for TCDD than AHRd (Poland et al. 1974; Okey et al. 1989). AHRd, AHRb1, AHRb2, and AHRb3 proteins are 104-kDa, 95-kDa,

104-kDa, and 105-kDa, respectively (Poland and Glover 1990; Thomas et al. 2002). Compared with AHRd, AHRb1 has a shorter length and five amino acid substitutions (Thomas et al. 2002).

The V375A substitution within the ligand binding domain is solely responsible for the enhanced ligand affinity of AHRb1 (Poland et al. 1994; Thomas et al. 2002). A nonsense mutation resulting in a premature stop codon is responsible for the shortened length of AHRb1 (Thomas et al. 2002).

The C57BL/6 strain possessing the Ahrb1 has been used predominantly in mice studies investigating AHR.

1.6 Rationale and Objective

AHR mediates the toxicities of TCDD and many other environmental pollutants, and is responsible for various lethal and sublethal conditions, including skin disorders, carcinogenesis, developmental abnormalities, and immunosuppression in humans, rats and mice (Bock and Köhle

2006). AHR also plays important roles in many physiological processes, such as detoxification, development, cell cycle regulation, and immune responses (Matthews and Ahmed 2013). Its

29 canonical mode of action as a transcription factor that recognizes its cognate AHRE sequence on

DNA to regulate the expression of target genes has been well established (Denison et al. 2011).

However, we and others hypothesize that rather than by direct binding to AHRE, AHR signalling may also occur through other unrevealed mechanisms, namely indirect DNA binding via tethering to other transcription factors, or direct DNA binding to regions not containing any

AHRE. Supporting non-AHRE binding, ChIP-chip studies from the Matthews laboratory have shown that around 50% AHR-bound regions across mouse genome do not contain any AHRE

(Dere et al. 2011). In these AHR-bound regions without any AHRE, motifs of other transcription factors were found, suggesting a list of candidate partners for AHR tethering (Dere et al. 2011).

DNA-protein and protein-protein interaction studies suggest that the activities of several transcription factors, such as ER and NFκB, were influenced by AHR tethering (Tian et al. 1999;

Beischlag and Perdew 2005; Matthews et al. 2005). Interestingly, as in the case of NFκB for example, some studies suggest transrepression as the result of AHR tethering, while others support coactivation (Tian et al. 1999; Vogel et al. 2007). Most, if not all, of such studies have been performed using cell lines, and the observed discrepancies between studies have been attributed to differences between cell lines and culturing conditions (Vogel et al. 2007). Since cell lines are typically transfected to overexpress the transcription factors of interest, the previous results may have limited physiological relevance; since AHR transrepression or coactivation has typically been shown using luciferase reporter assays, the previous results may not sufficiently reveal physiological conditions. Therefore, a study using intact animals would be valuable to demonstrate the presence of AHR tethering in vivo without overexpression, to determine if it leads to transrepression or coactivation, and identify which genes are transrepressed or coactivated.

Because further studies are required to demonstrate the presence of AHR tethering, Ahrdbd/dbd mice,

30 whose AHR is unable to bind AHRE in DNA (Bunger et al. 2008), is an excellent model for this purpose.

To reiterate, our hypothesis is that AHR may mediate gene regulation through unrevealed

AHRE-independent mechanisms, namely indirect DNA binding via tethering to other transcription factors, or direct DNA binding to regions not containing any AHRE. The objectives of this study are: (1) to determine the liver genomic binding sites for AHR in Ahrdbd/dbd mice using ChIP- seq; and (2) to confirm any observed changes in gene transcription due to tethering or non-

AHRE binding using RNA-seq. In other words, by eliminating all direct AHRE binding of AHR, we aim to observe indirect DNA binding and direct non-AHRE binding across the mouse genome, and reveal other as yet unknown cooperative transcription factors. In contrast to many related previous studies using transfected cell lines, this project is a novel attempt to demonstrate whether

AHR tethering truly exists in living intact organisms expressing physiological levels of transcription factors, and to identify which specific genes are transpressed or coactivated by AHR tethering. This information is essential for filling in possible missing parts of the AHR functional mechanism for regulating gene expression in health and diseases.

Chapter 2 Materials and Methods

2.1 Materials

2.1.1 Chemical and Biological Reagents

The chemical reagents used in the various buffers and their sources are described here. β- mercaptoethanol, bromphenol blue, IGEPAL® CA-630, protease inhibitor cocktail (PIC), sodium deoxycholate, sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic monohydrate

® - (NaH2PO4·H2O), Triton X-100, and xylene cyanole FF were from Sigma-Aldrich (St. Louis,

Missouri, USA). Ethylenediaminetetraacetic acid (EDTA), glycine, hydrochloric acid (HCl) 12.1

M, lithium chloride (LiCl), magnesium sulfate heptahydrate (MgSO4·7H2O), potassium chloride

(KCl), sodium chloride (NaCl), sodium dodecyl sulfate (SDS),

Tris(hydroxymethyl)aminomethane (Tris), and Tween® 20 were from BioShop (Burlington,

Ontario, Canada). Glacial acetic acid and glycerol were from Fisher Scientific (Fair Lawn, New

Jersey, USA). Dithiothreitol (DTT) was from Roche (Indianapolis, Indiana, USA). Methanol was from Caledon Laboratories (Georgetown, Ontario, Canada).

For mice genotyping, the REDExtract-N-Amp tissue PCR kit was from Sigma-Aldrich,

Invitrogen™ UltraPure™ distilled water was from Life Technologies (Carlsbad, California, USA), primers were ordered from Integrated DNA Technologies (Coralville, Iowa, USA). BamHI-HF® restriction enzyme and CutSmart™ buffer from New England BioLabs (Ipswich, Massachusetts,

USA) were used. Milli-Q water, Invitrogen UltraPure™ agarose from Life Technologies, and ethidium bromide from BioShop were used to make 2% gels. The samples were diluted with DNA loading buffer (6× DNA loading buffer contains 30% glycerol and 0.25% xylene cyanole) before loading them onto agarose gels and separating them by gel electrophoresis using TAE buffer (40 mM Tris, 1 mM disodium EDTA, and 20 mM glacial acetic acid).

For Ahr gene cloning and sequencing, Aurum™ total RNA mini kit from Bio-Rad

(Mississauga, Ontario, Canada) was used to extract RNA, Applied Biosystems™ high-capacity cDNA reverse transcription kit from Thermo Fisher Scientific (Waltham, Massachusetts, USA) was used to synthesize cDNA, Invitrogen™ Platinum® Pfx DNA polymerase from Thermo Fisher

Scientific was used to amplify Ahr, GenepHlow™ gel/PCR kit from Geneaid (New Taipei City,

Taiwan) was used for gel extraction and PCR purification, and Presto™ mini plasmid kit also from Geneaid was used to extract plasmids from transformed bacteria. Self-designed primers, distilled water, agarose, ethidium bromide, and CutSmart™ buffer were from the same source as abovementioned for mice genotyping. XhoI and NheI restriction enzymes as well as T4 DNA ligase and its buffer were from New England BioLabs. Invitrogen™ pcDNA™3.1 mammalian expression vector, DH5α competent E. coli, and Invitrogen™ S.O.C. medium were from Thermo

Fisher Scientific. Bacteriological grade agar and sodium salt ampicillin were from BioShop.

Multicell™ LB Miller’s broth was from Wisent Bioproducts (Montreal, Quebec, Canada). To maintain sterility, 95% ethyl alcohol from Commercial Alcohols Greenfield Global (Mississauga,

Ontario, Canada) was used to make 70% ethanol.

The luciferase reporter assay used the COS-1 cell line from the American Type Culture

Collection (Manassas, Virginia, USA). The cells were cultured in Dulbecco’s Modified Eagle’s

Medium (DMEM) high glucose from Sigma-Aldrich with 10% Gibco® fetal bovine serum and 1× penicillin-streptomycin, both from Thermo Fisher Scientific. Gibco® 0.05% trypsin-EDTA from

Thermo Fisher Scientific was used for cell dissociation. Trypan blue dye 0.40% from Bio-Rad was used for cell counting. Transfection was done using Lipofectamine® 2000 and Gibco® Opti-MEM®

I reduced serum medium, which were also from Thermo Fisher Scientific. The pGudLuc 4.1 plasmid was a generous gift from Dr. Michael Denison at University of California, Davis (Davis,

California, USA). The pCH110 and pEGFP plasmids were purchased from Pharmacia (Uppsala,

Sweden) and Clontech (Mountain View, California, USA), respectively. The 1×10-4 M TCDD in

DMSO was diluted from the 80.5 µg/mL TCDD in DMSO stock, and its preparation will be described in next paragraph. Dulbecco’s Phosphate Buffered Saline (PBS) was from Sigma-

Aldrich. Cell culture lysis 5× reagent, ONE-Glo™ luciferase assay substrate lyophilized, and

ONE-Glo™ luciferase assay buffer were from Promega (Madison, Wisconsin, USA). The β- galactosidase buffer consisted of 60 mM Na2HPO4, 40 mM NaH2PO4, 10 mm KCl, and 1 mM

MgSO4; 40 mM β-mercaptoethanol was added freshly before use. Sodium carbonate (Na2CO3) and o-Nitrophenyl-ß-D-Galactopyranoside (ONPG) were from BioShop.

To treat Ahrdbd/dbd and Ahrwt/wt mice, TCDD 100% 1 mg package was bought from

AccuStandard (New Haven, Connecticut, USA), and was dissolved in Hybri-Max™ sterile-filtered

DMSO ≥99.7% from Sigma-Aldrich at 80.5 µg/mL. Before use, it was heated at 60°C for at least

30 minutes to ensure complete dissolving. DMSO from the same bottle was used for the control treatment. Liquid nitrogen for flash freezing liver tissue was obtained from the MedStore at

University of Toronto (Toronto, Ontario, Canada).

For RT-qPCR, Aurum™ total RNA mini kit was used to extract RNA, Applied

Biosystems™ high-capacity cDNA reverse transcription kit was used to synthesize cDNA, and

KAPA SYBR® fast universal kit from Kapa Biosystems (Wilmington, Massachusetts, USA) was used to perform qPCR. The RNase-free DNase I from the RNA kit was reconstituted in autoclaved

10 mM Tris pH 7.5. Self-designed primers were ordered from Integrated DNA Technologies. To rinse the probe of the homogenizer in-between samples for the homogenization during RNA extraction, and protein extraction, 95% ethyl alcohol from Commercial Alcohols Greenfield

Global was diluted into 70%; PBS was from Sigma-Aldrich.

For Western blots, the RIPA buffer was composed of 150 mM NaCl, 5 mM EDTA pH 8.0,

50 mM Tris pH 8.0, 1% IGEPAL® CA-630, 0.5% sodium deoxycholate, and 0.1% SDS in Milli-

Q water; 2× PIC and 2 mM DTT were added to RIPA before use. PBS from Sigma-Aldrich was used to dilute protein samples. Laemmli sample buffer (5×) was composed of 25 mM Tris pH 8.0,

2.5 mM EDTA, 6.25% SDS, 25% glycerol, and 0.017% bromophenol blue in Milli-Q water. SDS-

PAGE running buffer (10×) contained 25 mM Tris, 192 mM glycine, and 0.1% SDS in Milli-Q water. Transfer buffer (10×) contained 25 mM Tris and 192 mM glycine; 1× transfer buffer was made of 10% 10× transfer buffer, 20% methanol, and 70% Milli-Q water. TBS buffer (10×) contained 0.2 M Tris and 1.5 M NaCl, and its pH was adjusted to 7.6 using HCl 12.1 M. Tween®

20 was added to 1× TBS buffer before use freshly at a concentration of 0.1%. Pierce™ BCA protein assay from Thermo Fisher Scientific was used for determining protein concentration. Skim milk powder was from BioShop. SA-210 anti-AHR polyclonal antibody was from Enzo

(Farmingdale, New York, USA). A2228 Anti-β-Actin monoclonal antibody was from Sigma-

Aldrich. HRP-linked anti-rabbit IgG antibody and HRP-linked anti-mouse IgG antibody were from

Cell Signaling Technology (Danvers, Massachusetts, USA). SuperSignal™ West Dura extended duration substrate, SuperSignal™ West Pico chemiluminescent substrate, and Restore™ western blot stripping buffer were all from Thermo Fisher Scientific.

For ChIP assays, several other buffers were made. Tris/Saline/EDTA (TSE) I buffer contained 20 mM Tris-HCl pH 8.0, 1% triton X-100, 150 mM NaCl, 0.1% SDS, and 2 mM EDTA.

TSE II buffer contained 20 mM Tris-HCl pH 8.0, 1% triton X-100, 500 mM NaCl, 0.1% SDS, and

2 mM EDTA. LiCl Buffer contained 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 1%

IGEPAL® CA-630, and 1% sodium deoxycholate. Formaldehyde 37% from Sigma-Aldrich was diluted to 1% with PBS from Sigma-Aldrich. To purify DNA samples, QIAquick® PCR

35 purification kit was from Qiagen (Hilden, Germany). To check fragment size, 1.5% agarose gel was made using reagents stated in mice genotyping. The samples were loaded onto gels with DNA loading buffer (6×) containing 30% glycerol only. The immunoprecipitation step used

Dynabeads™ protein A from Thermo Fisher Scientific, SA-210 anti-AHR polyclonal antibody from Enzo, and ab171870 polyclonal rabbit IgG from Abcam (Cambridge, UK). The qPCR was done using Applied Biosystems™ PowerUp™ SYBR® green master mix and UltraPure™ distilled water from Thermo Fisher Scientific, as well as self-designed primers from Integrated DNA

Technologies.

2.1.2 Plasticware and Other Consumables

DIAMOND® ECOPACK™ 1000 µL, 200 µL, and 20 µL pipette tips were from Gilson

(Middleton, Wisconsin, USA), while PROGENE® 10 µL pipette tips were from Ultident Scientific

(Montreal, Quebec, Canada). Axygen® 1.5 mL microtubes were from Corning (Corning, New

York, USA). Polypropylene conical centrifuge tubes 15 mL and 50 mL were from Froggabio

(Toronto, Ontario, Canada). All RNA work was done using Axygen® 1000 µL, 200 µL, and 20 µL filter pipette tips from Corning. Multiply®-µStrip 0.2ml chain and 8-Lid chain flat from Sarstedt

(Numbrecht, Germany) was used for cDNA synthesis and PCR reactions. The 2 mL microtubes from Aurum™ total RNA mini kit, which fits the tip of POLYTRON® homogenizer, were used for all tissue homogenization steps. Falcon® 15 mL polystyrene conical tube from Corning was used during the sonication step in Western blot and ChIP.

For Ahr gene cloning and sequencing, sterile 5 mL polypropylene tubes from Sarstedt were used to grow bacteria in LB medium, and Fisherbrand™ sterile 100 mm × 15 mm polystyrene petri dishes from Thermo Fisher Scientific were used to make LB agar plates. For luciferase

36 reporter assay, tissue culture flask T-75 from Sarstedt and Falcon® 12-well clear multiwell plate from Corning was used for cell culture. Liquid transfer was done using 5 mL, 10 mL, and 25 mL serological pipette from Sarstedt. Disposable Pasteur pipets from VWR (Radnor, Pennsylvania,

USA) was using for aspiration. CELLSTAR™ 96-well polystyrene black microplate with black bottom and UV-Star® 96-well clear microplate, both from Greiner Bio-One (Frickenhausen,

Germany), were used for luciferase and β-galactosidase reactions, respectively. Cell scraper 25 cm from Sarstedt was used to scrape cells in RIPA buffer for Western blot.

PrecisionGlide™ needle 27G × ½ (0.4 mm × 13 mm) from Becton Dickinson (Franklin

Lakes, New Jersey, USA) was used to deliver intraperitoneal injection of TCDD in mice.

Polypropylene 2 mL screw cap micro tube from Sarstedt was used for flash-freezing and preserving tissues from mice. For RT-qPCR, Thermo-fast 96 detection plate with black lettering and Applied Biosystems™ MicroAmp™ optical adhesive film from Thermo Fisher Scientific were used. For Western blot, mini-PROTEAN® TGX™ 10% 15-well precast gels and filter paper backing from Bio-Rad (Hercules, California, USA), 0.45 µm pore size Immobilon®-P PVDF membrane from EMD Millipore (Darmstadt, Germany), and half-speed blue-sensitive M Plus film from Agfa Healthcare (Mortsel, Belgium) were used. For ChIP-qPCR, Multiplate™ 96-well low- profile unskirted clear PCR plates from Bio-Rad and Applied Biosystems™ MicroAmp™ optical adhesive film were used.

2.1.3 Instruments

To make buffers, PB3002-S DeltaRange® balance from Mettler Toledo (Columbus, Ohio,

USA), SB20 pH meter from VWR, and Thermix® 220T stirrer from Fisher Scientific were used.

Milli-Q water for buffers and other purposes was made by Milli-Q® Direct 16 water purification system from EMD Millipore (Darmstadt, Germany).

Centrifugation was carried out by MicroCL 17 centrifuge from Thermo Fisher Scientific at room temperature and by Centrifuge 5424 from Eppendorf (Hamburg, Germany) at 4°C.

Incubation was done by Signature™ B.O.D. low temperature refrigerated incubator from VWR at

37°C and by Thermomixer R from Eppendorf at 95°C. PTC-225 Peltier thermal cycler from MJ

Research (Waltham, Massachusetts, USA) was used for PCR amplification and cDNA synthesis.

PowerPac™ basic power supply from Bio-Rad was used for electrophoresis of DNA and protein.

BioDoc-It™ system from Ultra-violet Products (Upland, California, USA) was used to visualize

DNA bands in agarose gels. POLYTRON® PT1200E handheld homogenizer from Kinematica

(Bohemia, New York, USA) was used to homogenize liver tissues for RNA extraction, Western blot, and ChIP. NanoDrop 1000 spectrophotometer from Thermo Scientific was used to determine the concentration of RNA and DNA. Sonication for Western blot and ChIP was done using

Bioruptor® from Diagenode (Denville, New Jersey, USA).

To clone Ahr gene, PCR products and plasmids on agarose gels were visualized by high performance ultraviolet transilluminator from Ultra-violet Products, allowing them to be cut for extraction. Pittsburg-Universal flame burner from Fisher Scientific was used to keep the working area sterile for bacteria transformation. G24 environmental incubator shaker from New Brunswick

Scientific (Edison, New Jersey, USA) was used for shaking bacteria at 37°C. For luciferase reporter assay, cell culture was done under SterilGARD hood class II type A/B3 from Baker

(Sanford, Maine, USA). Cells were incubated in Forma Series II water jacketed CO2 incubator

HEPA Class 100 from Thermo Fisher Scientific. Centrifuge 5702 from Eppendorf was used to collect cells. Trinocular inverted microscope from VWR and Bright-Line™ hemacytometer from

Sigma-Aldrich were used for cell counting. Liquid waste containing TCDD was collected by

Vacusafe comfort aspiration system from Integra Biosciences (Zizers, Switzerland). GloMax®-

Multi detection system from Promega and Synergy™ NEO microplate reader from BioTek

(Winooski, Vermont, USA) were used to read luciferase and β-galactosidase activities, respectively.

Two Gastight® #1710 glass syringes from Hamilton Company (Reno, Nevada, USA) were separately used for TCDD injection and DMSO injection. The weight of mice was measured using the CS200 balance from OHAUS (Parsippany, New Jersey, USA). The weight of dissected tissues was measured using the AB104-S/FACT analytical balance from Mettler Toledo. Applied

Biosystems™ QuantStudio 3 Real-Time PCR system from Thermo Fisher Scientific was used for

RT-qPCR. For Western blot, Synergy™ NEO microplate reader was used to read results of BCA protein assay. Mini-PROTEAN® II cell from Bio-Rad was used for electrophoresis and transfer.

MW-23 2/3 D Waver shaker from Major Science (Saratoga, California, USA) was used for washing and incubation at room temperature, and Rocker 25 from Labnet (Edison, New Jersey,

USA) was used for incubation at 4°C. SRX-101A medical film processor from Konica Minolta

Medical & Graphic (Hino, Japan) was used to develop films. The films were scanned by imageCLASS MF4880dw from Canon (Tokyo, Japan). For ChIP, B3D 1320 Super Nutation mixer from Southwest Science (Hamilton Township, New Jersey, USA) and DEL-1100 Lab

Revolution™ rotator from ManSci (Orlando, Florida, USA) were used to mix samples. Incubator

1500E from VWR was used for 65°C incubation. Dynal® Invitrogen™ bead separator 16 position rack from Thermo Fisher Scientific was used to collect Dynabeads™ protein A during immunoprecipitation. CFX96™ Real-Time system from Bio-Rad was used for the qPCR of ChIP samples.

2.2 Methods

2.2.1 Animal Facility and Colony Maintenance

The mice were maintained by the Division of Comparative Medicine at University of

Toronto, where certified technicians and veterinarians were employed to take care of animals for research. A maximum of four mice were housed in each cage. In addition to standard rodent chow and water, igloos and nesting material were provided as environmental enrichment. Everything was autoclaved and all procedures were done under biological safety cabinets to ensure sterility.

Food and water were inspected every day, and the caging were changed every two weeks. The health of the animals was monitored by technicians, and unusual signs were reported. At the facility, the temperature was maintained at 21°C, the humidity was controlled, and the light cycle was 12-hour light and 12-hour dark. The mice were ear-tagged and tail-clipped at 2 weeks, and weaned at 3 weeks. Selected breeders were paired at 5 weeks. Other mice selected for experiments were treated at 8 weeks. All procedures and experiments were approved by the Animal Ethics

Committee at University of Toronto in accordance with the guidelines of the Canadian Council on

Animal Care.

Ahrdbd/dbd mice were a kind gift from Dr. Carolyn Baglole at McGill University (Montreal,

Quebec, Canada). She obtained Ahrdbd/dbd mice from Dr. Chris Bradfield at University of

Wisconsin (Madison, Wisconsin, USA) who had originally generated this mouse line (Bunger et al. 2008). The Ahrdbd/dbd mice received were crossed with C57BL/6J wildtype mice from Envigo

(Mississauga, Ontario, Canada), raising Ahrwt/dbd heterozygous mice. The Ahrwt/dbd heterozygotes were bred together to give Ahrdbd/dbd mice and Ahrwt/wt littermates that were used for experiments.

2.2.2 Mouse Genotyping

Heterozygous Ahrwt/dbd male and female mice were bred in order to generate Ahrdbd/dbd and

Ahrwt/wt littermates. Tissues for genotyping were clipped from tails at 2 weeks old. DNA extraction and PCR reaction were performed using the REDExtract-N-Amp tissue PCR kit. For each reaction, the tail was incubated with 100 µL retraction solution and 25 µL tissue preparation solution first at room temperature for 10 minutes and then at 95°C for 3 minutes. DNA extraction was completed by adding 100 µL neutralization solution B. The PCR master mix was composed of 4 µL DNA,

10 µL REDExtract-N-Amp PCR reaction mix, 5 µL UltraPure™ distilled water, 0.5 µL forward primer at 20 µM, and 0.5 µL reverse primer at 20 µM per sample. The PCR cycling condition consisted of 40 cycles of the following: 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes. The restriction enzyme digestion was completed with 10 µL PCR product, 0.5 µL

BamHI-HF® restriction enzyme, 2.5 CutSmart™ buffer, and 12 µL UltraPureTM distilled water.

The digestion mix was incubated for a minimum of 1 hour at 37°C, and then run on 2% agarose gels.

Table 2.1 Primers for Ahrdbd genotyping

Primer Name Primer Sequence (5’ to 3’) Ahrdbd Genotyping Forward CTG AGG GGA CGT TTT AAT G Ahrdbd Genotyping Reverse AAC ATT TGC ACT CAT GGA TAG

2.2.3 Ahr Gene Cloning and Sequencing

The liver RNA of one Ahrdbd/dbd mouse and one Ahrwt/wt mouse was extracted using

Aurum™ total RNA mini kit, and cDNA was synthesized using Applied Biosystems™ high- capacity cDNA reverse transcription kit, both following procedures described in Section 2.2.6.

However, for cDNA synthesis here, 1,000 ng RNA instead of 500 ng was used; Ahr cloning reverse

41 primer, designed to target Ahr gene and have an XhoI restriction site, instead of the 10× RT random primers was used. Ahr cDNA was then PCR amplified using Platinum® Pfx DNA polymerase.

Each reaction contained: 1 µL Ahr cDNA, 10 µL 10× Pfx amplification buffer, 3 µL 10 mM dNTP mixture, 2 µL 50 mM MgSO4, 0.8 µL Platinum™ Pfx DNA polymerase, 3 µL 10 µM Ahr cloning forward primer which contains a Kozak sequence after a NheI restriction site, 3 µL 10 µM abovementioned Ahr cloning reverse primer, and 66.2 µL UltraPureTM distilled water. The cycling condition was: 94°C for 5 minutes, followed by 35 cycles of 94°C for 15 seconds, 72°C for 30 seconds, and 68°C for 3 minutes. After running the PCR products on a 2% gel, the fragment at approximately 2500 bp was excised. GenepHlow™ gel/PCR kit was used to extract Ahr PCR products following the standard protocol.

The Ahr PCR products and the pcDNA3.1 plasmids were separately digested overnight at

37°C. Each reaction contained appropriate amounts of PCR product or the plasmid, XhoI restriction enzyme, NheI restriction enzyme, and CutSmart™ Buffer. They were then purified using GenepHlow™ gel/PCR kit following the standard protocol. The Ahr PCR products were ligated to pcDNA3.1 plasmid overnight at room temperature using T4 DNA ligase. DH5α competent E. coli was transformed with the ligated plasmids. After the plasmids were added, the bacteria were sequentially incubated 30 minutes on ice, 1 minute at 42°C, and 1 minute on ice.

They were shaken for 1 hour at 37°C after S.O.C. medium was added. After centrifugation, the bottom part of the suspension was plated on LB agar plates containing ampicillin and incubated overnight at 37°C. Bacterial colonies were transferred into 5 mL LB containing ampicillin and incubated overnight with shaking at 37°C. Presto™ Mini plasmid kit was used to extract the plasmid. The extracted plasmids were digested with XhoI and NheI restriction enzymes, and run on 2% gels to check if the plasmids contained the Ahr insert. Selected plasmids were sent to be

42 sequenced at the Centre for Applied Genomics (TCAG) operated by the Hospital for Sick Children

(SickKids) (Toronto, Ontario, Canada). In addition to the T7 and BGHR primers provided by the

TCAG to target regions on pcDNA3.1 plasmid, Ahr sequencing primer 1 and Ahr sequencing primer 2 targeting the middle part of Ahr were used. The DNA sequences of Ahrdbd and Ahrwt were compared to published sequences of Ahrb1 in the C57BL/6J strain (AF405563.1) and Ahrd in the

129/SvJ strain (AF325111.1) using the GeneStudio™ Pro software. The DNA sequences were translated in silico using the translate tool from ExPASy SIB Bioinformatics Resource Portal

(ExPASy) (www.expasy.org). The amino acid sequences were aligned with AHR sequences in the

C57BL/6J strain (AAL89728.1) and the 129/SvJ strain (AAK13443.1) using BOXSHADE 3.21 from ExPASy.

Table 2.2 Primers for Ahr gene cloning and sequencing

Primer Name Primer Sequence (5’ to 3’) Ahr Cloning Forward Primer AAT TGC TAG CGC CAC CAT GAG CAG CGG CGC CAA CAT CAC Ahr Cloning Reverse Primer AAT TCT CGA GCT ACA GGA ATC CAC CAG GTG TGA TAT C Ahr Sequencing Primer 1 AAT TTC CAA GGG AGG TTA AAG TAT C Ahr Sequencing Primer 2 GTA TGC TGC AGG AGC GCC TGC AAC TAG

2.2.4 Luciferase Reporter Assay

COS-1 cells were plated on 12-well dishes at a density of 1.00×105 to 1.25×105 cells with

1 mL media per well. On the second day, each well was transfected with 1 µg DNA and 2 µL

Lipofectamine® 2000 dissolved in 100 µL Opti-MEM® media. Each well was given 300 ng pGudLuc 4.1, 100 ng pCH110, and 50 ng pEGFP. pGudLuc 4.1 is an AHR-driven reporter construct containing a luciferase gene downstream of the Cyp1a1 promoter. pCH110 with a lacZ gene coding for β-galactosidase was used as an internal control to normalize gene expression after

43 transfection. pEGFP enables the expression of enhanced green fluorescent protein for monitoring gene expression after transfection under microscope. In addition, the Ahrdbd transfected cells received 400 ng pcDNA3.1-Ahrdbd and 150 ng pcDNA3.1, the Ahrwt transfected cells received 400 ng pcDNA3.1-Ahrwt and 150 ng pcDNA3.1, and the control cells received 550 ng pcDNA3.1. After

6 hours, every well was treated with either 1.1 µL 100 µM TCDD in DMSO for a final concentration at 100 nM TCDD or 1.1 µL DMSO vehicle control. For each trial, each treatment was completed in duplicates.

On the third day, after washing once with PBS, cells were added with 250 µL cell culture lysis reagent each well and rocked for 10 minutes. Onto a black 96-well microplate, 25 µL lysate was loaded in duplicates and 25 µL ONE-Glo™ luciferase assay substrate was then added. After a 10-minute incubation, luciferase activity was measured using GloMax®-Multi detection system.

Onto a clear 96-well microplate, 10 µL lysate was loaded in duplicates and 100 µL β-galactosidase buffer was added. Then, 25 µL ONPG at 4 mg/mL was added to each well. The plates were incubated at 37°C until a pale-yellow color was seen. Lastly, 25 µL 1 M Na2CO3 was added to terminate the reaction. The plate was read at 420 nm. Luciferase activity was normalized to β- galactosidase activity. The luciferase reporter assay was repeated for three trials.

To confirm the expression of AHRdbd and AHRwt in COS-1 cells after transfection, COS-1 cells were again plated on 12-well dishes at a density of 1.00×105 to 1.25×105 cells with 1 mL medium per well. On each plate, 4 wells were transfected with each of pcDNA3.1, pcDNA3.1-

Ahrdbd, and pcDNA3.1-Ahrwt. The cells were washed with PBS once, and then scarped and suspended in 250 mL RIPA buffer. Samples from 4 wells were pooled together and used for

Western blot following procedures described in Section 2.2.7. The Western blot experiments were repeated three times.

2.2.5 Animal Treatment and Tissue Collection

TCDD was dissolved in DMSO at 80.5 µg/mL. Male 8-week-old Ahrdbd/dbd mice and

Ahrwt/wt littermates were intraperitoneally injected with 100 µg/kg TCDD or equivalent amount of

DMSO vehicle control. After 2, 6, or 24 hours, animals were sacrificed by cervical dislocation.

Liver tissues were flash frozen in liquid nitrogen immediately after collection and later stored at -

80°C. Four mice were included for each treatment.

2.2.6 RNA Extraction, cDNA synthesis, and RT-qPCR

Aurum™ total RNA mini kit was used for RNA extraction. For each sample, 700 µL lysis solution was added to 20 to 40 mg liver tissue, which was then homogenized by POLYTRON®

PT1200E handheld homogenizer at full speed for 10 seconds. Between each sample, the probe of the homogenizer was rinsed by sequentially spinning in Milli-Q water, 70% ethanol, Milli-Q water, and PBS. The rest steps were performed following the standard protocol of the kit. The RNA was eventually eluted by 80 µL elution solution and stored at -80°C.

Master mix of components from Applied Biosystems™ high-capacity cDNA reverse transcription kit was made with 4.2 µL Nuclease-free H2O, 2 µL 10× RT buffer, 0.8 µL 25× dNTP mix, 2 µL 10× RT random primers, and 1 µL MultiScribe™ reverse transcriptase per reaction. For each sample, 10 µL RNA that was diluted to 50 ng/µL with UltraPureTM distilled water was used.

The PCR cycling condition was: 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 min.

The cDNA samples were then diluted with 60 µL UltraPure™ distilled water for qPCR.

For the qPCR on cDNA, KAPA SYBR® fast universal kit, Thermo-fast 96 detection plate, and MicroAmp™ optical adhesive film were used. For each well, the total reaction volume was

10 µL, and the master mix composed of 5 µL KAPA SYBR fast qPCR master mix (2X) universal,

0.1 µL forward primer at 10 µM, 0.1 µL reverse primer at 10 µM, and 3.8 µL UltraPure™ distilled water. On Applied Biosystems™ QuantStudio 3 Real-Time PCR system, the cycling condition was 95°C for 3 minutes, followed by 45 cycles of 95°C for 10 seconds and 60°C for 20 seconds.

Each sample was done in triplicate. Data analysis was done using the ΔΔCt Method (Livak and

Schmittgen 2001). TATA-box binding protein (Tbp) was used as the reference gene for normalization.

Table 2.3 Primers for gene expression RT-qPCR

Primer Name Primer Sequence (5’ to 3’) Ahr Expression RT-qPCR Forward AGC ATC ATG AGG AAC CTT GG Ahr Expression RT-qPCR Reverse GGA TTT CGT CCG TTA TGT CG Cyp1a1 Expression RT-qPCR Forward TCC CCA AAC TCA TTG CTC AGA T Cyp1a1 Expression RT-qPCR Reverse CGT TAT GAC CAT GAT GAC CAA GA Cyp1b1 Expression RT-qPCR Forward GTG CGG CAA AAG CAT GTC TC Cyp1b1 Expression RT-qPCR Reverse GGG GAA AAG CAA CGT TCT GAC Tbp Expression RT-qPCR Forward GCA CAG GAG CCA AGA GTG AA Tbp Expression RT-qPCR Reverse TAG CTG GGA AGC CCA ACT TC

2.2.7 Western Blot

Approximately 50 to 80 mg frozen liver tissue was homogenized with POLYTRON® homogenizer at full speed for around 10 seconds, immediately after 400 µL RIPA buffer containing 2× PIC and 2 mM DTT was added. Between each sample, the probe of the homogenizer was rinsed by sequentially spinning in Milli-Q water, 70% ethanol, Milli-Q water, and PBS. The homogenate was sonicated with Bioruptor® on the low setting at 4°C for 5 minutes, 30 seconds on and 30 seconds off. After being rotated for 15 minutes at 4°C, it was centrifuged for 10 minutes at

20,000 ×g at 4°C. Protein concentration was determined with Pierce™ BCA protein assay kit.

Appropriate amounts of protein sample, PBS, and 5× laemmli sample buffer with DTT were mixed to generate 10 µg protein in 10 µL, which was then incubated at 95°C for 5 minutes.

The samples were loaded to precast gels. The gels were run at 135 V constant voltage for 75 minutes with ice. Protein was transferred to PVDF membranes at 105V constant voltage for 60 minutes with ice. Membranes were blocked with 5% milk in TBST for 2 hours at room temperature.

To blot for Ahr, the SA-210 anti-AHR antibody was diluted at 1:10,000 with 5% milk in

TBST. This primary antibody solution was used to incubate membranes overnight at 4°C. TBST was used to wash membranes 4 times, each for 10 minutes. Secondary antibody solution, containing HRP-linked Anti-rabbit IgG antibody diluted at 1:6,250 with 5% milk in TBST, was used to incubate membranes for 1 hour at room temperature. Membranes were then washed with

TBST again 4 times, each for 10 minutes. SuperSignal™ West Dura extended duration substrate was used for detection. Films were developed in dark with a medical film processor.

To blot for β-actin, membranes were incubated with stripping buffer for 10 minutes, washed once with TBST for 10 minutes, and blocked with 5% milk in TBST for 1 hour.

Membranes were then incubated for 1 hour at room temperature with A2228 β-actin antibody, which was diluted at 1:5,000 with 1% milk in TBST. After washing 3 times with TBST, membranes were incubated for 1 hour at room temperature with HRP-linked anti-mouse IgG antibody, which was diluted at 1:20,000 with 1% milk in TBST. After another 3 washings with

TBST, SuperSignal™ West Pico chemiluminescent substrate was used for detection.

The chemiluminescence was captured by films which were developed in a dark room. The films were color scanned at 600 dpi in TIFF file format. The original image was turned into grayscale in JPEG file format using GNU Image Manipulation Program 2.10.0 (www.gimp.org).

The program ImageJ (imagej.nih.gov/ij) was used to measure the pixel density of protein bands.

After the background signal was deducted, AHR signals were normalized with the corresponding

β-actin signals.

2.2.8 ChIP-seq and Data Analysis

Frozen liver tissues from four replicates of male 8-week-old mice treated with 100 µg/kg

TCDD or the DMSO vehicle control for 2 hours were used for ChIP. Immediately after 500 µL 1% formaldehyde in PBS was added, around 50 to 80 mg frozen liver tissues were homogenized with

POLYTRON® homogenizer at full speed for around 15 seconds. Between each sample, the probe of the homogenizer was rinsed by sequentially spinning in Milli-Q water, 70% ethanol, Milli-Q water, and PBS. Another 500 µL 1% formaldehyde in PBS was added after. The homogenate was then rotated at room temperature for 10 minutes. To quench the crosslinking, 2 M glycine solution was added to reach a final concentration of 0.125 M. The samples were then centrifuged at 8,000 rpm for 3 minutes at 4°C, and the supernatant was removed. The pellets were then washed twice by resuspending in 1 mL ice cold PBS and centrifuging. After removing PBS from the last wash, the pellet was resuspended in 500 µL ice old TSE I buffer with 2× PIC freshly added, and rotated for 20 minutes at 4°C. They were transferred to 15 mL polystyrene tubes, which were loaded into the Bioruptor® sonicator. Sonication was done at high intensity, 30 seconds on and 30 seconds off, for 15 minutes twice, with a 10-minute cooling period in-between. They were transferred to 2 mL microcentrifuge tubes and centrifuged at 15,000 rpm for 10 minutes at 4°C. The supernatant was the total chromatin sample collected. The samples were stored at -80°C if immunoprecipitation was not immediately performed.

To check the fragment size, 10 µL total chromatin was diluted with 90 µL 1% SDS in PBS, and was de-crosslinked with incubation at 95°C for 10 minutes. QIAquick PCR purification kit was used to purify DNA, following the standard protocol. The eluted DNA was run on 1.5% agarose gels. It was expected to have a smear with an average size of less than 500 bp.

Dynabeads™ protein A was equilibrated by washing with 1 mL TSE I buffer for 5 times, with 2-minute rotation each time. The beads were collected using a magnetic bead separator. The beads were resuspended in TSE I buffer to its original volume and stored overnight at 4°C. In new

2 mL microcentrifuge tubes, 3 µg SA-210 anti-AHR antibody was added to 200 µL total chromatin, which was then incubated overnight at 4°C on a rotating wheel. As a control, the same was done using 1 µg ab171870 polyclonal rabbit IgG. The next day, the beads were sequentially washed with TSE I buffer for 3 times, TSE II buffer for 1 time, LiCl buffer for 1 time, and TSE I buffer for another 1 time. For each washing, the beads were incubated at room temperature for 5 minutes on a rotating wheel. To elute the DNA, 100 µL 1% SDS in PBS was added to the beads, which were incubated at room temperature for 30 minutes on a rotating wheel and then at 65°C overnight.

As the input sample, 10 µL total chromatin was diluted to 100 µL with 1% SDS in PBS, and also incubated at 65°C overnight. The anti-AHR immunoprecipitated samples, the IgG immunoprecipitated samples, and the input samples were all purified using QIAquick PCR purification kit. The samples were eluted with 40 µL elution buffer from the kit. They were stored at -80°C before next steps.

To check the quality of the anti-AHR immunoprecipitation, qPCR was done to amplify the promoter regions of two AHR target genes, Cyp1a1 and Cyp1b1. Each reaction contained 1 µL

DNA sample, 0.1 µL forward primer at 10 µM, 0.1 µL reverse primer at 10 µM, 5 µL Applied

Biosystems™ PowerUp™ SYBR® Green master mix, and 3.8 µL UltraPure™ distilled water. On

C1000™ thermal cycler, the cycling condition was 95°C for 5 minutes, followed by 45 cycles of

95°C for 10 seconds and 60°C for 20 seconds. Each sample was done in triplicate. Data analysis was done using the percent input method and the fold enrichment method.

Table 2.4 Primers for ChIP-qPCR

Primer Name Primer Sequence (5’ to 3’) Cyp1a1 Promoter ChIP-qPCR Forward GAG GAT GGA GCA GGC TTA CG Cyp1a1 Promoter ChIP-qPCR Reverse GGG CTA CAA AGG GTG ATG CTT Cyp1b1 Promoter ChIP-qPCR Forward GGA GCC GAC TTC CCA GAA G Cyp1b1 Promoter ChIP-qPCR Reverse AAC AAA CGG TTG GGT TGA AAA G

After verifying the quality of immunoprecipitation, samples were further processed for library preparation using MicroPlex Library Preparation Kit v2 by Dr. Somisetty Satheesh from

Matthews Lab, Institute of Basic Medical Sciences, University of Oslo (Oslo, Norway). The four- step protocol consisted of template preparation which yields double-stranded DNA fragments with blunt ends, library synthesis which ligates adaptors to the blunt ends, library amplification which amplifies the fragments and adding barcodes using primers targeting the adaptors, and eventually library purification which purifies the amplified products using AMPure® XP beads. For template preparation, 2 µL template preparation buffer and 1 µL template preparation enzyme were added to 10 µL immunoprecipitated DNA. The reaction was done using a thermal cycler programmed at

22°C for 25 minutes and then at 55°C for 20 minutes. For library synthesis, 1 µL library synthesis buffer and 1 µL library synthesis enzyme were added to each reaction, which were then incubated at 22°C for 40 minutes using a thermal cycler. For library amplification, 25 µL library amplification buffer, 1 µL library amplification enzyme, 4 µL nuclease-free water, and 5 µL appropriate indexing reagent were added to each library synthesis reaction product. The amplification was run on a thermal cycler with the following program: 72°C for 3 minutes and

85°C for 2 minutes to extend and cleave the adaptors, 98°C for 2 minutes to denature the DNA, four repeats of 98°C for 20 seconds, 67°C for 20 seconds, and 72°C for 40 seconds to add the indexes, twelve repeats of 98°C for 20 seconds and 72°C for 50 seconds to amplify the library.

The library was then quantified by performing qPCR on Cyp1a1 promoter again. For library purification, after the library volume was adjusted to precisely 50 µL with nuclease-free water, 50

32.5 µL AMPure® XP beads were added. It was mixed well by pipetting 8 to 10 times and then incubated at room temperature for 10 minutes. After placing the sample on a magnetic rack for 2 minutes, the supernatant was transferred to a new tube, into which 12.5 µL AMPure® XP beads at room temperature was added. It was mixed well by pipetting 8 to 10 times, incubated at room temperature for 10 minutes, and placed on a magnetic rack for 2 minutes. After discarding the supernatant, the beads were washed for 2 times by repeating the following: incubating with 150

µL 80% ethanol for 30 seconds, aspirating the supernatant, and centrifuging at 2,000 ×g briefly to collect the beads. The beads were air dried by leaving the tube cap open for 5 minutes. To elute the DNA, 15 µL 1× low TE buffer, pH 8.0 was added. After being placed on a magnetic rack for

2 minutes, the supernatant containing purified library was collected and quantified using a bioanalyzer. Out of the four replicates for each treatment, three were chosen to be submitted for next-generation sequencing at the Norwegian Sequencing Centre (Oslo, Norway). The samples were sequenced on 2 lanes of the NextSeq 500 High-output, single reads at 75 bp.

Bioinformatic analysis of ChIP-seq samples was done by Dr. Aziz Khan at the Centre for

Molecular Medicine Norway, University of Oslo. The ChIP-seq pipeline implemented by the

ENCODE Data Coordinating Center (DCC) for the ENCODE Consortium (github.com/ENCODE-

DCC/chip-seq-pipeline2) (v.1.1.6) was used to analyze the ChIP-seq data (Figure 2.1) (Feingold et al. 2004). The raw reads were mapped to mouse reference genome (mm10) using BWA (v.0.7.13)

(Li and Durbin 2010). Samtools (v.1.2) was then used to filter and convert sam files to bam files

(Li et al. 2009). ChIP-seq peaks were called using MACS2 (v.2.1.1) at a relaxed p-value threshold of 1 x 10-2 for the individual biological replicates (Zhang et al. 2008). These relaxed peak sets from each biological replicate were then merged using Bedtools (v2.26.0) (Quinlan 2014). Intervene

(v.0.6.2) (github.com/asntech/intervene) was used to perform peaks overlap analysis, and to draw

Venn diagrams and UpSet plots (Khan and Mathelier 2017). Correlation plots and heatmaps were generated using deepTools (v.2.4). For motif analysis, Peak Motifs tool available in Regulatory

Sequence Analysis Tools (RSAT) (rsat.sb-roscoff.fr/peak-motifs_form.cgi) was used with the defult setting (Thomas-Chollier et al. 2013).

Analysis for overrepresented transcription factor binding sites was performed using

MatInspector available on Genomatix (www.genomatix.de) with the default parameter (Quandt et al. 1995; Cartharius et al. 2005). Overrepresentation was determined based on the number of matches in the immunoprecipitated DNA compared against the number of expected matches in the genomic background. To identify transcription factors associated with AHR, overrepresented transcription factor binding sites located within 10 to 50 bp from AHR binding sites was determined using the same tool. The TF family, AHR-ARNT heterodimers and AHR-related factors, is abbreviated as AHRR by Genomatix. In order to avoid the confusion with aryl hydrocarbon receptor repressor, this TF family is simply written as AHR in the results and discussion.

Figure 2.1 Schematic representation of the ChIP-seq pipeline from ENCODE.

2.2.9 RNA-seq and Data Analysis

Liver tissues from male 8-week-old mice treated with 100 µg/kg TCDD or the DMSO control for 6 hours were sent to Dr. Somisetty Satheesh in Matthews Lab at University of Oslo for sample preparation. Total RNA was isolated with the RNeasy Mini Kit from Qiagen, following the manufacturer’s protocol. The RNA yield was assessed with NanoDrop™ 2000 from Thermo

Fisher Scientific. The RNA quality was evaluated using the Agilent 2100 bioanalyzer from Agilent

(Palo Alto, California, USA) with the RNA 6000 Nano LabChip kit. The samples were submitted to TCAG at SickKids in Toronto for next-generation sequencing. The stranded poly(A) mRNA library preparation (NEBNext) protocol was used. The 16 samples were sequenced on 2 lanes of the HiSeq 2500 Rapid run flowcell, single-end sequencing, 100-bases. The reads were aligned to mm10 using TopHat available at UCSC Genome Browser (genome.ucsc.edu) with default parameters (Trapnell et al. 2009). Assembly of transcripts and differential gene expression analysis were performed using featureCounts and DESeq2 (Liao et al. 2014; Love et al. 2014).

2.2.10 Statistical Analysis

Prism Version 6.01 from GraphPad Software (San Diego, California, USA) was used for all statistical analyses. The observed sex ratio of mouse litters was compared with the expected

50% to 50% ratio using two-tailed binomial test. The observed frequency of Ahrwt/wt, Ahrwt/dbd, and

Ahrdbd/dbd pups was compared with the Mendelian ratio using two-tailed chi-square test. Data of luciferase reporter assay, Western blot, RT-qPCR, and ChIP-qPCR experiments, presented as mean ± SEM, were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test.

After Tukey’s test, two-tailed unpaired t-test was also used to calculate the significant difference of Egfr expression between DMSO-treated Ahrdbd/dbd mice and TCDD-treated Ahrdbd/dbd mice. For all analyses, the significance threshold was set at P ≤ 0.05.

Chapter 3 Results

3.1 Characterization of Ahrdbd/dbd Mice

This thesis aimed to demonstrate AHR gene regulation through tethering to other transcription factors or binding directly to non-AHRE regions using ChIP-seq and RNA-seq analysis of liver tissue isolated from Ahrwt/wt or Ahrdbd/dbd mice treated with TCDD. This mouse line carries an Ahr with mutated DNA-binding domain, as described in Sections 1.5 and 2.2.1.

Before proceeding with the next generation sequencing studies, I first performed experiments to confirm that the AHRdbd does not bind to AHRE sequences nor does it regulate canonical AHR target genes, such as Cyp1a1. The cloning and sequencing of the Ahr gene in Ahrdbd/dbd mice showed that the gene contained the expected mutation in the DNA binding domain that prevents its binding to AHREs (Figure 3.3). RT-qPCR and Western blots showed that Ahr mRNA and AHR protein were expressed in Ahrdbd/dbd mice, although at lower levels compared with Ahrwt/wt mice

(Figures 3.4 and 3.5). Luciferase reporter assays showed in vitro that TCDD-activated AHRdbd protein was unable to increase Cyp1a1-regulated luciferase activity (Figure 3.6). Hepatic RT- analyses showed TCDD-activated AHRdbd was unable to upregulate two canonical target genes, namely Cyp1a1 and Cyp1b1 (Figures 3.8 and 3.9).

3.1.1 The Mouse Colony

Heterozygous Ahrwt/dbd pairs were used for breeding. Neonatal deaths due to parental infanticide or cannibalism, aside from other unknown causes, were common. Identification numbers were assigned to the newborns by the veterinary technicians at 1 week of age, after neonatal deaths were frequently observed. Therefore, the number of actual births and neonatal deaths were not documented; the numbers below refer to newborns that survived their first week.

Out of 559 mice born from heterozygous Ahrwt/dbd breeding pairs, 279 (49.9%) were female

(Figure 3.1A). Among the 134 Ahrwt/wt pups, 72 (53.7%) were female (Figure 3.1B). Among the

331 Ahrwt/dbd pups, 162 (48.9%) were female (Figure 3.1C). Among the 94 Ahrdbd/dbd pups, 45

(47.9%) were female (Figure 3.1D). No sex ratios were significantly different from the expected sex ratio of 50% to 50%.

Among the 559 mice, 134 (24.0%) were Ahrwt/wt, 331 (59.2%) were Ahrwt/dbd, and only 94

(16.8%) were Ahrdbd/dbd, statistically different from the Mendelian ratio (P ≤ 0.0001) (Figure 3.2A).

Among the 279 female mice, 72 (25.8%) were Ahrwt/wt, 162 (58.1%) were Ahrwt/dbd, and only 45

(16.1%) were Ahrdbd/dbd, also statistically different from the Mendelian ratio (P ≤ 0.01) (Figure

3.2B). Among the 280 male mice, 62 (22.1%) were Ahrwt/wt, 169 (58.1%) were Ahrwt/dbd, and only

49 (16.1%) were Ahrdbd/dbd, again statistically different from the Mendelian ratio (P ≤ 0.01) (Figure

3.2C).

B C D

Figure 3.1 The sex ratio of the offspring of Ahrwt/dbd breeders. A. Females accounted for 49.9% (279/559) of all the offspring of Ahrwt/dbd breeders, while males accounted for 50.1% (280/559). B. Females accounted for 53.7% (72/134) of Ahrwt/wt pups, while males accounted for 46.3% (62/134). C. Females accounted for 48.9% (162/331) of Ahrwt/dbd pups, while males accounted for 51.1% (169/331). D. Females accounted for 47.9% (45/94) of Ahrdbd/dbd pups, while males accounted for 52.1% (49/94). There was no statistical difference found between all these observed sex ratios and the expected 50% to 50%.

B C

Figure 3.2 The genotypic ratio of the offspring of Ahrwt/dbd breeders. A. Among all the offspring of Ahrwt/dbd breeders, 24.0% (134/559) were Ahrwt/wt, 59.2% (331/559) were Ahrwt/dbd, and only 16.8% (94/559) were Ahrdbd/dbd. This frequency was statistically different from the Mendelian ratio (P ≤ 0.0001). B. Among the female, 25.8% (72/279) were Ahrwt/wt, 58.1% (162/279) were Ahrwt/dbd, and only 16.1% (45/279) were Ahrdbd/dbd, statistically different from the Mendelian ratio (P ≤ 0.01). C. Among the male, 22.1% (62/280) were Ahrwt/wt, 58.1% (169/280) were Ahrwt/dbd, and only 16.1% (49/280) were Ahrdbd/dbd, again statistically different from the Mendelian ratio (P ≤ 0.01).

3.1.2 Gene Sequencing of Ahrwt and Ahrdbd

Ahrdbd/dbd mice were generated by mutating the DNA-binding domain of the Ahr gene from

129/SvJ mice, but were then crossed with C57BL/6J mice (Bunger et al. 2008). In other words, the Ahrdbd gene came from the 129/SvJ strain, but the background of the mouse line was C57BL/6J.

To confirm the genotype of the mice received, the Ahr genes carried by Ahrwt/wt and Ahrdbd/dbd mice were cloned and sequenced. Their DNA sequences were aligned with sequences of Ahr in

C57BL/6J and 129/SvJ that are available on GenBank as AF405563.1 and AF325111.1, respectively. The cloned Ahrwt had an exact match with the Ahrb1 from the C57BL/6J strain.

Compared with the Ahrd from 129/SvJ, the cloned Ahrdbd had a substitution mutation from ATC to GGG at nucleic acids 73 to 75, and an insertion of GGATCC between nucleic acids 117 and

118. As expected, these were the two mutations introduced to disable the DNA-binding domain

(Bunger et al. 2008). The rest of the cloned Ahrdbd sequence was identical with the Ahrd gene from the 129/SvJ strain. In addition to these two artificial mutations, all other differences between the cloned Ahrwt and the cloned Ahrdbd were due to differences between Ahrb1 from the C57BL/6J and

Ahrd from the 129/SvJ. Compared with Ahrd from 129/SvJ, the Ahrb1 from C57BL/6J had 13 nucleotide differences: 753A>G, 960C>T, 972G>A, 1124T>C, 1389A>G, 1412C>T, 1598A>G,

1680C>T, 1765A>C, 1773C>G, 1833G>A, 2184A>G, and 2416C>T. The 2416C>T change results in a premature stop codon, making the AHR in C57BL/6J (Ahrb1) shorter than that of

129/SvJ (Ahrd).

The DNA sequences of the cloned Ahrwt and the cloned Ahrdbd genes were translated in silico, and then aligned with sequences of the AHR proteins from the C57BL/6J and the 129/SvJ strains available on GenBank as AAL89728.1 and AAK13443.1 (Figure 3.3). As expected, the two artificial mutations in Ahrdbd result in an I25G substitution as well as a GS insertion after

59 amino acid 39, which are responsible for producing a non-functional DNA-binding domain. All other differences between AHRwt and AHRdbd are due to differences between the C57BL/6J and

129/SvJ strains. Since several point mutations at the DNA level are synonymous, there were only six amino acid replacements. Compared with the AHR in 129/SvJ mice, the AHR in C57BL/6J mice had: M324I, V375A, P471L, N533S, M589L, and R806X. M324I and M589L are conservative mutations where the replaced amino acids have similar biochemical properties.

R806X results in an AHR protein with only 805 amino acids in C57BL/6J, compared with the 848 amino acids in 129/SvJ. V375A has been shown to be responsible for the enhanced ligand affinity of AHR in C57BL/6J, while all other changes have no or very limited impact at the functional level (Poland et al. 1994; Thomas et al. 2002). In summary, DNA sequencing confirmed that

Ahrwt/wt and Ahrdbd/dbd mice in our colony have the expected alleles of the Ahr gene.

Figure 3.3 Amino acid sequence alignment of the cloned AHRwt and AHRdbd proteins. AHR sequences in the C57BL/6J strain (AHRb1) and the 129/SvJ strain (AHRd) are available at GenBank as AAL89728.1 and AAK13443.1, respectively. Conserved residues are in black, conservative changes are in grey, and non-conservative mutations are in white.

3.1.3 mRNA and Protein Expression of AHR in Mouse Liver

The ultimate goal of this thesis was to identify gene targets of the AHRdbd receptor by performing ChIP-seq and RNA-seq on livers from Ahrwt/wt or Ahrdbd/dbd mice treated with TCDD. I first determined the relative hepatic expression levels of AHR in Ahrwt/wt and Ahrdbd/dbd mice. RT- qPCR was used to quantify expression of Ahr transcripts (Figure 3.4). In vehicle-treated mice, liver Ahr mRNA levels were significantly lower in Ahrdbd/dbd mice than in Ahrwt/wt mice at all time points; specifically, the relative numbers on average were 0.35 versus 1.03 at 2 hours (P ≤ 0.05),

0.34 versus 1.04 at 6 hours (P ≤ 0.01), and 0.32 versus 0.95 at 24 hours (P ≤ 0.05). After treatment with 100 µg/kg TCDD, Ahr expression in Ahrwt/wt mice was variable at 2 hours and 6 hours, but no significant differences were found between Ahrwt/wt and Ahrdbd/dbd mice. However, 24 hours after TCDD treatment Ahrwt/wt mice had significantly higher Ahr mRNA expression than Ahrdbd/dbd mice (P ≤ 0.01). For both Ahrwt/wt and Ahrdbd/dbd mice at all time points, TCDD treatment did not significantly alter the expression of Ahr mRNA.

Western blotting was used to quantify AHR protein levels in the same mice (Figure 3.5).

Similar to the mRNA results, AHR protein levels were significantly lower in DMSO-treated

Ahrdbd/dbd mice than in Ahrwt/wt mice at all time points; specifically, the average numbers were 0.29 versus 1.00 at 2 hours (P ≤ 0.0001), 0.23 versus 0.98 at 6 hours (P ≤ 0.0001), and 0.39 versus 1.14 at 24 hours (P ≤ 0.0001). These differences were not altered at 2 hours after TCDD treatment. In contrast, TCDD treatment reduced AHR protein expression in Ahrwt/wt mice, from 0.98 to 0.39, by

6 hours (P ≤ 0.0001), and from 1.14 to 0.71 after 24 hours (P ≤ 0.01). Due to this reduction, there were no significant differences in AHR protein levels between Ahrdbd/dbd mice and Ahrwt/wt mice treated with TCDD for 6 and 24 hours. For Ahrdbd/dbd mice, no significant differences in AHR protein levels were observed between DMSO and TCDD-treated animals.

Figure 3.4 Hepatic Ahr mRNA levels in Ahrwt/wt and Ahrdbd/dbd mice after treatment with DMSO or 100 µg/kg TCDD for 2, 6, and 24 hours. Data represent mean ± SEM (N = 4). RT- qPCR was done using liver cDNA. Ahr fold expression was calculated using the ΔΔCt method, normalized with Tbp as the reference gene for normalization, and expressed relative to Ahrwt/wt mice treated with DMSO for 2 hours. All meaningful comparisons with statistical significance are indicated (* P ≤ 0.05, ** P ≤ 0.01; two-way ANOVA, Tukey’s multiple comparisons test).

Figure 3.5 Hepatic AHR protein levels in Ahrwt/wt and Ahrdbd/dbd mice after treatment with DMSO or 100 µg/kg TCDD for 2, 6, and 24 hours. The pixel density of protein bands was normalized to β-actin. All normalized AHR/β-actin signals were expressed relative to Ahrwt/wt mice treated with DMSO for 2 hours. A. Data represent mean ± SEM (N = 4). All meaningful comparisons with statistical significance are indicated (** P ≤ 0.01, **** P ≤ 0.0001; two-way ANOVA, Tukey’s multiple comparisons test). B. Representative scanned image of a Western blot.

3.1.4 Comparison of AHRwt and AHRdbd for Activation of AHRE-driven Reporter

This project aimed to use Ahrdbd/dbd mice as a tool to identify genes that AHR regulates indirectly by tethering to other transcription factors, or genes that AHR regulates directly by binding to non-AHRE regions. Thus, it was essential to demonstrate that AHRdbd is unable to regulate gene expression via direct DNA binding to AHREs. To this end, I employed a Cyp1a1- regulated luciferase reporter assay.

COS-1 cells were transfected with pGudLuc 4.1, pCH110, and pEGFP, as well as either empty pcDNA3.1, pcDNA3.1-Ahrwt, or pcDNA3.1-Ahrdbd plasmid. Cells were then treated with either 1×10-7 M TCDD or DMSO vehicle. Three independent experiments were performed.

Luciferase activity was first normalized to β-galactosidase activity, and then the normalized luciferase activity was expressed as a percentage relative to the group exhibiting the highest activity, namely AHRwt with TCDD (Figure 3.6). Compared to empty pcDNA3.1, Ahrwt transfection alone increased luciferase activity from 2.5% to 25.4% (P ≤ 0.0001. TCDD treatment increased luciferase activity of Ahrwt transfected cells from 25.37% to 100.00% (P ≤ 0.0001).

Luciferase activity of Ahrwt transfected cells was higher than Ahrdbd transfected cells, without and with TCDD (P ≤ 0.0001). No significant differences in luciferase activity were observed in the presence of Ahrdbd transfected cells compared to empty pcDNA3.1. TCDD did not affect luciferase activity in Ahrdbd transfected cells.

Western blots were used to confirm that the AHRwt and AHRdbd proteins were equally expressed after transfection. Three independent experiments were done. Pixel density of AHR was normalized with that of β-actin, and then calculated relative to the AHRwt group. Compared to empty pcDNA3.1, both pcDNA3.1-Ahrwt and pcDNA3.1-Ahrdbd increased AHR expression, from

0 to 1.00 and 1.04, respectively (P ≤ 0.05; Figure 3.5A). There was no significant difference between AHRwt and AHRdbd protein expression levels.

Figure 3.6 Relative Cyp1a1-regulated luciferase activity of COS-1 cells transfected with empty pcDNA3.1, pcDNA3.1-Ahrwt, and pcDNA3.1-Ahrdbd plasmids. Data represent mean ± SEM (N = 3). In addition to one of the pcDNA3.1 plasmids, all groups received pGudLuc 4.1, pCH110, and pEGFP. They were then treated with either DMSO or 1×10-7 M TCDD. For each treatment, the luciferase activity was normalized to the corresponding β-galactosidase activity. The normalized luciferase activity was then converted into a percentage, relative to pcDNA3.1-Ahrwt with TCDD. All meaningful comparisons with statistical significance were indicated (**** P ≤ 0.0001; two-way ANOVA, Tukey’s multiple comparisons test).

Figure 3.7 Relative AHR protein expression of COS-1 cells transfected with empty pcDNA3.1, pcDNA3.1-Ahrwt, and pcDNA3.1-Ahrdbd plasmids. In addition to one of the pcDNA3.1 plasmids, all groups received pGudLuc 4.1, pCH110, and pEGFP. Western blot was performed using protein extracted. The pixel density of protein bands was normalized with the corresponding β-actin. The normalized AHR/β-actin signal was expressed relative to the pcDNA3.1-Ahrwt group. A. Data represent mean ± SEM (N = 3). Comparisons with statistical significance were indicated (* P ≤ 0.05; one-way ANOVA, Tukey’s multiple comparisons test). B. A representative scanned image of a Western blot. 67

3.1.5 mRNA Expression of Cyp1a1 and Cyp1b1 in Mouse Liver

To demonstrate that AHRdbd is unable to regulate genes via direct DNA binding to the

AHRE in vivo, the effect of TCDD treatment on hepatic mRNA expression of two canonical AHR target genes, Cyp1a1 and Cyp1b1, was compared in Ahrwt/wt and Ahrdbd/dbd mice. As expected, treatment with 100 µg/kg TCDD increased Cyp1a1 mRNA expression in Ahrwt/wt mice from 1.1 to

2,332 at 2 hours (P ≤ 0.0001), from 3.5 to 25,343 at 6 hours (P ≤ 0.0001), and from 1.5 to 16,318 at 24 hours (P ≤ 0.0001) (Figure 3.8). In contrast, TCDD-treated Ahrdbd/dbd mice did not have significantly different Cyp1a1 mRNA expression compared with the DMSO-treated at any time point. In response to TCDD, Ahrwt/wt mice had higher Cyp1a1 mRNA expression than Ahrdbd/dbd mice at 2 hours (P ≤ 0.0001), 6 hours (P ≤ 0.0001), and 24 hours (P ≤ 0.0001).

TCDD treatment also increased Cyp1b1 mRNA expression in Ahrwt/wt mice from 1.2 to 7.4 at 2 hours (P ≤ 0.01), from 1.1 to 48.5 at 6 hours (P ≤ 0.0001), and from 1.1 to 910.5 at 24 hours

(P ≤ 0.05) (Figure 3.9). Consistent with the regulation of Cyp1a1 mRNA levels, no significant increase was observed in Cyp1b1 mRNA expression in TCDD-treated Ahrdbd/dbd mice compared with the DMSO-treated. In response to TCDD, Ahrwt/wt mice had higher Cyp1b1 mRNA expression levels than Ahrdbd/dbd mice at 6 hours (P ≤ 0.0001) and 24 hours (P ≤ 0.05).

Figure 3.8 Hepatic Cyp1a1 mRNA expression levels in Ahrwt/wt and Ahrdbd/dbd mice after treatment with DMSO or 100 µg/kg TCDD for 2, 6, or 24 hours. Data represent mean ± SEM (N = 4). RT-qPCR was done using liver cDNA. Cyp1a1 fold expression was calculated using the ΔΔCt method, normalized with Tbp as the reference gene for normalization, and expressed relative to Ahrwt/wt mice treated with DMSO. A. 2-hour treatment. B. 6-hour treatment. C. 24-hour treatment. All meaningful comparisons with statistical significance were indicated (**** P ≤ 0.0001; two-way ANOVA, Tukey’s multiple comparisons test).

Figure 3.9 Hepatic Cyp1b1 mRNA expression in Ahrwt/wt and Ahrdbd/dbd mice treated with DMSO or 100 µg/kg TCDD for 2, 6, or 24 hours. Data represent mean ± SEM (N = 4). RT- qPCR was done using liver cDNA. Cyp1b1 fold expression was calculated using the ΔΔCt method, normalized with Tbp as the reference gene for normalization, and expressed relative to Ahrwt/wt mice treated with DMSO. A. 2-hour treatment. B. 6-hour treatment. C. 24-hour treatment. All meaningful comparisons with statistical significance were indicated (* P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001; two-way ANOVA, Tukey’s multiple comparisons test).

3.2 ChIP-seq

ChIP-seq was performed to identify DNA regions where AHR binds indirectly to DNA via tethering to other transcription factors, or directly to DNA sequences that do not contain an AHRE.

Ahrdbd/dbd mice were treated with DMSO vehicle or 100 µg/kg TCDD for 2 hours, and Ahrwt/wt mice were used as a positive control. This 2-hour timepoint was chosen because it is the approximate time required for maximal DNA-binding of AHR, and large numbers of AHR-bound regions have been observed (Dere et al. 2011). Bioinformatic analysis of the ChIP-seq revealed AHR-bound regions in mouse genome and the transcription factor binding sites present in the bound peaks.

3.2.1 ChIP-qPCR

ChIP samples were checked by qPCR prior to submission for sequencing, to confirm that the immunoprecipitation was successful. For the qPCR, primers targeting sequences in the promoters of Cyp1a1 and Cyp1b1, two canonical targets of AHR, were used. Average Ct values of immunoprecipitated and input samples were calculated into percent input, or simply the percentage of the DNA sequence of interest being precipitated by the antibody from an input sample (Figure 3.10). For both Cyp1a1 and Cyp1b1, samples from TCDD-treated Ahrwt/wt mice immunoprecipitated by anti-AHR were significantly higher than the IgG background controls.

They were, on average, 0.5450 versus 0.0068 for Cyp1a1 and 0.9996 versus 0.0060 for Cyp1b1 (P

≤ 0.0001). As expected, for anti-AHR immunoprecipitated samples, TCDD-treated Ahrwt/wt mice had significantly higher signals in both genes than DMSO-treated Ahrwt/wt mice and TCDD-treated

Ahrdbd/dbd mice (P ≤ 0.0001). For Cyp1a1, the average anti-AHR signals of DMSO-treated Ahrwt/wt,

DMSO-treated Ahrdbd/dbd, and TCDD-treated Ahrdbd/dbd were 0.0244, 0.0113, and 0.0098, respectively; for Cyp1b1, they were 0.0217, 0.0085, and 0.0202, respectively. For these three

71 groups of mice, Cyp1a1 and Cyp1b1 signals were not significantly different between anti-AHR antibody and IgG control, indicating low background noise.

Another method to present ChIP-qPCR results is fold enrichment, which is essentially anti-

AHR signal over IgG background for each sample. Since IgG background signals were all extremely low, any slight increase or decrease across different samples would magnify the variation of fold enrichment, and this is the major reason why percent input is often preferred over fold enrichment. Although fold enrichment of TCDD-treated Ahrwt/wt had greater variation than percent input, significance was still found (Figure 3.11). For both Cyp1a1 and Cyp1b1, the fold enrichment of TCDD-treated Ahrwt/wt was significantly higher than that of DMSO-treated Ahrwt/wt and TCDD-treated Ahrdbd/dbd (P ≤ 0.01). For Cyp1a1, the average fold enrichments for DMSO- treated Ahrwt/wt, TCDD-treated Ahrwt/wt, DMSO-treated Ahrdbd/dbd, and TCDD-treated Ahrdbd/dbd were respectively 2.2, 112.4, 1.5, and 1.1. For Cyp1b1, they were respectively 2.2, 211.2, 1.1, and

2.5. For both genes, there were no significant differences among DMSO-treated Ahrwt/wt, TCDD- treated Ahrdbd/dbd, and DMSO-treated Ahrdbd/dbd mice.

Figure 3.10 qPCR analysis of ChIP samples using Cyp1a1 and Cyp1b1 promoters, presented as percent input. Data represent mean ± SEM (N = 4). Ahrwt/wt and Ahrdbd/dbd mice were treated with DMSO or 100 µg/kg TCDD for 2 hours. DNA and protein molecules in liver tissues were crosslinked and sheared. An anti-AHR antibody was used to immunoprecipitate DNA fragments that were directly or indirectly bound to AHR protein, while IgG was used as control. qPCR was done to check the presence of Cyp1a1 and Cyp1b1 promoter sequences in the immunoprecipitated samples. Percent input represents the percentage of the DNA sequence of interest being precipitated by the antibody from a sample. A. Percent input of Cyp1a1. B. Percent input of Cyp1b1. All meaningful comparisons with statistical significance were indicated (**** P ≤ 0.0001; two-way ANOVA, Tukey’s multiple comparisons test).

Figure 3.11 qPCR analysis of ChIP samples using Cyp1a1 and Cyp1b1 promoters, presented as fold enrichment. Data represent mean ± SEM (N = 4). Ahrwt/wt and Ahrdbd/dbd mice were treated with DMSO or 100 µg/kg TCDD for 2 hours. DNA and protein molecules in liver tissues were crosslinked and sheared. An anti-AHR antibody was used to immunoprecipitate DNA fragments that were directly or indirectly bound to AHR protein, while IgG was used as control. qPCR was done to check the presence of Cyp1a1 and Cyp1b1 promoter sequences in the immunoprecipitated samples. Fold enrichment represents the fold increase in signal relative to the IgG background signal. A. Fold enrichment of Cyp1a1. B. Fold enrichment of Cyp1b1. All meaningful comparisons with statistical significance were indicated (** P ≤ 0.01; two-way ANOVA, Tukey’s multiple comparisons test).

For the ChIP-Seq, the library preparation of the anti-AHR and IgG immunoprecipitated

DNA samples was performed by Dr. Somisetty Satheesh in the Matthews laboratory at the

University of Oslo. After library amplification, qPCR was repeated not only to estimate the library yield, but also to confirm that the amplification was linear. In other words, the difference among the treatments should be the same before and after library amplification. Since the input samples did not go through library preparation, the post-amplification qPCR result is presented here as fold enrichment or anti-AHR over IgG, rather than percent input.

As expected, the Cyp1a1 fold enrichment was almost identical between pre- and post- amplification (Figure 3.11A and Figure 3.12). After library amplification, the fold enrichment of

TCDD-treated Ahrwt/wt was significantly higher than that of DMSO-treated Ahrwt/wt and TCDD- treated Ahrdbd/dbd (Figure 3.12; P ≤ 0.001). On average, DMSO-treated Ahrwt/wt, TCDD-treated

Ahrwt/wt, DMSO-treated Ahrdbd/dbd, and TCDD-treated Ahrdbd/dbd were respectively 3.9, 109.3, 4.3, and 2.3. There was no significant difference among DMSO-treated Ahrwt/wt, TCDD-treated

Ahrdbd/dbd, and DMSO-treated Ahrdbd/dbd. After confirming the post-amplification fold enrichment, the libraries were purified by Dr. Somisetty Satheesh and submitted for sequencing at the

Norwegian Sequencing Centre (Oslo, Norway).

Figure 3.12 qPCR analysis of the amplified libraries using Cyp1a1 promoter, presented as fold enrichment. Data represent mean ± SEM (N = 4). The DNA samples immunoprecipitated with anti-AHR and IgG were prepared into libraries by Dr. Somisetty Satheesh in the Matthews Lab at University of Oslo. Libraries were amplified and unique barcodes were added to the samples. Post-amplification qPCR was used to estimate the library yield and confirm the linearity of the amplification. (*** P ≤ 0.001; two-way ANOVA, Tukey’s multiple comparisons test).

3.2.2 AHR-bound Regions

Results from the ChIP-seq were analyzed using the ENCODE Transcription Factor ChIP- seq Processing Pipeline to determine AHR-bound regions, also referred to as peaks. The AHR- bound regions were compared among all groups. A Venn diagram was used to demonstrate common and unique peaks of DMSO-treated Ahrwt/wt mice, TCDD-treated Ahrwt/wt mice, DMSO- treated Ahrdbd/dbd mice, and TCDD-treated Ahrdbd/dbd mice (Figure 3.13A). An UpSet plot was also constructed to represent the same information, since it directly indicates and visualizes the number of genes in every intersection (Figure 3.14).

There were 268 AHR-bound peaks identified in DMSO-treated and 16,853 AHR-bound peaks identified in TCDD-treated Ahrwt/wt mice, with 194 peaks common to both groups. For

Ahrdbd/dbd mice, there were AHR-bound 1,504 peaks with DMSO and AHR-bound 3,689 peaks with TCDD; 862 peaks were common between the groups. For DMSO treatment, Ahrdbd/dbd mice shared AHR-bound 184 peaks with Ahrwt/wt mice. Unique AHR-bound regions identified in Ahrwt/wt and Ahrdbd/dbd were 84 and 1,320, respectively. Upon the TCDD treatment, the number of shared

AHR-bound peaks between the two genotypes was 2,074; the AHR-bound peaks unique to Ahrwt/wt and Ahrdbd/dbd and were 14,779 and 1,615, respectively (Figure 3.13B).

Figure 3.13 Venn diagrams comparing AHR-bound regions determined by ChIP-seq among all groups. DMSO-treated Ahrwt/wt mice had 268 peaks, TCDD-treated Ahrwt/wt mice had 16,853 peaks, DMSO-treated Ahrdbd/dbd mice had 1,504 peaks, and TCDD-treated Ahrdbd/dbd mice had 3,689 peaks. A. Comprehesive diagram comparing all groups. B. Simplified diagram focusing on TCDD- treated Ahrwt/wt mice and TCDD-treated Ahrdbd/dbd mice.

Figure 3.14 UpSet plot comparing AHR-bound regions determined by ChIP-seq among all groups. DMSO-treated Ahrwt/wt mice had 268 peaks, TCDD-treated Ahrwt/wt mice had 16,853 peaks, DMSO-treated Ahrdbd/dbd mice had 1,504 peaks, and TCDD-treated Ahrdbd/dbd mice had 3,689 peaks. On this graph, each row corresponds to a set, while each column corresponds to one intersection in the Venn diagram (Figure 3.13). Filled and connected cells indicate the participation of the set in the intersection, while empty cells mean the set is not in this intersection.

3.2.3 De novo and Enriched Transcription Factor Binding Site Analysis

We first performed a de novo motif analysis on the top 5000 AHR-bound peaks from

TCDD-treated Ahrwt/wt mice and all of the AHR-bound peaks from TCDD-treated Ahrwt/wt mice.

The top 6 motifs for both datasets are shown in Table 3.1. As expected, an AHRE-like motif was present in 3 of the 6 predicted motifs from the AHR-bound peaks from TCDD-treated Ahrwt/wt mice, but completely absent from the TCDD-treated Ahrdbd/dbd mice.

We next performed transcription factor binding site analysis in order to identify transcription factor binding motifs enriched in the AHR-bound regions. The top 10 transcription factor binding motifs ranked by Z-Score are listed in Table 3.2 for DMSO-treated and TCDD- treated Ahrwt/wt mice, and in Table 3.4 for DMSO-treated and TCDD-treated Ahrdbd/dbd mice. TF families are not official gene symbols, but they follow the definition at Genomatix and may include several functionally similar transcription factors. TF Description was provided for information.

As expected, the AHR binding motif was one of the top sites in the AHR-bound regions of

TCDD-treated Ahrwt/wt mice, but not observed in the DMSO treated samples. Moreover, the AHR binding motif was absent from the lists of top 10 for Ahrdbd/dbd mice, even with TCDD treatment.

There were 8 TF families, namely ZF5F, NRF1, HDBP, TF2B, E2FF, CDEF, MTEN, and CTCF, common for all four groups of mice. HNFP and SP1F were the other 2 common in three groups,

DMSO-treated Ahrwt/wt, DMSO-treated Ahrdbd/dbd, and TCDD-treated Ahrdbd/dbd mice. TCDD- treated Ahrwt/wt mice had the above-mentioned AHR and EGRF instead. Each TF family had similar over-representation, the ratio of the actual number of matches to the expected, across all four groups. In addition, transcription factor binding motifs located within 10 to 50 bp of the AHRE were identified for TCDD-treated Ahrwt/wt mice, as they could reveal candidate cooperative transcription factors and tethering partners of AHR. The top 10 are presented on Table 3.3.

Table 3.1 Top 6 de novo motifs discovered in AHR-bound regions of TCDD-treated Ahrwt/wt mice and TCDD-treated Ahrdbd/dbd mice.

De Novo Motif Discovered Known Similar Motifs TCDD-treated Ahrwt/wt mice (AHRE: TnGCGTG) SP2 KLF5 SP1

THAP1 HIC2 NR4A2

TCFL5 NRF1 AHR_ARNT

SP1

KLF14 KLK13 AHR_ARNT

AHR_ARNT

TCDD-treated Ahrdbd/dbd mice

SP2 KLF5 SP1

ZIC3 ZIC1

NR2F2 ESRRB CREM

GATA6 GATA1 GATA2

Table 3.2 Top 10 enriched transcription factor binding sites in AHR-bound regions of DMSO-treated and TCDD-treated Ahrwt/wt mice.

TF TF Description Nr. of Expected Over- Z- Family Matches representation Score DMSO-treated Ahrwt/wt mice ZF5F ZF5 POZ domain zinc finger 1188 48.93 24.28 162.79 NRF1 Nuclear respiratory factor 1 969 57.64 16.81 119.99 HDBP Huntington's disease gene regulatory region binding proteins 322 9.01 35.74 104.08 TF2B RNA polymerase II transcription factor II B 155 2.57 60.31 94.82 E2FF E2F-myc activator/cell cycle regulator 2154 402.32 5.35 87.38 CDEF Cell cycle regulators: Cell cycle dependent element 255 8.81 28.94 82.76 MTEN Core promoter motif ten elements 511 39.14 13.06 75.35 HNFP Histone nuclear factor P 366 36.12 10.13 54.81 CTCF CTCF and BORIS gene family, transcriptional regulators with 883 170.73 5.17 54.49 11 highly conserved zinc finger domains SP1F GC-Box factors SP1/GC 1160 303.49 3.82 49.17 TCDD-treated Ahrwt/wt mice ZF5F ZF5 POZ domain zinc finger 26835 1387.80 19.34 683.14 NRF1 Nuclear respiratory factor 1 23138 1634.84 14.15 531.87 HDBP Huntington's disease gene regulatory region binding proteins 6234 255.69 24.38 373.85 E2FF E2F-myc activator/cell cycle regulator 47634 11411.93 4.17 339.36 TF2B RNA polymerase II transcription factor II B 2893 72.82 39.73 330.42 AHR AHR-ARNT heterodimers and AHR-related factors 19358 2605.03 7.43 328.29 CDEF Cell cycle regulators: Cell cycle dependent element 4691 250.01 18.76 280.84 MTEN Core promoter motif ten elements 10116 1110.29 9.11 270.28 CTCF CTCF and BORIS gene family, transcriptional regulators with 20953 4842.71 4.33 231.58 11 highly conserved zinc finger domains EGRF EGR/nerve growth factor induced protein C & related factors 36684 12451.76 2.95 217.36

Table 3.3 Top 10 enriched transcription factor binding sites within 10 to 50 bp from AHR binding motifs in TCDD-treated Ahrwt/wt mice.

Module with TF Description Nr. of Expected Over- Z- AHR Matches representation Score TCDD-treated Ahrwt/wt mice AHR_ZF5F ZF5 POZ domain zinc finger 14163 339.03 41.78 750.77 AHR_E2FF E2F-myc activator/cell cycle regulator 19041 655.39 29.05 718.19 AHR_NRF1 Nuclear respiratory factor 1 12945 439.07 29.48 596.82 AHR_EGRF EGR/nerve growth factor induced protein C & related 15335 799.28 19.19 514.16 factors AHR_SP1F GC-Box factors SP1/GC 10877 465.14 23.38 482.76 AHR_CTCF CTCF and BORIS gene family, transcriptional 8238 299.34 27.52 458.82 regulators with 11 highly conserved zinc finger domains AHR_KLFS Krueppel like transcription factors 14244 1080.86 13.18 400.4 AHR_ZF02 C2H2 zinc finger transcription factors 2 10455 615.75 16.98 396.51 AHR_BEDF BED subclass of zinc-finger proteins 5834 215.14 27.12 383.05 XCPE_AHR X gene core promoter element 1 3265 76.47 42.70 364.58

Table 3.4 Top 10 enriched transcription factor binding sites in AHR-bound regions of DMSO-treated and TCDD-treated Ahrdbd/dbd mice.

TF TF Description Nr. of Expected Over- Z- Family Matches representation Score DMSO-treated Ahrdbd/dbd mice ZF5F ZF5 POZ domain zinc finger 4497 152.19 29.55 352.19 NRF1 Nuclear respiratory factor 1 3735 179.28 20.83 265.56 HDBP Huntington's disease gene regulatory region binding proteins 1270 28.04 45.29 234.46 TF2B RNA polymerase II transcription factor II B 616 7.99 77.10 214.98 E2FF E2F-myc activator/cell cycle regulator 8442 1251.44 6.75 203.42 CDEF Cell cycle regulators: Cell cycle dependent element 906 27.42 33.04 167.70 MTEN Core promoter motif ten elements 1916 121.76 15.74 162.57 CTCF CTCF and BORIS gene family, transcriptional regulators with 3714 531.06 6.99 138.15 11 highly conserved zinc finger domains HNFP Histone nuclear factor P 1479 112.35 13.16 128.90 SP1F GC-Box factors SP1/GC 4888 944.01 5.18 128.43 TCDD-treated Ahrdbd/dbd mice ZF5F ZF5 POZ domain zinc finger 12280 540.77 22.71 504.85 NRF1 Nuclear respiratory factor 1 9977 637.03 15.66 370.08 HDBP Huntington's disease gene regulatory region binding proteins 3763 99.63 37.77 366.97 TF2B RNA polymerase II transcription factor II B 1712 28.38 60.32 315.97 E2FF E2F-myc activator/cell cycle regulator 23472 4446.76 5.28 285.54 MTEN Core promoter motif ten elements 5507 432.64 12.73 243.96 CDEF Cell cycle regulators: Cell cycle dependent element 2480 97.42 25.46 241.35 CTCF CTCF and BORIS gene family, transcriptional regulators with 10947 1887.00 5.80 208.63 11 highly conserved zinc finger domains HNFP Histone nuclear factor P 4204 399.21 10.53 190.42 SP1F GC-Box factors SP1/GC 13720 3354.35 4.09 179.08

3.3 RNA-seq

RNA-seq was then performed to identify whether ligand-activated AHRdbd, with its mutated DNA-binding domain incapable of binding the AHRE, was able to influence changes in target gene transcription. Ahrwt/wt and Ahrdbd/dbd mice were treated with either 100 µg/kg TCDD or

DMSO vehicle for 6 hours. This time was chosen to capture the initation of AHR-mediated transcription and avoid potential secondary regulations. Bioinformatic analysis identified genes that were regulated differently under the different treatments with Padj < 0.05 (Figures 3.15 and

3.16; Tables 3.5, 3.6, 3.7, and 3.8). The mRNA expression levels of 12 selected genes from the lists were then confirmed using RT-qPCR (Figures 3.17 and 3.18).

3.3.1 Genes Revealed by RNA-seq

Changes in gene expression levels were compared between Ahrdbd/dbd mice and Ahrwt/wt mice (Figure 3.15). There were 410 genes overexpressed in Ahrwt/wt mice compared to Ahrdbd/dbd mice after DMSO treatment. This value increased to 501 with TCDD treatment. There were 171 genes that appeared in both lists. On the other hand, 516 genes had lower expression in DMSO- treated Ahrwt/wt mice than in DMSO-treated Ahrdbd/dbd mice. This value was increased to 566 when comparing TCDD-treated animals. Of these genes, 261 were common to both datasets. The top 25 overexpressed genes and the top 25 repressed genes in DMSO-treated Ahrwt/wt mice relative to

DMSO-treated Ahrdbd/dbd mice are listed in Table 3.5. The top 25 overexpressed genes and the top

25 repressed genes in TCDD-treated Ahrwt/wt mice relative to TCDD-treated Ahrdbd/dbd mice are listed in Table 3.6.

Changes in gene expression levels were compared between TCDD-treated mice and

DMSO-treated mice (Figure 3.16). TCDD treatment induced 58 genes in Ahrwt/wt mice. In contrast,

85 only epidermal growth factor receptor (Egfr), which was not one of the 58, was induced in

Ahrdbd/dbd mice. There were 5 genes downregulated in TCDD-treated Ahrwt/wt mice compared to

DMSO controls, while no gene was downregulated in Ahrdbd/dbd mice. The top 25 upregulated genes and 5 downregulated genes in TCDD-treated Ahrwt/wt mice relative to DMSO-treated Ahrwt/wt mice are listed on Table 3.7. The only gene differently expressed in TCDD-treated Ahrdbd/dbd mice relative to DMSO-treated Ahrdbd/dbd mice is shown on Table 3.8.

A Overall

B Overexpressed C Repressed

Figure 3.15 Overlap of genes differently expressed in Ahrwt/wt mice versus Ahrdbd/dbd mice. A. Genes differently expressed in DMSO-treated Ahrwt/wt mice than in DMSO-treated Ahrdbd/dbd mice (yellow) versus genes differently expressed in TCDD-treated Ahrwt/wt mice than in TCDD-treated dbd/dbd Ahr mice (green). B. Genes overexpressed only. C. Genes repressed only. All genes with Padj < 0.05 were included. The graphs were constructed using InteractiVenn (interactivenn.net) (Heberle et al. 2015).

A Overall

B Upregulated C Downregulated

Figure 3.16 Overlap of genes differently expressed with TCDD versus with DMSO. A. Genes regulated differently upon TCDD treatment in Ahrwt/wt mice (blue) were compared with those in Ahrdbd/dbd mice (purple). B. Genes upregulated upon TCDD treatment. C. Genes downregulated upon TCDD treatment. All genes with Padj < 0.05 were included. The graphs were constructed using InteractiVenn (interactivenn.net) (Heberle et al. 2015).

Table 3.5 Top 25 upregulated genes and top 25 downregulated genes in DMSO-treated Ahrwt/wt mice relative to DMSO-treated Ahrdbd/dbd mice

Gene log2FoldChange Padj Gene log2FoldChange Padj Moxd1 6.79 0.000823 Cyp2a4 -8.44 1.86E-15 Ism2 4.95 0.004058 Tpo -6.55 2.35E-07 Mup9 4.10 7.03E-07 Cyp2b9 -5.83 0.035817 Mup15 4.05 0.026263 Cspg4 -5.66 4.75E-05 Mup12 3.71 3.90E-15 Olfr558 -5.29 0.000545 Mup1 3.56 1.75E-09 Scn2a1 -4.77 0.002324 Serpina12 3.43 7.92E-12 Tcf24 -4.63 1.96E-70 Cyp2c53-ps 3.26 0.027421 Ppp1r42 -4.05 0.000101 Capn8 3.15 1.84E-22 Sema3g -3.75 2.79E-09 Mup7 3.11 1.09E-07 Cyp4a14 -3.63 0.00113 Ces2b 3.06 1.71E-05 Esm1 -3.49 3.34E-38 Orm3 2.81 0.032415 Scn8a -3.23 1.47E-07 Slc22a7 2.55 2.24E-45 Tmprss4 -2.96 0.009449 Mup13 2.51 1.48E-10 Spink1 -2.88 0.013975 Mup14 2.50 4.10E-06 Gsta1 -2.82 0.001678 Serpina3c 2.42 0.01071 Jag2 -2.73 0.02992 Gna14 2.36 9.13E-15 Gucy2c -2.72 3.86E-13 Dct 2.31 1.52E-15 Tff3 -2.62 2.14E-07 Crip3 2.27 0.001117 Nipal1 -2.62 1.20E-07 Ces2c 2.24 2.65E-32 Bmper -2.60 0.001626 Acot11 2.24 4.31E-17 Slc22a26 -2.59 0.020501 Obp2a 2.16 0.000715 Gldn -2.58 0.002644 Rarres1 2.10 6.24E-51 Defb1 -2.55 0.000463 Susd4 2.09 3.12E-38 Hcar2 -2.54 1.12E-13 Cxcl14 2.09 2.83E-10 Lepr -2.50 3.08E-05

Table 3.6 Top 25 upregulated genes and top 25 downregulated genes in TCDD-treated Ahrwt/wt mice relative to TCDD-treated Ahrdbd/dbd mice

Gene log2FoldChange Padj Gene log2FoldChange Padj Cyp1a1 9.07 1.28E-06 Cyp2a4 -6.32 1.84E-07 Ecel1 8.40 0.019092 Tpo -5.92 2.52E-06 Col9a2 5.59 0.001433 Cspg4 -5.11 0.000287 Moxd1 5.29 0.003052 Tcf24 -4.19 8.34E-40 Tnfaip8l3 5.13 2.76E-05 Gldn -4.11 1.88E-05 Nptx1 5.11 0.020227 Sema3g -3.93 1.99E-08 Cyp1a2 4.88 1.32E-11 Tff3 -3.84 1.30E-09 Fabp12 4.48 0.005044 Ppp1r3g -3.73 0.000256 Esrrg 4.28 0.000111 Ppp1r42 -3.63 7.42E-05 Ahrr 4.22 1.04E-05 Esm1 -3.42 2.62E-45 Insl6 3.89 0.000263 Gucy2c -3.41 7.96E-20 Mup9 3.84 4.70E-38 H19 -3.40 8.81E-05 Serpine1 3.78 0.023324 Cux2 -3.38 0.000304 Ptges 3.64 0.000194 Prelid2 -2.96 0.000137 Pcp4l1 3.59 2.55E-07 Lepr -2.96 7.10E-21 Cyp1b1 3.52 0.001699 Mmd2 -2.83 2.11E-15 Lrtm1 3.52 3.01E-13 Bmper -2.71 0.035881 Tiparp 3.34 8.41E-06 Ephx3 -2.67 0.028623 Sun3 3.31 0.039636 Nipal1 -2.66 0.013607 Kcnk1 3.08 1.36E-05 Fam19a2 -2.65 0.00214 4931440P22Rik 3.08 0.000384 Dnaic1 -2.58 0.000125 Pmm1 2.97 0.000461 Atp6v0d2 -2.56 2.94E-08 Fmo3 2.94 0.009544 Pi16 -2.53 0.00119 Slc34a2 2.90 1.90E-06 Cldnd2 -2.44 0.012649 Apol7c 2.76 0.010053 Hcar2 -2.41 3.24E-06

Table 3.7 Top 25 upregulated genes and the only 5 downregulated genes in TCDD-treated Ahrwt/wt mice relative to DMSO-treated Ahrwt/wt mice

Gene log2FoldChange Padj Gene log2FoldChange Padj Cyp1a1 9.91 1.83E-06 Ccnd1 -1.19 0.023284 Serpine1 6.44 0.000311 Kcnk5 -0.86 0.033912 Fabp12 4.76 0.000155 Paqr9 -0.66 0.020976 Ahrr 4.28 0.028766 Zfpm1 -0.58 0.027113 Insl6 4.18 0.004424 Cldn3 -0.57 0.018420 Cyp1b1 3.94 0.006701 4931440P22Rik 3.54 0.004424 Tiparp 3.27 0.001154 Esrrg 3.27 0.033912 Cyp1a2 3.25 0.001154 Nqo1 3.10 0.014395 Cbr3 2.80 0.005041 Selenbp1 2.70 0.005041 Pmm1 2.69 0.014395 Pcp4l1 2.55 0.023284 Rtn4rl2 2.44 0.004691 Osbpl3 2.33 0.005038 Ier3 2.23 0.001154 Ackr3 2.17 0.016481 Myom1 2.04 0.020976 Acpp 1.89 0.016481 Srxn1 1.88 0.044721 Htatip2 1.88 0.043530 Atg9b 1.85 0.022571 Ccno 1.72 0.023023

Table 3.8 The only gene differently expressed in TCDD-treated Ahrdbd/dbd Mice relative to DMSO-treated Ahrdbd/dbd mice

Gene log2FoldChange Padj Egfr 0.32 0.044722

3.3.2 Validation by RT-qPCR

The expression of 12 genes chosen from the RNA-seq lists was confirmed by RT-qPCR

(Figures 3.17 and 3.18). Monooxygenase DBH like 1 (Moxd1), Cyp1a2, and estrogen related receptor gamma (Esrrg) were overexpressed on the list of TCDD Ahrwt/wt versus TCDD Ahrdbd/dbd and the list of DMSO Ahrwt/wt versus DMSO Ahrdbd/dbd; Cyp1a2 and Esrrg were also induced on the list of TCDD Ahrwt/wt versus DMSO Ahrwt/wt. For Moxd1, although its relative expression on average measured by RT-qPCR was much higher in TCDD-treated Ahrwt/wt mice (0.191) than in

TCDD-treated Ahrdbd/dbd mice (0.026), and much higher in DMSO-treated Ahrwt/wt mice (2.473) than in DMSO-treated Ahrdbd/dbd mice (0.016), no statistical significance was found. The expression of Cyp1a2 was higher in TCDD-treated Ahrwt/wt mice (9.761) than in TCDD-treated

Ahrdbd/dbd mice (0.284) with significance (P ≤ 0.0001), higher in DMSO-treated Ahrwt/wt mice

(1.048) than in DMSO-treated Ahrdbd/dbd mice (0.258) without significance, and higher in TCDD- treated Ahrwt/wt mice (9.761) compared with DMSO-treated Ahrwt/wt mice (1.048) with significance

(P ≤ 0.0001). Similarly, the expression of Esrrg was higher in TCDD-treated Ahrwt/wt mice (15.243) than in TCDD-treated Ahrdbd/dbd mice (0.305) with significance (P ≤ 0.0001), higher in DMSO- treated Ahrwt/wt mice (1.039) than in DMSO-treated Ahrdbd/dbd mice (0.363) without significance, and higher in TCDD-treated Ahrwt/wt mice (15.243) compared with DMSO-treated Ahrwt/wt mice

(1.039) with significance (P ≤ 0.0001). Endothelin converting enzyme like 1 (Ecel1) appeared exclusively on the RNA-seq list of TCDD Ahrwt/wt versus TCDD Ahrdbd/dbd as an overexpressed gene. RT-qPCR indicated its expression was significantly higher in TCDD-treated Ahrwt/wt mice

(360.038) compared with TCDD-treated Ahrdbd/dbd mice (1.400) (P ≤ 0.001), but also significantly higher in TCDD-treated Ahrwt/wt mice (360.038) than in DMSO-treated Ahrwt/wt mice (1.702) (P ≤

0.001). Serpin family E member 1 (Serpine1) was overexpressed on the list of TCDD Ahrwt/wt

91 versus TCDD Ahrdbd/dbd, but repressed on the list of DMSO Ahrwt/wt versus DMSO Ahrdbd/dbd, and did not appear on the list of TCDD Ahrwt/wt versus DMSO Ahrwt/wt. With RT-qPCR, its expression was significantly higher in TCDD-treated Ahrwt/wt mice (173.129) than in TCDD-treated Ahrdbd/dbd mice (6.825) (P ≤ 0.01), non-significantly lower in DMSO-treated Ahrwt/wt mice (0.600) than in

DMSO-treated Ahrdbd/dbd mice (2.984), but significantly higher in TCDD-treated Ahrwt/wt mice

(173.129) compared with DMSO-treated Ahrwt/wt mice (0.600) (P ≤ 0.01).

According to the RNA-seq data, Cyp2a4, Thyroid peroxidase (Tpo), and Chondroitin sulfate proteoglycan 4 (Cspg4) were repressed in the TCDD Ahrwt/wt versus TCDD Ahrdbd/dbd and in the DMSO Ahrwt/wt versus DMSO Ahrdbd/dbd samples. For Cyp2a4 RT-qPCR, one outlier with extremely high expression was excluded from each of DMSO-treated Ahrdbd/dbd mice and TCDD- treated Ahrdbd/dbd mice. Cyp2a4 expression was lower in TCDD-treated Ahrwt/wt mice (2.447) compared with TCDD-treated Ahrdbd/dbd mice (310.040) without significance, while significantly lower in DMSO-treated Ahrwt/wt mice (2.051) than in DMSO-treated Ahrdbd/dbd mice (719.429) (P

≤ 0.01). Tpo expression was significantly lower in TCDD-treated Ahrwt/wt mice (1.346) compared with TCDD-treated Ahrdbd/dbd mice (78.990) (P ≤ 0.01), and also significantly lower in DMSO- treated Ahrwt/wt mice (1.028) than in DMSO-treated Ahrdbd/dbd mice (93.298) (P ≤ 0.01). Similarly,

Cspg4 expression was significantly lower in TCDD-treated Ahrwt/wt mice (0.750) compared with

TCDD-treated Ahrdbd/dbd mice (4.279) (P ≤ 0.05), and also significantly lower in DMSO-treated

Ahrwt/wt mice (1.124) than in DMSO-treated Ahrdbd/dbd mice (5.677) (P ≤ 0.01). Cut like homeobox

2 (Cux2) appeared exclusively on the RNA-seq list of TCDD Ahrwt/wt versus TCDD Ahrdbd/dbd as a repressed gene. Although it was much less expressed in TCDD-treated Ahrwt/wt mice (0.607) than in TCDD-treated Ahrdbd/dbd mice (6.258), no significance was found with RT-qPCR.

Based on the RNA-seq data, Ahrr and Tiparp were among the most strongly induced genes in TCDD-treated Ahrwt/wt mice relative to DMSO-treated Ahrwt/wt mice. They were both overexpressed in TCDD-treated Ahrwt/wt mice relative to TCDD-treated Ahrdbd/dbd mice, and both were not present on the list of DMSO-treated Ahrwt/wt mice relative to DMSO-treated Ahrdbd/dbd mice. RT-qPCR showed that Ahrr expression was significantly higher in TCDD-treated Ahrwt/wt mice (23.254) compared with both DMSO-treated Ahrwt/wt mice (1.021) (P ≤ 0.01) and TCDD- treated Ahrdbd/dbd mice (1.214) (P ≤ 0.01). Similarly, Tiparp expression was also significantly higher in TCDD-treated Ahrwt/wt mice (11.280) compared with both DMSO-treated Ahrwt/wt mice

(1.025) (P ≤ 0.0001) and TCDD-treated Ahrdbd/dbd mice (0.907) (P ≤ 0.0001).

RNA-seq revealed Egfr as the only gene on the list of TCDD Ahrdbd/dbd versus DMSO

dbd/dbd Ahr with a log2FoldChange of 0.32 and a Padj of 0.045. It was observed as repressed on the

wt/wt dbd/dbd list of TCDD Ahr versus TCDD Ahr with a log2FoldChange of -0.42 and a Padj of 0.006.

RT-qPCR analysis revealed that its expression was significantly lower in TCDD-treated Ahrwt/wt mice (0.727) than in TCDD-treated Ahrdbd/dbd mice (1.233) (P ≤ 0.01). Egfr expression was higher in TCDD-treated Ahrdbd/dbd mice (1.233) than in DMSO-treated Ahrdbd/dbd mice (0.982) with very low variation, but no statistical significance was observed with Tukey’s test, while two-tailed unpaired t-test exclusively focusing on these two groups found a significance (P ≤ 0.05).

Figure 3.17 mRNA expression confirmed by RT-qPCR of genes selected from the RNA-seq result – Part one. Data represent mean ± SEM (N = 4). Fold expression for every gene was calculated using the ΔΔCt method, normalized with Tbp as the reference gene for normalization, and expressed relative to DMSO-treated Ahrwt/wt mice. All meaningful comparisons with statistical significance were indicated (** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001; two-way ANOVA, Tukey’s multiple comparisons test). For Cyp2a4, one outlier with extremely high expression was excluded from each of DMSO-treated Ahrdbd/dbd and TCDD-treated Ahrdbd/dbd.

Figure 3.18 mRNA expression confirmed by RT-qPCR of genes selected from the RNA-seq result – Part two. Data represent mean ± SEM (N = 4). Fold expression for every gene was calculated using the ΔΔCt method, normalized with Tbp as the reference gene for normalization, and expressed relative to DMSO-treated Ahrwt/wt mice. All meaningful comparisons with statistical significance were indicated (* P ≤ 0.05, ** P ≤ 0.01, and **** P ≤ 0.0001; two-way ANOVA, Tukey’s multiple comparisons test). For Egfr, Tukey’s test did not find a significant difference between DMSO-treated Ahrdbd/dbd and TCDD-treated Ahrdbd/dbd, but two-tailed unpaired t-test focusing on these two groups found a significance at P ≤ 0.05.

Chapter 4 Discussion

Ahrdbd/dbd mice, expressing an AHR protein that is unable to bind to AHREs, are a powerful tool to investigate whether AHR is able to regulate gene transcription via tethering to other transcription factors or binding to non-AHRE regions. To ensure that the Ahrdbd/dbd mice carry and express the expected allele, the Ahr gene was sequenced and its expression was verified by RT- qPCR and Western blotting. To ensure that the AHRdbd lacked the ability to regulate genes via direct DNA-binding to AHREs, in vitro Cyp1a1-regulated luciferase reporter assays and TCDD- induced Cyp1a1 and Cyp1b1 mRNA levels in Ahrdbd/dbd mice were tested. ChIP-seq was then performed to identify AHRdbd-bound regions in the mouse genome, and RNA-seq was performed to identify genes that are regulated by ligand-activated AHRdbd.

4.1 The Impact of AHR on Reproduction, Fertility and Sex Ratio.

As described in Chapter 1.2.3, AHR activation by TCDD is known to disrupt normal reproduction and in utero development. In addition, specific spatial and temporal expression of

AHR was found in developing mouse embryos (Abbott and Probst 1995). Since Ahr-/- mice were generated, their breeding capability had attracted attention. The Ahrwt/- heterozygous breeding pairs of the Ahr-/- mouse line generated by the group of Dr. Fernandez-Salguero produced offspring with the Mendelian ratio, but between 40% to 50% Ahr-/- homozygous mutants died or were selectively cannibalized by 4 days of age (Fernandez-Salguero et al. 1995). Another group made a different observation on this same mouse line: Ahr-/- homozygous pups had a 16% death rate during the 2- week lactation period and a 26.5% death rate within 2 weeks after weaning (Abbott et al. 1999).

In contrast, another two Ahr-/- mouse lines generated by different groups have been reported to produce pups at the expected Mendelian ratio (Schmidt et al. 1996; Mimura et al. 2003).

In this study, we obtained 559 pups from heterozygous Ahrwt/dbd breeders that survived their first week of life, offering us an opportunity to assess the strain’s reproduction on a scale unprecedented for all Ahr-/- or Ahrdbd/dbd mouse lines. Since activated AHR disrupts hormone signalling, including estrogen receptor pathways, mutations on Ahr could possibly impact the sex ratio of offspring. However, the sex ratio was normal (Figure 3.1). Interestingly, regardless of the sex, the relative frequencies of Ahrwt/wt, Ahrwt/dbd, and Ahrdbd/dbd were approximately 24%, 59%, and 17%, statistically different from the Mendelian ratio (Figure 3.2). Unfortunately, since the newborns were first counted at 1 week of age and genotyped at 2 weeks according to the general rules at our animal facility, we do not know whether the decreased frequency of Ahrdbd/dbd homozygotes is due to reduced births or increased neonatal death. Parental cannibalism and deaths from unknown causes before 1 week of age were frequently observed by veterinary technicians.

The group of Dr. Bradfield, who originally generated this mouse line, have reported the relative frequencies of Ahrwt/wt, Ahrwt/dbd, and Ahrdbd/dbd at birth to be 27%, 52%, and 21%, closer to the

Mendelian ratio (Bunger et al. 2008). Therefore, it is possible that our Ahrdbd/dbd homozygotes were initially born at slightly less than the expected 25%, with neonatal deaths from selective cannibalism or other unknown causes further reducing the homozygote frequency. This is similar to Dr. Fernandez-Salguero’s Ahr-/- homozygotes, which were born in accordance with a Mendelian ratio but either died of unknown causes or were selectively cannibalized within 4 days.

Postnatal deaths of the Ahr-/- pups from Dr. Fernandez-Salguero’s mouse line were found to be unrelated to the genotype of the female breeders, and thus unrelated to maternal factors such as lactation and nursing behavior (Abbott et al. 1999). Necropsy showed that dead Ahr-/- pups had lymphocyte infiltration in various organs and tissues, especially the gastrointestinal tract, urinary tract, and lung (Fernandez-Salguero et al. 1995). Therefore, the poor survival of pups with Ahr

97 mutations is likely related to abnormal immune responses in these animals. Since the Ahrdbd/dbd mouse line demonstrated a similarly high incidence of neonatal deaths, the AHRE-binding capability and hence the canonical pathway of AHR is implicated in this phenomenon.

4.2 The Genotype and Functionality of AHRdbd

The Ahr genes in the Ahrdbd/dbd mice and Ahrwt/wt littermates were sequenced, and their expression was assessed at mRNA and protein levels. The Ahrdbd gene matched perfectly with the

Ahrd allele from the 129/SvJ strain except for one three-nucleotide substitution and one six- nucleotide insertion which were intentionally delivered to disable the DNA-binding domain

(Figure 3.3). This is consistent with the original design and generation of the Ahrdbd/dbd mice, comprising homologous recombination induced in the ES1 stem cells from the 129/SvJ strain and backcrossing with the C57BL/6J strain (Bunger et al. 2008). However, the fact that Ahrwt/wt littermates carry the Ahrb1 allele from the C57BL/6J strain can cause complications for interpreting differences between the two genotypes. As previously mentioned, the AHRd protein is less responsive to ligand activation than the AHRb1 protein (Thomas et al. 2002). To overcome this,

TCDD was given to Ahrdbd/dbd mice and Ahrwt/wt littermates at a dose of 100 µg/kg throughout this study. This dose is sufficient to fully activate the less responsive AHRd, so that the difference between the ligand activation of AHRd and AHRb1, meaning AHRdbd and AHRwt, is minimized

(Nebert et al. 2004). A dose-response study done in DBA/2 and C57BL/6 mice has shown that the function of both AHRd and AHRb1, measured as specific CYP1A1 activity, reached a plateau at

100 µg/kg TCDD (Niwa et al. 1975; Nebert et al. 2004).

In order to confirm that the AHRdbd protein does not have the ability to regulate the canonical downstream genes by binding the AHRE, luciferase reporter assays were used to

98 compare AHRdbd and AHRwt activities in vitro. Ahrdbd and Ahrwt genes were cloned into the pcDNA3.1 plasmid, and transfected into the COS-1 cells along with pGudLuc 4.1, an AHR-driven reporter construct with a Cyp1a1 promoter. In the presence or absence of TCDD, cells receiving

Ahrdbd demonstrated relative luciferase activities at around 2%, not different from those of the negative control (Figure 3.6). Cells receiving Ahrwt were at 25.37% with DMSO and 100.00% with

TCDD (Figure 3.6). These results are consistent with previous studies in 3T3 cells, although the activities of their AHRdbd and negative control with and without TCDD were around 10% of that observed in TCDD-treated AHRwt (Bunger et al. 2008). As an explanation for the high activity of

AHRwt even without TCDD, COS-1 and 3T3 cells are both Ahr-/-, so that the presence of any AHR activity upon transfection would be prominent. Since there is no AHR in these cell lines, they cannot upregulate metabolizing enzymes downstream of AHR to deplete natural AHR ligands existing in the culture medium, and these ligands may become activators once functional AHR is expressed.

The in vivo function of the AHRdbd protein was also checked by assessing hepatic mRNA expression of two prototypical AHR targets, Cyp1a1 and Cyp1b1, following TCDD injection. At

2, 6, and 24 hours, the induction of Cyp1a1 in Ahrwt/wt mice was orders of magnitude higher compared with that of Ahrdbd/dbd mice (Figure 3.8). For Cyp1b1, significant difference started at 6 hours with 48.52 versus 2.28, and peaked at 24 hours with 910.50 versus 1.83 (Figure 3.9). Cyp1b1 has high basal expression, while Cyp1a1 is negligible (Nebert et al. 2004), which partly explains the substantial fold induction of Cyp1a1 compared to Cyp1b1. In summary, the luciferase assay and RT-qPCR confirmed in vitro and in vivo that the AHRdbd is unable to regulate its canonical targets by direct DNA binding. This information is essential to ensure that any DNA-bound regions detected by ChIP-seq and any differentially regulated genes revealed by RNA-seq in Ahrdbd/dbd

99 mice would be due to AHR tethering to cooperative transcription factors or through binding to non-AHRE DNA sequences.

4.3 AHR Expression and Conjectural Self-regulation

AHR expression in mice was checked by RT-qPCR and Western blot. The primary goal was to confirm the AHRdbd protein is expressed in Ahrdbd/dbd mice, which is a precondition for the

ChIP experiment. Western blot analyses confirmed AHR expression in both genotypes (Figure

3.5). Interestingly, for all timepoints, DMSO-treated Ahrdbd/dbd mice expressed approximately 3 times less AHR compared with DMSO-treated Ahrwt/wt littermates at both mRNA and protein levels with statistical significance (Figures 3.4 and 3.5). Because of the similar difference for both mRNA and protein, the reduced AHR expression is likely to be caused by transcriptional regulation, not translational. The current data are not able to determine whether this difference is due to the disabled DNA-binding domain or due to the pre-existing allelic difference between Ahrd and Ahrb1.

There are two AHRE motifs contained in the promoter region of mouse Ahr gene, indicating that

AHR may either upregulate itself to amplify the induction of drug metabolizing enzymes for protection, or downregulate itself to avoid overactivity (Garrison and Denison 2000). In one study,

SKH1 mice used in skin research were selectively crossed to bear either Ahrd or Ahrb1; although quantification and statistics were not performed, SKH1-Ahrd mice express less AHR than SKH1-

Ahrb1 mice. However, this difference was far less than that previously reported between Ahrdbd/dbd and Ahrwt/wt mice (Smith et al. 2018). It is likely that without exogenous ligand treatment, AHR upregulates itself, so that the less responsive AHRd exerts a smaller effect than AHRb1. Thus, an explanation for the reduced AHR expression of Ahrdbd/dbd mice is that AHRdbd fails to upregulate its own expression. In addition, the pre-existing allelic difference between Ahrdbd and Ahrwt may

100 also be a factor, as the AHR expression of Ahrwt/wt mice would probably be less if they expressed

Ahrd instead of Ahrb1. Admittedly, the difference of AHR expression between Ahrdbd/dbd and Ahrwt/wt mice may influence the comparison of ChIP-seq results. Nonetheless, with a high 100 µg/kg

TCDD treatment, any indirect DNA binding of AHRdbd would be easily detected if it exists.

The AHR expression study revealed another interesting phenomenon about the downregulation of AHR in response to TCDD. Western blot studies demonstrate that AHR expression decreased significantly at 6 hours after TCDD treatment in Ahrwt/wt mice. This decrease was reduced by 24 hours, as the proteins levels recovered slightly (Figure 3.5). This is consistent with experiments done in Sprague-Dawley rats treated with 10 µg/kg [3H]TCDD (Pollenz 1998).

AHR downregulation following TCDD exposure has been attributed to ubiquitin-mediated degradation through the 26S proteasome (Pollenz 2002). Ahrdbd/dbd mice, on the other hand, did not show any sign of AHR downregulation 6 and 24 hours after 100 µg/kg TCDD injection (Figure

3.5). This suggests that the DNA-binding capability is essential for the eventual downregulation of TCDD-activated AHR. These data also agree with a previous report showing that Ahr-defective mouse hepatoma cells transfected with an Ahr unable to bind DNA had less AHR downregulation than cells transfected with wildtype Ahr following TCDD treatment (Ma and Baldwin 2000).

Moreover, Ahr in Ahrwt/wt mice was not significantly reduced at the mRNA level following TCDD treatment, supporting the established ubiquitin-proteasome mediated degradation of AHR (Figure

3.4) (Ma and Baldwin 2000; Pollenz 2002). However, there seemed to be a non-significant trend for lower Ahr mRNA in Ahrwt/wt mice after 2 and 6 hours of TCDD injection, suggesting a possibility for transcriptional regulation to slightly contribute (Figure 3.4). Nonetheless, the AHR self-regulation suggests that under physiological conditions with only the weak endogenous ligands, AHR upregulates itself to better exert its function, whereas under toxicological stresses

101 with strong exogenous or synthetic ligands, AHR downregulates itself to dampen down adverse effects.

4.4 AHR-bound Regions Revealed by ChIP-seq

The major objective of this study was to demonstrate whether AHR regulates gene expression via tethering to other transcription factors, or independent from its canonical direct

DNA binding to AHREs. To this end, we performed ChIP-seq studies with Ahrdbd/dbd mice to identify AHR-bound regions.

First, qPCR was performed to verify the quality of the ChIP samples before sequencing.

The abundance of the promoter regions of Cyp1a1 and Cyp1b1, two prototypical AHR canonical targets, were compared across anti-AHR and IgG immunoprecipitated samples from DMSO- treated Ahrwt/wt mice, TCDD-treated Ahrwt/wt mice, DMSO-treated Ahrdbd/dbd mice, and TCDD- treated Ahrdbd/dbd mice. Strong signals were detected in the anti-AHR immunoprecipitated DNA of

TCDD-treated Ahrwt/wt mice, and they were significantly higher than the signals of the IgG background control (Figure 3.10). The fold enrichments of AHR recruitment to Cyp1a1 and

Cyp1b1 after TCDD-treatment in Ahrwt/wt mice were more than 100 times higher than those of

Ahrdbd/dbd mice (Figure 3.11). These differences were maintained after library preparation and linear amplification (Figure 3.12). These results indicate that the ChIP procedure was successful in obtaining highly enriched AHR-bound DNA fragments with minimal non-specific background.

Since the AHR-bound regions from the TCDD-treated Ahrdbd/dbd mice did not contain Cyp1a1 and

Cyp1b1, this supports the data that AHRdbd is unable to directly bind AHRE containing DNA sequences.

102

A Venn diagram and an UpSet plot were constructed to compare all groups (Figures 3.13 and 3.14). There were 268 and 16,853 AHR-bound peaks identified in DMSO- and TCDD-treated

Ahrwt/wt mice, respectively. The 268 AHR-bound peaks may represent the low level of AHR action by natural endogenous AHR ligands (Denison and Nagy 2003). As expected, TCDD treatment drastically increased the number of AHR-bound peaks. There were 194 AHR-bound peaks that were common to both DMSO and TCDD treatments, and these represented 72% of AHR-bound regions with DMSO treatment. Although the majority of peaks in DMSO-treated Ahrwt/wt mice also appeared with TCDD treatment, 28% of them were bound by AHR only in the absence of TCDD.

In other words, a considerable portion of the DNA regions bound to AHR may be the result of activation by endogenous ligands and represent distinct gene targets from TCDD-activated AHR.

Gene microarray studies revealed that differential gene expression profiles are induced by different

AHR ligands (Ovando et al. 2010; Hrubá et al. 2011; Goodale et al. 2013). Therefore, AHR ligands with different structures may induce minor conformational changes in AHR, which allow activated

AHR to recognize different DNA sequences or interact with different coactivators in a ligand- specific manner (Soshilov and Denison 2014). It would be interesting to further analyze the 28% of AHR-bound regions in DMSO-treated Ahrwt/wt mice, since they may be important mediators of beneficial outcomes from the consumption of foods containing natural AHR ligands, such as cruciferous vegetables (Matthews and Ahmed 2013).

Interestingly, 1,504 AHR-bound peaks were identified in liver tissue isolated from DMSO- treated Ahrdbd/dbd mice. Ahrdbd mice express the Ahrd allele with mutations disabling its DNA- binding, while Ahrwt is the unmutated Ahrb1. Compared with AHRd, AHRb1 is 43 amino acids shorter. An explanation for the increased peaks in Ahrdbd/dbd mice than in Ahrwt/wt mice could be that the different amino acid sequence and length of AHRd may enable it to interact with more

103 coactivators or tethering partners, and thus bind to more DNA regions. Since the C-terminus of

AHR is known to be important for coactivator binding, a longer sequence may result in increased or differential interactions with coactivators or other transcriptional regulators. The function of these 43 amino acids has not been extensively studied, so it is not known whether they allow substantial coactivator binding or transcription factor tethering. However, if this is true, then the unmutated AHRd, with a functional DNA-binding domain, should bind to more DNA regions than

AHRdbd. Also, although AHR was expressed less in Ahrdbd mice than in Ahrwt/wt mice, the fact that

AHRdbd protein is constitutively localized in nucleus may contribute to its increased bound regions compared to AHRwt as well (Figures 3.4 and 3.5) (Bunger et al. 2008). Another possibility is that the mutated DNA binding domain alters the conformation of AHR, allowing it to interact with more transcripton factors and thus bind to more DNA sequences than AHRwt in the absence of

TCDD. The I25G point mutation is located within and the GS insertion is located at the end of the basic region of AHR, which is known to exclusively convey DNA binding, rather than in the basic

HLH region which is involved in protein-protein interactions (Figure 1.1) (Bunger et al. 2008).

Therefore, it is unclear whether the DNA binding domain mutation increases protein-protein interactions. Further investigations are required to elucidate this issue.

4.5 Transcription Factor Binding Sites Enriched in AHR-bound Regions

De novo motif analysis of the AHR-bound regions revealed by ChIP-seq found the AHRE sequence, 5’-TnGCGTG-3’, in the AHR-bound regions of TCDD-treated Ahrwt/wt mice, but not

TCDD-treated Ahrdbd/dbd mice, confirming the inability of AHRdbd to bind the AHRE (Table 3.1).

GC-rich binding motifs were highly overrepresented in Ahrwt/wt mice, indicating the promoter- centric binding preference of AHR. The de novo motifs found in Ahrdbd/dbd mice showed similarity

104 with motifs of other transcription factors, such as SP1 and ESRRB, representing candidate partners for AHR tethering.

The AHR-bound regions were also analyzed for enriched transcription factor binding sites.

The top 10 TF families for all groups are listed on Tables 3.2 and 3.4. AHR-bound regions of the positive control, TCDD-treated Ahrwt/wt mice, were very similar to published ChIP-seq results obtained from TCDD-treated MCF-7 cells; all the 89 TF families in MCF-7 cells were included in the 215 TF families of Ahrwt/wt mice (Lo and Matthews 2012). Our ChIP-seq results may have even better quality, since the Z-score and over-representation of TF families were in many cases higher than the previous MCF-7 cell results (Lo and Matthews 2012).

A notable feature of our data is that TCDD-treated Ahrwt/wt mice and TCDD-treated

Ahrdbd/dbd mice had many more matches than their DMSO-treated counterparts. This corresponds to the numbers of AHR-bound peaks detected in each group, and again demonstrates the massive mobilization of AHR to the genome in response to TCDD. As expected, the TF family AHR appeared on the list of top 10 for TCDD-treated Ahrwt/wt mice, but not for any other group.

Interestingly, the AHR motif only ranked 6th, less than five other TF families on this list, suggesting that other transcription factors may also play prominent roles in AHR function. Notably, with the exception of the AHR, almost all other top enriched TF families appeared with similar rankings and over-representations across all four groups, regardless of being Ahrwt/wt or Ahrdbd/dbd.

Importantly, this suggests the enrichment of all TF families other than the AHR is highly likely to be caused by AHR tethering to these transcription factors and the resultant indirect DNA-binding.

Also, the DNA-binding domain mutation of AHR may not have resulted in apparent conformational changes that would create new interactions with transcription factors.

105

In addition, focusing on TCDD-treated Ahrwt/wt mice, analysis was performed to find out the transcription factor binding motifs located within 10 to 50 bp from AHR motifs (Table 3.3).

Again, the result from TCDD-treated Ahrwt/wt mice was highly similar to that of MCF-7 cells, since

188 out of the 190 TF families in MCF-7 cells were included in the 214 TF families in Ahrwt/wt mice (Lo and Matthews 2012). The overrepresentative sites of TF families identified in this way provide strong support for AHR tethering to other transcription factors and indirect DNA-binding.

The TF families, except AHR, that appeared at least once in all the abovementioned lists were ZF5F, NRF1, E2FF, CDEF, CTCF, EGRF, SP1F, KLFS, ZF02, BEDF, and XCPE. Among them, XCPE (X gene core promoter element 1) is a core promoter element important for transcription by RNA polymerase II mediating mRNA synthesis (Tokusumi et al. 2007; Carter and

Drouin 2009). The others, including E2FF (E2F-myc activator/cell cycle regulator), CDEF (Cell cycle regulators: Cell cycle dependent element), CTCF (CTCF and BORIS gene family, transcriptional regulators with 11 highly conserved zinc finger domains), EGRF (EGR/nerve growth factor induced protein C & related factors), SP1F (GC-Box factors SP1/GC), KLFS

(Krueppel like transcription factors), and ZF02 (C2H2 zinc finger transcription factors 2) are families of transcription factors involved in the regulation of cell proliferation, growth, differentiation, or apoptosis, and are thus also related to embryonic development and carcinogenesis (Sukhatme 1990; Attwooll et al. 2004; Safe and Abdelrahim 2005; Xie et al. 2007;

Matsumura 2009; Müller and Engeland 2010; McConnell and Yang 2010; Martin-Kleiner 2012).

EGRF, and BEDF (BED subclass of zinc-finger proteins) include transcription factors involved in or at least associated with physiology or pathology of the nervous system (Poirier 2008; Nyegaard et al. 2010). These TF families may influence AHR’s role in cell cycle regulation, development,

106 and cancer described in Chapters 1.2.3, 1.3.3, and 1.3.4, as well as its function in the nervous system (Juricek and Coumoul 2018).

The transcription factor, ZF5, from the ZF5F TF family (ZF5 POZ domain zinc finger) is probably best known as a repressor of the fragile X-mental retardation 1 (Fmr1) gene (Orlov et al.

2007). Mutations in Fmr1 impair female reproductivity, causing premature ovarian failure and primary ovarian insufficiency (Pastore and Johnson 2014). This could be associated with the fact that Ahr-/- females have reduced success in maintaining conceptuses and surviving pregnancy; their litter size is smaller and the survival rate of pups is lower than the wildtype mothers (Abbott et al.

1999). ZF5 is also a transcriptional activator of the dopamine transporter (DAT) (Lee et al. 2004).

It may relate to the ability of TCDD to increase dopamine levels in different regions of brains

(Juricek and Coumoul 2018). Lastly, researchers have used computational methods to look for transcription factors involved in adipogenesis; ZF5 and AHR, along with other transcription factors in the top 10 TF families, including CTCF, EGR1, E2F, KLF4, and SP1 were all identified in this study (Ambele and Pepper 2017). Thus, the cooperation of AHR with these other transcription factors may be important in the wasting syndrome of TCDD-treated rodents introduced in Section 1.2.3.

Transcription factor NRF1 is known for regulating metabolic genes, similar to AHR. A study using Nrf2-/- mice found that nuclear respiratory factor 2 (NRF2), another member of the same family, is required for TCDD-induced expression of many drug metabolizing enzymes, including isoforms of glucuronosyltransferases and glutathione-S-transferase (Yeager et al. 2009).

Compared with NRF2, NRF1 exhibits high structural similarity, recognizes the same DNA- binding element, and has functional overlaps (Leung et al. 2003). The cooperation of AHR with

NRF1 may also likely have important implications in the expression of metabolizing enzymes.

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For all of the abovementioned TF families, it is important to note that their enriched presence in AHR-bound regions may or may not merely be a reflection of sharing common targets and regulating common processes compared with AHR. The transcription factor binding site analysis on AHR-bound regions does not necessarily mean that an interaction occurs between

AHR and these other transcription factors. In addition, even if a physical interaction or tethering exists between AHR and these other transcription factors, it may or may not cause alterations in gene transcription.

4.6 Alteration in Gene Expression Revealed by RNA-seq

ChIP-seq revealed AHR-bound regions across the genome of Ahrdbd/dbd mice and the transcription factor binding sites contained in those peaks, suggesting the presence of AHR tethering to other transcription factors and its indirect binding to DNA. To investigate the impact of this indirect binding on gene transcription, RNA-seq was performed using Ahrdbd/dbd mice and

Ahrwt/wt littermates treated with 100 µg/kg TCDD or DMSO vehicle for 6 hours. This time point was chosen with the intention of identifying direct AHR target genes. RT-qPCR was used to check the expression of 12 selected genes from the RNA-seq result.

Overall, results from the RT-qPCR analysis supported the RNA-seq data (Figures 3.17 and

3.18). However, for some genes statistical significance was not established. For example, RNA- seq showed that Moxd1 was overexpressed on the list of TCDD Ahrwt/wt versus TCDD Ahrdbd/dbd and the list of DMSO Ahrwt/wt versus DMSO Ahrdbd/dbd, but no statistical significance was found by

RT-qPCR. It has been recognized that RNA-seq does not always agree with the traditional RT- qPCR, as one study found about 80% exon-exon junctions found by RNA-seq were validated by

RT-qPCR (Su et al. 2014). It is also possible for RNA-seq to count one long mRNA molecule

108 more than once if it results in over one matching read, while RT-qPCR always counts one mRNA only once. Considering that most comparisons identified by RNA-seq were validated by RT-qPCR with significance and other non-significant ones clearly showed the same trends, the RNA-seq result is trustworthy overall.

The intention of the RNA-seq analysis was to identify specific gene targets regulated by

TCDD in the Ahrdbd/dbd mice. If AHRdbd changes gene expression upon ligand activation, it would suggest the indirect DNA-binding of AHR via tethering to other transcription factors indeed affects gene transcription. Even though the number of AHR-bound regions was higher in Ahrdbd/dbd mice upon TCDD treatment, only one gene, Egfr, was found to be differently expressed in TCDD

dbd/dbd dbd/dbd Ahr versus DMSO Ahr with a low log2FoldChange of 0.32 and a high Padj of 0.045

(Table 3.8). RT-qPCR showed a slight difference between DMSO-treated Ahrdbd/dbd (0.982) and

TCDD-treated Ahrdbd/dbd (1.233). This comparison was not significant with Tukey’s test comparing

Egfr expression among all four groups of mice, but was significant with t-test focusing on DMSO- and TCDD-treated mice only (P ≤ 0.05) (Figure 3.18). As only one gene has slightly influenced mRNA expression, the ChIP-seq and the RNA-seq together suggest that although AHR may indirectly bind to many regions in genome via tethering to other transcription factors, this indirect binding may not cause much alteration in gene transcription.

If AHR tethering does not affect gene transcription, then Ahrdbd/dbd mice should be physiologically identical to Ahr-/- mice. One study performed RNA expression array on 10-week- old male Ahr-/- mice injected with 1,000 µg/kg TCDD or corn oil vehicle for 19 hours (Tijet et al.

2006). Our RNA-seq result in Ahrdbd/dbd mice is generally consistent with their result. There were

926 genes either more or less expressed in Ahrwt/wt mice than in Ahrdbd/dbd mice with DMSO vehicle with Padj < 0.05, while the number was 1067 with TCDD treatment; only 435 genes were

109 commonly affected without and with TCDD (Figure 3.15). This was consistent with the phenomenon observed in Ahrwt/wt mice and Ahr-/- mice, where AHR regulates distinct gene batteries without and with TCDD, with less than half of the genes in common (Tijet et al. 2006).

Out of the 435 common genes in our experiments, only 3 were regulated by AHR in different directions without TCDD than with TCDD, also agreeing with the observation in Ahrwt/wt versus

Ahr-/- mice (Tijet et al. 2006). Since Ahr-/- mice had only a few genes upregulated or downregulated after TCDD injection, the authors concluded that the effects of TCDD were predominantly mediated through the action of AHR (Tijet et al. 2006). Similarly, since Ahrdbd/dbd mice had only one gene differently expressed upon TCDD treatment based on RNA-seq and it was not validated by Tukey’s test of the RT-qPCR results, the suggestion would be that all effects of TCDD are mediated through not only the action of AHR in general, but also through its AHRE-binding activity. As only very few changes in RNA expression levels was found by RNA-seq in Ahrdbd/dbd mice upon TCDD treatment, if not nothing at all, our study showed the binding of the Ahrdbd to

DNA mediated through tethering to other transcription factors or binding to non-AHRE regions was non-productive.

4.7 Limitations

Several limitations of the study have been considered in previous parts of the discussion.

The major limitation with this study is that Ahrdbd/dbd mice carry the Ahrd allele with a DNA-binding domain mutation, while their Ahrwt/wt littermates carry the Ahrb1 allele, due to reasons explained in

Chapter 1.5. Compared with the AHRd protein, the AHRb1 protein has 5 substitutions and is 43 amino acids shorter (Thomas et al. 2002). The V375A substitution alone renders AHRb1 to be 10 times more responsive to ligand activation than AHRd. Therefore, the 100 µg/kg TCDD dosage,

110 at which both AHRd and AHRb1 reach maximal plateau, was chosen to try to obtain similar levels of AHR activation in Ahrdbd/dbd mice and Ahrwt/wt littermates. This dose was shown to induce similar amounts of hepatic Cyp1a1 activity via the two different alleles, but might result in differential regulation of other AHR target genes (Niwa et al. 1975; Nebert et al. 2004). Another concern is that the different amino acid sequences and lengths between AHRd and AHRb1 may cause different protein-protein interactions. This is especially important as the study focuses on AHR tethering to other transcription factors. The C-terminus of AHR is known as the coactivator/corepressor binding domain. Although there is no direct evidence, it is possible that AHRd, with its 43 additonal amino acids in the C-terminus, interacts with more tethering partners than AHRb1. This could be a contributing factor for DMSO-treated Ahrdbd/dbd mice to have more AHR-bound regions than

DMSO-treated Ahrwt/wt mice (Figures 3.13 and 3.14). Since the transcription factor binding site analysis of the AHR-bound peaks suggested almost the same candidate tethering partners between

Ahrdbd/dbd mice and Ahrwt/wt mice, this issue is unlikely to play a major role or impact the conclusions from our experiments.

Second, the fact that AHR protein is expressed at a lower level in Ahrdbd/dbd mice than in

Ahrwt/wt mice is another factor to be considered for any comparison between them (Figure 3.5).

TCDD was delivered at 100 µg/kg to ensure that all existing AHR in Ahrdbd/dbd mice was activated.

The quality of Ahrdbd/dbd mice ChIP-seq results was most likely not adversely affected by its lower

AHR expression, as more peaks were found in DMSO-treated Ahrdbd/dbd mice than the Ahrwt/wt.

Even if Ahrdbd/dbd mice and Ahrwt/wt mice are not ideal groups to be compared, the comparison of

DMSO-treated Ahrdbd/dbd mice versus TCDD-treated Ahrdbd/dbd mice still reveals necessary information regarding tethering or non-AHRE binding of AHR.

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Last, it could be argued that the DNA-binding domain mutation, which includes an I25G substitution and a GS insertion after the 39th amino acid, may alter protein-protein interactions with AHR. Since the mutations target the short DNA-binding domain and this AHRdbd protein has been shown to retain interactions with chaperones, this is theoretically unlikely. Admittedly, it would be informative if results from this study could be replicated using another system with a different DNA-binding domain mutation in the future. For example, a R39A mutation has also been found to prevent DNA-binding of AHR (Dong et al. 1996).

4.8 Future Directions

ChIP-seq identified more AHR-bound regions in TCDD-treated Ahrdbd/dbd mice than in untreated mice, while RNA-seq and subsequent RT-qPCR did not find any genes to be differentially expressed. It may still be interesting to verify whether AHR indeed tethers to the transcription factors identified by the binding site analysis of AHRdbd-bound regions, such as ZF5 and NRF1, through protein-protein interaction studies, such as co-immunoprecipitation. If association between AHRdbd and these candidate partners is not present, then the DNA binding of

AHRdbd would likely to be due to direct binding to non-AHRE regions.

Second, the RNA-seq experiment used mice 6 hours after treatment with TCDD or DMSO vehicle. This timepoint was chosen to focus on the primary effects of AHR-mediated transcriptional regulation. Since samples were also obtained from mice 24 hours after exposure, sequencing of those 24-hour samples may provide further information regarding genes that are more slowly regulated. For example, although Cyp1a1 mRNA expression peaked at 6 hours,

Cyp1b1 mRNA expression had a 10-fold increase from 6 hours to 24 hours (Figures 3.8 and 3.9).

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Last, the role of AHR in immune responses has been suspected to be heavily mediated through AHR tethering and indirect DNA binding, since important immune regulators, such as

NFκB and AP-1, are major candidate tethering partners of AHR (Beischlag et al. 2008). One important aspect of AHR function in the immune system is its protective effect against colitis. Ahr-

/- mice have more severe symptoms and higher lethality compared with Ahrwt/wt mice when they are treated with dextran sodium sulfate (DSS), which induces colitis, while TCDD treatment alleviates symptoms and lethality in Ahrwt/wt mice (Takamura et al. 2010; Arsenescu et al. 2011).

Ahrdbd/dbd mice are currently being treated with DSS to test whether loss of canonical AHR signalling via direct binding to its cognate AHRE sequences is sufficient to increase the sensitivity of mice to colitis.

4.9 Conclusion

AHR is a transcription factor which, upon ligand activation, translocates into the nucleus and heterodimerizes with ARNT. Although AHR gene regulation is traditionally known to be mediated by direct DNA binding following the recognition of AHRE, recent evidence suggests it may also bind to DNA indirectly for gene regulation by tethering to other transcription factors and function through non-AHRE mechanisms. Ahrdbd/dbd mice, whose AHR is not able to directly bind an AHRE, provide a great tool to study the effect of AHR tethering and non-AHRE binding. ChIP- seq showed that Ahrdbd/dbd mice indeed had an increased number of AHR-bound regions across the genome upon activation by TCDD. However, this non-AHRE binding of AHR may not have much impact on gene transcription. Egfr was the only differently expressed gene in Ahrdbd/dbd mice after

TCDD treatment identified by RNA-seq. From RT-qPCR, its slight upregulation in TCDD-treated

Ahrdbd/dbd mice was statistically significant with t-test comparing to DMSO-treated Ahrdbd/dbd mice

113 alone, but was not with Tukey’s test which also included Ahrwt/wt mice. This thesis provides critical and novel information regarding the mechanism of AHR-mediated transcriptional regulation, which plays roles in numerous physiological and pathological processes, including development and carcinogenesis. It supports the existence of, but refutes the physiological significance of

TCDD activated AHR causing changes in gene expression even in the absence of a DNA binding domain capable of binding to AHREs. Thus, these findings agree with the traditional view that the majority of AHR-mediated gene regulation requires its direct DNA binding to its cognate AHRE sequences.

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