Regulation of Eukaryotic Transcription
by bHLH/PAS Transcription Factors
AhR and Arnt
Nan Hao
B.Sc. (Biomedical Science) (Honours)
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy
Discipline of Biochemistry School of Molecular and Biomedical Science The University of Adelaide, Australia August, 2012
Table of Contents
Abstract ...... iii
Original Publications ...... v
Declaration ...... vi
Acknowledgements ...... vii
Abbreviations ...... ix
Chapter 1: Introduction ...... 1
1.1 History at a glance ...... 1
1.2 The modular nature of bHLH/PAS transcription factors ...... 2
1.2.1 The bHLH domain ...... 2
1.2.2 The PAS domain ...... 3
1.2.3 The transactivation/transrepression domain ...... 7
1.3 Mammalian bHLH/PAS proteins ...... 7
1.3.1 Activation and regulation of AhR ...... 8
1.3.2 The endogenous function of AhR ...... 13
1.3.2.1 Cross talk between AhR and ER ...... 16
1.3.2.2 AhR and immunity ...... 19
1.3.3 Evolutionary diversity of AhR-- What have we learnt? ...... 26
1.3.4 AhR ligands ...... 31
1.3.5 The LBD of AhR ...... 35
1.4 Aims and Approaches ...... 41
Chapter 2: Results ...... 43
2.1 Discovery that the dimerization specificity bHLH/PAS factor Arnt resides in its N-terminal
PAS domain (Paper II) ...... 43
2.2 Discovery that Xenobiotic and Suspension Activation of AhR regulates both overlapping
and distinct sets of gene expression (Paper III) ...... 44
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2.3 Discovery that the mode of interaction between AhR and atypical ligand YH439 is distinct
from that of HAH/PAH ligands (Paper I) ...... 46
Chapter 3: Discussion ...... 49
Chapter 4: References ...... 59
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Abstract
The Aryl hydrocarbon Receptor (AhR) is a Class I basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) transcription factor essential for adaptive response to xenobiotics. As for all bHLH/PAS proteins, it has a uniform molecular design that is composed of three functional domains, including a highly conserved N-terminal bHLH domain, a pair of degenerate PAS repeats (designated PAS.A and
PAS.B), and a poorly conserved C-terminal transactivation domain. However, in contrast to the other proteins in the family, AhR is the only member known to bind xenobiotic ligand found in nature.
The ligand binding domain (LBD) of AhR resides in its PAS.B region. Structure activity relationship analysis suggested that the ligand binding pocket of AhR is promiscuous, which can accommodate a large number of planar and hydrophobic compounds. Polycyclic Aromatic Hydrocarbons (PAHs) and Halogenated Aromatic Hydrocarbons (HAHs) are by far the most common classes of AhR ligands. In addition, pharmaceutical compounds such as the hepatoprotective agent YH439 that fall outside the aromatic classification, have also been shown to activate AhR, presumably by functioning as AhR agonists.
To better characterize the LBD of mouse AhR (mAhR), rational site-directed mutagenesis was performed based on the LBD sequences of zebrafish (Danio rerio). Unexpectedly, the mAhR H285Y mutant as well as previously identified A375I mutant were found to discriminate between ligands, suggesting that in contrast to the PAH/HAH ligands, the atypical ligand YH439 has a novel mode of interaction that does not require full access of the ligand binding cavity.
All bHLH/PAS proteins function as obligate dimers. In order to form an active, DNA binding complex, the AhR have to dimerize with Class II bHLH/PAS protein Aryl hydrocarbon receptor nuclear translocator (Arnt). This is mediated primarily via the N terminal bHLH and PAS.A domains.
Furthermore, the data presented in this thesis suggest that both the α-helical connector and
β-strand structure of the PAS.A domains are required for AhR/Arnt heterodimerization, which is distinct from the β-scaffold surfaces proposed for dimerization between the PAS.B domains of
HIF-2α (Hypoxia Inducible Factor-2α) and Arnt. Intriguingly, interaction between Arnt and other
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Class I bHLH/PAS proteins were found to occur via the same dimerization interface, suggesting that the dimerization selectivity of common partner factor Arnt resides on a small number of key amino acids within a single dimerization interface of Arnt.
In addition to the canonical AhR activation by xenobiotics, switching cells from adherent to suspension culture also activates the AhR, representing a non-xenobiotic, physiological activation of
AhR signaling. This is further supported by the observation that AhR is recruited to the xenobiotic response element (XRE) of prototypical AhR target genes Cyp1a1, Cyp1b1 and Tiparp following both xenobiotic and suspension culture induced AhR activation. However, genome wide microarray analysis revealed significant differences between the two activation mechanisms in modulating target gene expression, implying the existence of a fine-tuning control to define the target gene specificity of AhR.
Taken together, the work presented in this thesis explores the various mechanisms underlying AhR regulation with a special emphasis on specificity, which not only advances our current understanding on the non-canonical pathways of AhR activation, but also lends novel insights into how eukaryotic genes are regulated at the transcriptional level.
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Original Publications
This thesis is based on the following publications, which will be referred to by their roman numerals in the text:
Paper I Whelan F, Hao N, Furness SG, Whitelaw ML, Chapman-Smith A. (2010)
Amino acid substitutions in the aryl hydrocarbon receptor ligand binding domain
reveal YH439 as an atypical AhR activator. Mol. Pharmacol. 77(6):1037-1046.
Paper II Hao N, Whitelaw ML, Shearwin KE, Dodd IB, Chapman-Smith A. (2011)
Identification of residues in the N-terminal PAS domains important for dimerization of
Arnt and AhR. Nucleic Acids Res. 39(9):3695-3709.
Paper III Hao N, Lee KL, Furness SG, Poellinger L, Whitelaw ML. (2012)
Xenobiotics and Loss of Cell Adhesion Drives Distinct Transcriptional Outcomes by
Aryl Hydrocarbon Receptor Signaling. Mol. Pharmacol.
Manuscript under peer-review
All publications have been reproduced with the permission from the copyright holders.
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Declaration
This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution to Nan Hao and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.
I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968.
The author acknowledges that copyright of published works contained within this thesis (as listed in the previous page) resides with the copyright holder(s) of those works.
I also give permission for the digital version of my thesis to be made available on the web, via the
University’s digital research repository, the Library catalogue, and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.
Nan Hao
July 2012
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Acknowledgements
Time flies, four years of my PhD study has now come to an end. Doing a PhD is by no means an easy task, and I could not have done it without help from many wonderful people.
First and foremost, I want to thank my principle supervisor A/Prof. Murray Whitelaw, for welcoming me to work in your lab, and for always being supportive and encouraging. You are a truly inspirational model to many students like me, both as a passionate scientist and as a human being.
I also want to acknowledge my co-supervisor Dr. Anne Chapman-smith, for her patience, guidance and encouragement, and Dr. Dan Peet, for his support and friendship. It is a great opportunity to learn from you.
I am also grateful to my collaborators Dr. Keith Shearwin, Dr. Ian Dodd at School of Molecular and
Biomedical Science (Biochemistry), The University of Adelaide, Dr. Kian Leong Lee, Prof. Lorenz
Poellinger at Cancer Science Institute of Singapore, National University of Singapore, and Prof. Jeff
Gorman at Protein Discovery Centre, Queensland Institute of Medical Research. Thank you all for your advice and valuable insights on the project.
My Thanks also go to my fellow colleagues in Whitelaw and Peet labs – Adrienne, Alix, Anne R.,
Bec, Colleen, Dave, Fiona, Freya, Jacqueline, Karolina, Margo, Matt, Natalia, Navdeep, Nick,
Rachel, Sam, Sarah L., Sarah W., Scott, Sebastian, Shweetha, Teresa, Tom, Veronica, and Wei Li.
Thank you for your thoughtful discussions and advice throughout my PhD.
To all members in Thomas and Jensen labs, for being good neighbors, and to all other people in
Biochemistry discipline and school support staffs, for providing such an enjoyable and inspiring environment to learn.
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To all my friends, especially Ang Zhou, Jennifer Liu, Peter Feng, Fay Qiao, and Danni Yang for their friendship and support, and for making my life in Adelaide colorful and enjoyable.
To my wife, for her love, support, encouragement, and understanding.
And finally, to my parents for their love and support. I am forever grateful for them in supporting me to pursue my interest.
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Abbreviations
3',4'-DMF 3',4'-Dimethoxyflavone
3MC 3-Methylcholanthrene
6-MCDF 6-Methyl-1,3,8-trichlorodibenzofuran
AhR Aryl hydrocarbon Receptor
AhRE-II AhR Response Element like sequence
AhRR AhR Repressor
AIP AhR-Interacting Protein
Aldh3a1 Aldehyde dehydrogenase 3 family, member a1
APC Antigen Presenting Cell
Arnt Aryl hydrocarbon receptor nuclear translocator
α-NF α-Naphthoflavone
B[α]P Benzo[α]pyrene bHLH basic Helix-Loop-Helix bHLH.Zip basic Helix-Loop-Helix Zipper
β-NF β-Naphthoflavone
CAT Chloramphenicol Acetyltransferase
CD Circular Dichroism
Cdk Cyclin dependent kinase cDNA complementary DNA
C/EBPβ CCAAT/Enhancer-Binding Protein β
CH-223191 2-Methyl-2H-pyrazole-3-carboxylic Acid (2-methyl-4-o-tolylazo-phenyl)-amide
CRM1 Chromosome Region Maintenance
CRP C-Reactive Protein
CTL Cytotoxic T Lymphocyte
Cyp Cytochrome P450
DBD DNA Binding Domain
DC Dendritic Cell
DIM Diindolylmethane
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DiMNF 3’,4’-Dimethoxy-α-naphthoflavone
DMSO Dimethyl Sulfoxide
DN Double Negative
DNA Deoxyribonucleic Acid
DV Ductus Venous
E1 Estrone
E2 17β-Estradiol
EAE Experimental Autoimmune Encephalomyelitis
E-box Enhancer Box
EMSA Electrophoretic Mobility Shift Assays
ER Estrogen Recepter
ERE Estrogen Responsive Elements
FCS Fetal Calf Serum
FICZ 6-formylindolo[3,2-b]carbazole
FoxP3 Forkhead box P3
GNF351 N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine
GST-Ya Glutathione-S-transferase
GVH Graft-vs-Host
HAH Halogenated Aromatic Hydrocarbon
HDC Histidine Decarboxylase
HES-1 Hairy and Enhancer of Split homology-1
HIF Hypoxia Inducible Factor
H-NOXA Heme-Nitric oxide/Oxygen binding Associated
HP Haptoglobin
HSC Hematopoietic Stem Cell
Hsp90 Heat shock protein 90
H/W Han/Wistar
IDO Indoleamine-2, 3-dioxygenases iDRE inhibitory Dioxin Response Element
IFN-γ Interferon-γ x |
IGFBP-1 Insulin-like Growth Factor Binding Protein-1
IL Interleukin
IMDM Iscove's Modified Dulbecco's Medium
KA Kynurenic Acid kb kilo base
Kd Dissociation constant
KLF2 Kruppel-Like Factor 2
KO Knock Out
LBD Ligand Binding Domain
LD50 Lethal Dose 50
LDL Low Density Lipoprotein
L-E Long-Evans
LMB Leptomycin B
LOV light, oxygen, voltage
LPS Lipopolysaccharide
MD Molecular Dynamics
MHC-II Major Histocompatibility Complex class II
MNF 3’-methoxy-4’-nitroflavone
MOG35-55 Myelin Oligodendrocyte Glycoprotein peptide 35-55
NES Nuclear Export Sequence
NF-κB Nuclear Factor-kappa B
NK Natural Killer
NLS Nuclear Localization Sequence
NMT2 N-Myristoyltransferase 2
NOD Nonobese Diabetic
NPAS Neuronal PAS protein
Nqo1 NAD(P)H quinone oxidoreductase 1
NR Nuclear Hormone Receptor
PAH Polycyclic Aromatic Hydrocarbons
Pai-2 Plasminogen activator inhibitor-2
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PAS Per-Arnt-Sim homology pCA p-Coumaric Acid
PCDD Polychlorinated Dibenzo-p-dioxins
PCDF Polychlorinated Dibenzofuran pentaCB 3,3’,4,4’,5-pentachlorobiphenyl
PIA Pancreatic Islets Allotransplantation pM picomolar
Por P450 (cytochrome) oxidoreductase
PPAR α Peroxisome proliferator-activated receptor α pRb Retinoblastoma protein
PTM Post Translational Modification
PYP Photoactive Yellow Protein qRT-PCR quantitative Real Time Polymerase Chain Reaction
RevB2H Reverse Bacterial two Hybrid
RMSD Root-Mean-Square Deviation
RNA Ribonucleic Acid
RNAPII RNA polymerase II
RPMI Roswell Park Memorial Institute
SAA Serum Amyloid Associated
SAhRM Selective AhR Modulator
SAR Structure Activity Relationship
SGA360 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole
Sim Single minded
SNP Single Nucleotide Polymorphism
SSD Signal Sensing Domain
Stat1 Signal transducer and activator of transcription 1
TAD Transactivation Domain
TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
TCDF 2,3,7,8-tetrachlorodibenzofuran xii |
TDO Tryptophan-2,3-dioxygenase
TGF-β Tansforming Growth Factor-β
Tiparp TCDD-inducible poly(ADP-ribose) polymerase
TK Thymidine Kinase
TNF-α Tumour Necrosis Factor-α
TRAMP Transgenic Adenocarcinoma of the Mouse Prostate
Treg Regulatory T cell
Trh Trachealess
Ugt1a6 UDP glucuronosyltransferase 1 family, polypeptide A6
UTR Untranslated Region
Way-169916 4-[1-allyl-7-(trifluoromethyl)-1H-indazol-3-yl] benzene-1 wt wild type
XRE Xenobiotic Response Element
YH439 isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate
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Chapter 1: Introduction
1.1 History at a glance
It is believed that human civilization began with the advancement of science.
Since about 3500 years ago (14th-11th centuries B.C.), in the ancient China, people had begun to practice experimental medicine for therapeutic purpose (Unschuld, 2010). At around the same time, there was also a rapid development of a body of knowledge concerning the organization of the human body in the Western world, mostly through the scientific dissection of animals, which led to the identification of the major organs in the body, including heart, liver, lungs and kidneys. One of the most notable figures during this period is the great Greek physician Hippocrates of Cos (ca. 460-370
B.C.), who is also regarded as the “Father of Medicine”. Other pioneers in this period include eminent natural philosophers and physicians Alcmaeon of Croton (5th century B.C.) in Italy and
Empedocles (490-430 B.C.) in Sicily (Malomo et al., 2006 and references therein). Together, their works led to the emergence of a new subject of science, which is what we now call “anatomy”.
Within the next 20 centuries, the organ remained to be the smallest unit of structure known for any living organism. This view held true until the development of cell theory by two German biologists
Theodor Schwann and Matthias Jakob Schleiden, who proposed that the cell is the fundamental unit of structure in all living organisms (Schleiden, 1838; Schwann, 1839; Turner, 1890).
However, what constitutes a cell was still a mystery at that time. In 1897, Eduard Buchner from
Berlin University found that the yeast extracts were able to ferment sucrose even when there were no living yeast cells in the mixture (Buchner, 1897). He named the “magic” ingredient in the yeast
“Zymase”, or what is now known as enzymes. Stemming from Eduard’s work, in 1926, James B.
Sumner successfully isolated and crystallized the enzyme urease from jack beans and showed for the first time that urease is in fact a protein (Sumner, 1926).
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Following the isolation and crystallization of protein urease by James B. Sumner, in 1953, James D.
Watson and Francis Crick published the first crystal structure of DNA (Wilkins et al., 1953), and soon after, the first protein structure, of hemoglobin, was solved by X-ray crystallography (Kendrew et al.,
1958). Structural analysis of hemoglobin and later myoglobin (Kendrew et al., 1958; Muirhead and
Perutz, 1963) revealed that proteins are folded into unique 3-dimensional structures, while the shape or the conformation of the protein determines their biological functions.
Looking back through the history of the life science discoveries, it took more than 2000 years to reshape our understanding of life from the organ level down to the cellular level, and another 100 years from cellular to molecular level (e.g. proteins). It is now generally accepted that proteins are the major constituents of the cells, and (arguably) the most important building blocks of life.
Furthermore, it has been suggested that proteins are modular in nature (Koide, 2009; Pawson and
Nash, 2003), constructed from domains, which are loosely defined as evolutionally conserved modules of 25 to 500 amino acid in length that can be folded independently from the rest of protein
(Wetlaufer, 1973). Intriguingly, the functions of most proteins can often be predicted by their choice of domains/modules. To this end, the bHLH/PAS (basic Helix-Loop-Helix/Per-Arnt-Sim Homology) family proteins provide excellent examples of such modular protein designs, which will be the focus of this thesis.
1.2 The modular nature of bHLH/PAS transcription factors
All bHLH/PAS proteins have a uniform molecular design that is composed of three functional modules, including a highly conserved N-terminal bHLH domain, adjacent PAS repeats (mostly two), and a poorly conserved C-terminal transactivation domain, or transrepression domain (Figure 1.1).
1.2.1 The bHLH domain
The bHLH domain was first identified from studies of murine transcription factors E12 and E47
(Murre et al., 1989). Since its initial discovery, there has been a rapid expansion in the numbers of bHLH proteins, with now over 8678 eukaryotic and viral bHLH sequences documented in the Pfam
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A NOTE: This figure/table/image has been removed to comply with copyright regulations. It is included in the print copy of the thesis held by the University of Adelaide Library.
Figure 1.1 Schematic representation of the domain architecture of the mammalian bHLH/PAS proteins. The bHLH/PAS proteins can be subdivided into two classed based on their dimerization potential. Class I bHLH/PAS factors lack the ability to either homodimerize or heterodimerize with other class I factors, while class II bHLH/PAS proteins can both homodimerize and heterodimerize with other class I bHLH/PAS family proteins. Coding scheme: grey, basic domain; blue, Helix-loop-Helix domain; red, PAS domains; orange, transactivation domain; green, transrepression domain; purple, oxygen dependent degradation domain. Figure adapted from the PhD thesis of Dr. Anne Raimondo, the University of Adelaide, 2011.
Database (Version 26.0, November 2011 Build), which play pivotal roles in a wide range of developmental processes, including cellular differentiation, lineage commitment, and sex determination (for reviews, see Atchley and Fitch, 1997; Massari and Murre, 2000; Ross et al., 2003;
Skinner et al., 2010).
The bHLH domain is characterized by having a short stretch of 8-15 predominantly basic amino acid residues directly adjacent to two amphipathic α helices, separated by a loosely structured loop of various lengths. The basic region of bHLH domain is the primary site for DNA contact. Thus, having a bHLH domain automatically places any associated protein in the DNA binding protein category. In contrast, the HLH motif forms a dimerization interface that promotes homo- and hetero-dimerization between bHLH proteins.
Most bHLH proteins recognize a hexanucleotide DNA sequence commonly known as E-box sequence (CANNTG) or a closely related E-box variant (Chaudhary and Skinner, 1999; Swanson et al., 1995; Wang et al., 1995; Wharton et al., 1994). However, the exact binding sequence varies considerably among bHLH proteins, depending on the topology of the proteins and also the presence or absence of additional domains, e.g. leucine zipper domain, PAS domain, or Orange domain. Even within the same bHLH protein clade (e.g. among different bHLH/PAS proteins), there is also a marked difference in the binding affinity between different bHLH proteins and different
E-box variations (Swanson et al., 1995; Wang et al., 1995; Wharton et al., 1994).
1.2.2 The PAS domain
In contrast to the bHLH domain, the function of the PAS domain is much less understood. However, it becomes increasingly evident that the PAS domain is multi-functional.
The PAS domain has been identified in all kingdoms of life (Figure 1.2), arguing for an ancient conserved role of this domain during evolution. The name “PAS” was originally used to describe the sequence homology shared by three proteins in which it was first identified: the Drosophila clock protein PER (Period), vertebrate bHLH/PAS protein Arnt, and Drosophila Sim (single minded)
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Protein (Crews et al., 1988; Hoffman et al., 1991; Reddy et al., 1984). Now, over 10,000 PAS sequences and 64 experimentally determined PAS structures have been deposited in the Pfam
Database (Version 26.0, November 2011 Build). All PAS sequences fold to a highly conserved structure known as the PAS fold (Taylor and Zhulin, 1999), although the sequence homology among different PAS sequences at amino acid levels is extremely low, generally less than 20%.
Studies of Ectothiorhodospira halophila photoactive yellow protein (PYP) provided the structural basis of a PAS fold (Pellequer et al., 1998), which is characterized by having a 5 stranded antiparallel β-sheet with a central α-helix core and a α-helical connector spanning the two halves of the β-scaffold (Figure 1.3). Intriguingly, structural superimposition of 47 experimentally determined
PAS domains indicated that the central β-scaffold is well conserved among PAS folds, with the root-mean-square deviation (RMSD) for backbone alpha-carbons (Cα) of less than 2Å. In contrast, the length and orientation of the α-helices as well as the connection loops appear to be more flexible
(Moglich et al., 2009b). Similar results have also been proposed from computer based molecular dynamics (MD) simulations (Pandini and Bonati, 2005; Vreede et al., 2003; Xing et al., 2012). Thus, the flexible and rigid nature of PAS α/β-scaffold may underpin the promiscuous ligand binding ability of different PAS domains.
Prokaryotic proteins may contain single, dual or multiple PAS domains, whereas most of the eukaryotic PAS proteins carry a pair of PAS domains (Taylor and Zhulin, 1999). Sequence analysis among mammalian bHLH/PAS proteins suggested that positionally conserved PAS domains in general have a higher degree of homology (McIntosh et al., 2010). For example, the N-terminal PAS domains of murine bHLH/PAS proteins share 35% sequence identity to one another, and that of the
C-terminal PAS domains are 31%. In contrast, the homology between the N- and C-terminal PAS domains is generally less than 20%. Thus, the two mammalian PAS domains may have evolved independently, presumably resulting from an early divergence in eukaryote evolution. In support of this idea, bacterial sensor kinase is known to carry multiple PAS domains with different evolutionary origins (reviewed in Taylor and Zhulin, 1999). At first glance, it may appear that having multiple PAS domains in a single protein is redundant; however on the other hand, having repeated domains of diverse evolutionary origins could be a cost effective way to extend the functional repertoire of a
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Archaea Bacteria Eukaryota Unclassified
Figure 1.2 Sunburst visualisation of species distribution of all PAS sequences in the Pfam database (Version 26.0, November 2011 built). In this view, each arc represents a node in the phylogenetic tree. The radius of the arc (i.e. the distance from the edge of each arc to its root center) shows the taxonomic level, in the hierarchical clades of: superkingdom, kingdom, phylum, class, order, family, genus, and species. There is a total of 10,555 PAS sequences documented in the Pfam database, the majority (7,711 sequences) of them are found in Bacteria. In addition, there were also 2538 PAS sequences identified in Eukaryota, and a small proportion of them in either Archaea (290 sequences) or unclassified sources (16 sequences). No PAS sequences have yet been identified from Viruses or Viroids. Archaea Q5V5P7 PAS.C
Bacteria
PYP FixL H-NOXA
Fungi & Plantae
VVD Phy3 LOV2
Animalia
dPER PAS.A dPER PAS.B mPER2 PAS.A
mPER2 PAS.B NCoA HERG
hPASK HIF2α PAS.B Arnt PAS.B
Figure 1.3 Cartoon representation of PAS domain structures from different species. Three- dimensional structure information were retrieved from Protein Data Bank with the following accession numbers: Haloarcula marismortui HTR-like protein Q5V5P7 PAS.C (3BWL), Halrhodospira halophila photoactive yellow protein PYP (1NWZ), Bradyrhizobium japonicum oxygen sensor FixL (1DRM), Nostoc punctiforme H-NOXA (2P04), Neurospora crassa Vivid PAS protein VVD (2PD7), Adiantum capillus-veneris phytochrome-like protein Phy3 PAS.B (1G28), Avena sativa LOV2 PAS.B (2V0U), Drosophila melanogaster and Mus musculus PERIOD proteins dPER and mPER2 (1WA9 & 3GDI), as well as Homo sapiens PAS containing transcription cofactor NCoA (1OJ5), potassium channel HERG (1BYW), PAS kinase hPASK (1LL8), PAS.B domain of HIF2α (1P97) and Arnt (1X0O). All structures were draw with Pymol with α-helices highlighted in red, β-strands in yellow and loops in green.
given protein. This is even more pronounced when applied to sensory proteins such as PAS containing proteins, allowing cells to integrate signals from complex microenvironments (Kyndt et al.,
2010). Taken together, the divergent functions of two PAS domains in mammalian proteins could stem from independent evolutionary origins, resulting from early split and subsequent specialization of PAS domains.
The majority of prokaryotic PAS domains are found in sensor histidine kinases, where they act as the signaling modules for changes in cell microenvironment, such as light, oxygen levels, redox potential, and the overall energy level of a cell (Taylor and Zhulin, 1999). Similarly, eukaryotic PAS domains also function as signaling modules and are known to bind small molecules, either naturally or in experimental conditions (Denison and Nagy, 2003; Scheuermann et al., 2009). There are 33
PAS domain containing proteins in mouse, most of which also possess a bHLH domain, and thus function as sequence specific transcription factors. The exceptions of this are PERIOD proteins
Per1-3, NCoA/SRC-1/p160 family transcription coactivators NCoA1-3, potassium voltage-gated channel proteins Kcnh1-8, phophodiesterases Pde8a and Pde8b, and serine/threonine kinase Pask
(reviewed in McIntosh et al., 2010).
In addition to roles in signal sensing, the eukaryotic PAS domain can also determine target gene specificity. This is demonstrated by domain swap experiment, in which replacing the PAS domain of
Drosophila bHLH/PAS protein Trachealess (dTrh) with the equivalent region from Drosophila Sim
(dSim) is sufficient to convert dTrh into a functional dSim, activating dSim target genes expression without affecting the dTrh target genes (Zelzer et al., 1997). These differences in target gene specificity are postulated to result from differential recruitments of transcription cofactors by the PAS domains of dTrh and dSim, suggesting a role of the PAS domain in interaction with transcription co-factors. Consistent with this notion, the C-terminal PAS domain of aryl hydrocarbon receptor nuclear translocator (Arnt) is known to interact with a number of coiled-coil coactivators such as
CoCoA and TACC3, when dimerizing with Hypoxia Inducible Factor-2α (HIF-2α) (Partch and
Gardner, 2011).
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As will be discussed later in this thesis, the mammalian bHLH/PAS proteins function as obligate dimers, where the N-terminal PAS domain determines dimerization specificity. Isolated bHLH domain of aryl hydrocarbon receptor (AhR) displays a broad dimerization capability with other bHLH containing proteins, including the unrelated bHLH Leucine Zipper (bHLH-Zip) protein USF. Addition of the PAS domain however restricts the dimerization potential of AhR, preventing the formation of both AhR homodimers and AhR/USF heterodimers (Pongratz et al., 1998).
Studies in our lab also suggest that the bHLH and the N-terminal PAS domains function as a single module in folding and dimerization for mammalian bHLH/PAS proteins (Chapman-Smith and
Whitelaw, 2006). In contrast to AhR and HIF-1α bHLH domain alone, addition of N-terminal PAS domain enhances DNA binding in in vitro electrophoretic mobility shift assays (EMSA)
(Chapman-Smith et al., 2004). This is further supported by DNase I footprinting experiments where the core response element and immediately adjacent sequences were similarly protected from
DNase I cleavage with either AhR/Arnt bHLH fused to leucine zipper domains of Max/Mac or native versions of the AhR/Arnt PAS domains, the presence of PAS domains extended the protection region to nearby sequence (Chapman-Smith and Whitelaw, 2006). Thus, it appears that the PAS domain of both AhR and HIF-α alter DNA conformation when bound to their cognate DNA sequences, suggesting a possible contact between the PAS domain and DNA (A. Chapman-Smith, unpublished data).
It became increasingly evident that most PAS domains are also flanked by short α-helices at the N- and C-termini, known as α-helical caps (Moglich et al., 2009b). Studies of Escherichia coli aerotaxis protein Aer suggested that the α-helical cap may function to protect the central β-barrel, and thus stabilizes the PAS core (Watts et al., 2006). However, the presence of an α-helical cap structure at this position is not absolutely required, and other structures that can fulfill a similar function may replace the cap (Taylor and Zhulin, 1999). In addition, studies of Nostoc punctiforme histidine kinase
H-NOXA (heme-nitric oxide/oxygen binding associated) domain and Neurospora crassa photoreceptor Vivid PAS domain suggest that the N-terminal α-helical cap facilitates PAS dimer formation (Ma et al., 2008; Zoltowski et al., 2007). Furthermore, the α-helical cap of prokaryotic histidine kinase PAS domains may also play important roles in relaying the signal originated from
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the PAS domain to other effector domains (for a review, see Moglich et al., 2009b).
1.2.3 The transactivation/transrepression domain
The C-terminal transregulatory domains of bHLH/PAS proteins are the least conserved regions among bHLH/PAS proteins. In general, these domains provide a dimerization interface for transcription cofactors such as CBP/p300 and NCoA1-3 (Beischlag et al., 2002; Carrero et al., 2000;
Hankinson, 2005; Kallio et al., 1998; Kobayashi et al., 1997). However, due to their large sequence divergence, different bHLH/PAS proteins could potentially recruit a different set of transcription cofactors, leading to distinct sets of target gene activation.
Interestingly, the bHLH/PAS proteins HIF-1α and HIF-2α contain two transactivation domains, known as N-terminal transactivation domain (NTD) and C-terminal transactivation domain (CTD), respectively. Both NTD and CTD are required for optimal transcription (Hu et al., 2007). However, the N-terminal transactivation domain also controls target gene specificity, as replacement of HIF-2α
NTD with equivalent region of HIF-1α is sufficient to convert HIF-2α into a functional HIF-1α (Hu et al., 2007).
In contrast to most bHLH/PAS proteins which function to enhance transcription, the bHLH/PAS protein Sim2 contains two transrepression domains at its C terminus. Fusion of Sim2 C-terminal domains with Gal4 DNA binding domain (DBD) strongly repressed a thymidine kinase (TK)/Gal4
DNA binding site hybrid promoter driven CAT (chloramphenicol acetyltransferase) reporter gene expression (Moffett et al., 1997). Moreover, mammalian two hybrid assay with a Gal4-DBD/Arnt fusion protein and full length Sim2 suggested that the transrepression domains Sim2 were also able to override the strong activation signal exerted by Arnt.
1.3 Mammalian bHLH/PAS proteins
The mammalian bHLH/PAS family transcription factors include AhR (also known as DR or dioxin receptor), AhR repressor (AhRR), Hypoxia inducible factor-αs (HIF-1α, HIF-2α, and HIF-3α), Single
| 7
minded proteins (Sim1 and 2), Neuronal PAS factors (NPAS 1, NPAS 3, and NPAS 4/Nxf), circadian clock proteins (Clock, NPAS2/Clock2, BMAL1, and BMAL2) and their heterodimeric partner proteins
Arnt and Arnt2.
The bHLH/PAS proteins can be further divided into two classes (Figure 1.1) based on their dimerization potentials (Kewley et al., 2004). Class I bHLH/PAS factors include AhR, AhRR, HIF-αs,
Sims, NPAS(s), and Clock proteins, which lack the ability to either homodimerize or heterodimerize with other Class I factors. In contrast, Class II bHLH/PAS factors such as Arnt and Arnt2 can both homodimerize and heterodimerize with other Class I bHLH/PAS proteins, although the functional significance of Arnt homodimerization is unknown (Hirose et al., 1996; Sogawa et al., 1995). As mentioned above, all bHLH/PAS proteins work as obligate dimers, and require dimerization with
Class II factor for their functions (Ema et al., 1996; Hoffman et al., 1991; Mimura et al., 1999; Ooe et al., 2004; Wang et al., 1995).
A comprehensive review of each of these factors is beyond the scope of this thesis. The rest of this thesis will focus only on AhR; however, readers interested in the other bHLH/PAS factors are referred to a recent review by McIntosh B., Hogenesch J., and Bradfield C (McIntosh et al., 2010).
1.3.1 Activation and regulation of AhR
The aryl hydrocarbon receptor (Poland et al., 1976) represents one of the best studied examples of bHLH/PAS protein, which mediates cellular response to environmental toxins such as
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) and other structurally related HAHs/PAHs
(halogenated aromatic hydrocarbons/polycyclic aromatic hydrocarbons).
The AhR possesses both nuclear localization (NLS) and nuclear export (NES) sequences, and is continuously shuttled between cytoplasm and nucleus (Ikuta et al., 2000; Kawajiri and Ikuta, 2004).
In its latent state (i.e. ligand unbound state), AhR is localized predominately in the cytoplasm of the cell, and is associated with a dimer of Hsp90 (heat shock protein 90) and other scaffold proteins including 23 kDa heat shock protein p23 and immunophilin-like AhR-interacting protein AIP (also
8 |
known as XAP2 or Ara9) (Furness and Whelan, 2009 and references therein). Interaction between
Hsp90/p23/AIP and AhR masks the N-terminal nuclear localization sequences of AhR, favoring its cytoplasmic localization (Berg and Pongratz, 2002; Petrulis et al., 2003; Pollenz et al., 2006;
Pongratz et al., 1992). Furthermore, interaction between AhR and chaperones also keep the
C-terminal PAS domain of AhR in an open conformation, ready for ligand binding (Pongratz et al.,
1992).
Interaction between AhR and ligands leads to rapid AhR nuclear translocation and promotes
AhR/Arnt dimerization in exchange of Hsp90/AIP/p23 chaperon proteins, a process commonly referred to as AhR transformation (Figure 1.4). However, the sequence of events underlying ligand dependent AhR transformation is not well understood. It was initially hypothesized that ligation of
AhR by AhR agonist induces a localized conformational change at the N terminal bHLH region of
AhR (Ikuta et al., 1998), resulting in partial dissociation of Hsp90 and thus the exposure of NLS.
However, recent evidence suggested that this model is likely to be over-simplified, as addition of
Hsp90 binding stabilizing agent sodium molybdate does not inhibit ligand dependent nuclear translocation of AhR (Heid et al., 2000). Furthermore deletion of the C-terminal part of AhR, which plays no role in Hsp90 binding, leads to constitutive AhR nuclear localization (Pollenz et al., 2006;
Ramadoss and Perdew, 2005). Taken together, these studies point to a new model of AhR transformation, involving not only previously proposed re-orientation of the N-terminal bHLH domain, but also the N terminal PAS domain and C terminal portion of the protein, potentially via increased dimerization potential of AhR PAS domain with Arnt (Soshilov and Denison, 2008). Consistent with this model, artificial disruption of Hsp90 and AhR interaction by salt or geldanacycin treatment resulted in less stable AhR/Arnt dimer formation (Lees and Whitelaw, 1999; Pongratz et al., 1992).
In addition to xenobiotic ligands, increasing evidence has pointed to the existence of endogenous
AhR ligands (Chang and Puga, 1998; Chiaro et al., 2007; Wincent et al., 2009). Interestingly, the level of the endogenous ligands seem to be inversely proportional to the level of xenobiotic metabolizing enzymes such as Cyp1a1 and Cyp1b1. Loss of Cyp1a1 gene expression in mouse hepatoma c37 cells led to constitutive AhR activation even in the absence of exogenous ligand, whereas overexpression of Cyp1b1 in African green monkey kidney epithelium CV-1 cell, which has
| 9
minimal level of endogenous AhR, repressed AhR activation in reporter gene assays. Exposure of cells to hydrodynamic shear stress (Mufti et al., 1995) or shear-conditioned serum (McMillan and
Bradfield, 2007a) also activates AhR, presumably through production of modified low density lipoproteins (LDLs) (McMillan and Bradfield, 2007a). In addition, switching adherent cells to suspension cultures, as well as disrupting cell-cell contacts have also been shown to activate AhR in a number of cell culture systems, including hepatoma Hepa1c1c7 cells, keratinocytes and fibroblasts (Cho et al., 2004; Ikuta et al., 2004; Monk et al., 2001; Sadek and Allen-Hoffmann, 1994a;
Sadek and Allen-Hoffmann, 1994b). Together, these alternative AhR activation pathways provide further experimental models for investigating the endogenous functions of AhR, which may be distinct from that of xenobiotic mediated adaptive responses, although the molecular detail underlying this ligand-independent AhR activation is yet to be identified.
Following AhR transformation, the AhR/ARNT complex binds to its cognate DNA consensus
(TNGCGTGA) sequence known as xenobiotic response elements (XRE) (Lusska et al., 1993; Shen and Whitlock, 1992; Yao and Denison, 1992) in regulatory regions of target genes, and initiates gene transcription. In an attempt to identify global AhR target genes following ligand dependent AhR activation, microarray and ChIP-on-chip based experiments were performed by a number of research groups using different combinations of ligands/cell types (Adachi et al., 2004; Ahmed et al.,
2009; de Waard et al., 2008; Dere et al., 2011; Henry et al., 2010; Pansoy et al., 2010; Tijet et al.,
2006). In addition, time course experiments were employed to study the temporal expression patterns of AhR target genes following AhR activation (Boverhof et al., 2005; Hayes et al., 2007).
Although these large scale gene profiling experiments provide valuable information towards understanding the biological functions of AhR in adaptive xenobiotic responses, results from these experiments were often quite variable, and in general do not agree with each other. Furthermore, experiments comparing AhR targets from cells of different species or even between cell lines of the same species yielded very different results, with little overlap between target genes (Boutros et al.,
2008; Dere et al., 2006; Flaveny et al., 2010; Kim et al., 2009). Thus, the spectrum of genes regulated by AhR is thought to be strongly dependent on the ligand and cell context such as cell type and developmental stage.
10 |
e.g. 3MC, β-NF Cell membrane 1) S68 Hsp90 Hsp90 NES AhR Hsp90 Hsp90 AhR 2) Hsp90 AhR 3) Hsp90 E3 ubiquitin CRM1 ligase Nuclear
pore
Nuclear Hsp90
S68 P AhR membrane Hsp90 Arnt NES AhR
4)
AhRR
Cyp1a1,Cyp1b1
Arnt
Arnt AhR AhRR AhR XRE XRE
Figure 1.4 Mechanism of AhR activation and its regulation. In the latent state, AhR is found predominately in the cytoplasm of the cell, and it is associated with a dimer of molecular chaperones Hsp90. Interaction between AhR and ligands induce a conformational change of AhR, results in its rapid nuclear translocation, where it dimerizes common partner factor Arnt, and induces target genes expression, e.g. Cyp1a1, Cyp1b1 and AhRR. Following activation, the AhR activities are tightly controlled by several negative feedback loops, including 1) degradation of activating ligands by xenobiotic metabolizing enzymes, 2) proteasomal degradation of the receptors, 3) CRM-1 (Chromosome Region Maintenance) mediated nuclear export of S68 de-phosphorylated AhR, 4) induction of AhR repressors. See main text for details.
Despite the variability observed among AhR target gene discovery experiments, a small subset of
AhR target genes including Cyp1a1 (cytochrome P450 1 family, member a1), Cyp1a2, Cyp1b1,
Nqo1 (NAD(P)H quinone oxidoreductase 1), Aldh3a1 (Aldehyde dehydrogenase 3 family, member a1), Ugt1a6 (UDP glucuronosyltransferase 1 family, polypeptide A6), and GST-Ya (glutathione
S-transferase), collectively referred to as “AhR genes battery”, are commonly upregulated by AhR upon ligand induction (Nebert et al., 2000 and references therein). These AhR battery genes encode phase I and phase II xenobiotic metabolizing enzymes, which function to metabolize activating ligands.
In addition to the xenobiotic battery genes, other experimentally validated AhR targets include: 1) genes involved in cell signaling such as NMT2 (N-myristoyltransferase 2) (Kolluri et al., 2001) and
IGFBP-1 (insulin-like growth factor binding protein-1) (Marchand et al., 2005); 2) cell cycle control genes p27kip1 (Kolluri et al., 1999), p21CIP1 (Barnes-Ellerbe et al., 2004), and HES-1 (Hairy and
Enhancer of Split homology-1) (Thomsen et al., 2004); 3) tissue remodeling genes Slug (Ikuta and
Kawajiri, 2006), Ecto-ATPase (Gao et al., 1998), and Pai-2 (Plasminogen activator inhibitor-2)
(Sutter et al., 1991) and 4) genes with less studied functions such as TiParp (Ma et al., 2001).
However, the expressions as well as the regulation of these genes are often cell type specific, and it is likely that the regulation of these genes only occur in a small number of specialized cells.
Environmental toxins such as TCDD and related HAHs have long been known to elicit a broad spectrum of toxicological endpoints including severe wasting syndrome, thymic involution, teratogenicity, hepatotoxicity and tumor promotion (Bock and Kohle, 2006; Poland and Knutson,
1982). This TCDD mediated toxicity is almost exclusively AhR dependent, as mice expressing low affinity AhR allele are partially resistant to TCDD and AhR null mice are completely immune to the
TCDD toxicity (Fernandez-Salguero et al., 1996; Mimura et al., 1997; Okey et al., 1989; Schmidt et al., 1996). Furthermore, AhR knock in mice with mutant NLS (designated AhRnls) have been developed. Consistent with that of AhR null mice, the AhRnls/nls mice are resistant to TCDD mediated toxicity (Bunger et al., 2003). Conversely, transgenic mice expressing a constitutively active form of
AhR have reduced life span, with increased incidence of developing stomach and liver tumors
(Andersson et al., 2002; Moennikes et al., 2004), which highlights the importance of maintaining an
| 11
adequate AhR levels for a healthy cell physiology. To this end, cells have evolved a number of negative feedback systems to dampen down the activating AhR signaling (Figure 1.4).
Being a signal (i.e. ligand) regulated transcription factor, the best way to terminate AhR signaling is to remove the upstream activating signals. Activation of AhR by AhR ligands upregulates phase I and phase II xenobiotic metabolizing enzymes, which oxidize xenobiotic ligands such as PAHs
(Guengerich, 2001), and facilitate their active removal by ATP dependent membrane transporters.
However, not all AhR ligands are metabolized. For example, the prototypical AhR ligand TCDD has an estimated half life of approximately 2 weeks in mice and about 7.1 years in humans (Miniero et al., 2001; Pirkle et al., 1989). Thus, as an alternative pathway to prevent persistent AhR signaling, activated AhR itself is subjected to proteasome dependent degradation (Ma and Baldwin, 2000;
Pollenz, 1996). However, the exact location where this occurs is still unclear. Davarinos and Pollenz showed that blocking of AhR export by nuclear exporter inhibitor leptomycin B (LMB) inhibited proteasome mediated AhR degradation in mouse Hepa1c1c7 cells, suggesting that AhR must be transported from the nucleus back to the cytoplasm for degradation (Davarinos and Pollenz, 1999).
In contrast, another study took advantages of a mutant AhR protein that is constitutively nuclear
(DRNLS), and showed that AhR can be degraded within the nucleus independent of AhR ligand
(Roberts and Whitelaw, 1999).
The third built-in negative feedback circuit for preventing excessive AhR activity relies on the NES of
AhR. Two independent NES motifs have been identified in AhR, located either within the bHLH region (N-NES) or in the N-terminal PAS region (C-NES). A study by Berg and Pongratz suggested that the C-NES is required to maintain the cytosolic localization of unliganded AhR, whereas the
N-NES is important for exporting ligand activated AhR (Berg and Pongratz, 2001). Consistent with this study, Ikuta and colleagues showed that nuclear localization of “endogenous” signal activated
AhR from subconfluent HaCaT cells is partially controlled by a single serine residue (Ser68) in the
C-NES, with phosphorylation of Ser68 inhibiting CRM1 (Chromosome Region Maintenance) mediated nuclear export of AhR (Ikuta et al., 2004). In human keratinocytes, the phosphatase inhibitor okadaic acid that specifically inhibits serine/threonine de-phosphorylation reduced nuclear
12 |
export of AhR (Ikuta et al., 2004), although the molecular mechanism leading to Ser68 dephosphorylation and thus its nuclear export has not been fully elucidated.
Finally, ligand dependent activation of AhR is known to induce the expression of AhRR, a repressor protein of AhR (Mimura et al., 1999). AhRR is an atypical member of bHLH/PAS protein which possesses only a single PAS domain. However, phylogenetic tree analysis suggests that this protein is more closely related to AhR than any other mammalian bHLH/PAS proteins, and may arise from an early duplication of the AhR gene. At the protein level, the AhR repressor shares a high degree of sequence homology with AhR especially at the N-terminal third of protein, but the C-terminal part of the two proteins are quite divergent.
As its name suggests, AhRR functions as a negative regulator of AhR (Mimura et al., 1999).
However, the exact mechanism by which this occurs is not well understood. The initial study by
Mimura and colleagues showed that AhRR is capable of dimerizing with common bHLH/PAS dimerization partner protein Arnt and binding to cognate XRE sequence in vitro (Mimura et al., 1999).
Thus, it seems logical to assume a direct competition model between AhR and AhRR, where increased expression of AhRR inhibits AhR by either sequestering Arnt away from AhR or competing for XRE binding. A contradictory study by Evans and colleagues however showed that transient overexpression of Arnt in monkey kidney fibroblast COS-7 cells did not relieve AhRR mediated repression. Furthermore, a point mutant of AhRR (AhRR Y9F), which retains its full Arnt dimerization ability but fails to bind XRE, was still capable of repressing AhR (Evans et al., 2008).
Given these results, it is possible that AhRR forms higher order complex with AhR/Arnt heterodimers at the target XRE, which inhibit AhR target gene transcription by recruiting transcription co-repressors via the C-terminal half of the protein (Oshima et al., 2007), much as has been suggested for another bHLH/PAS protein Sim2 (Moffett et al., 1997).
1.3.2 The endogenous function of AhR
AhR-deficient mice have been independently generated from three laboratories by excision of either the first (Fernandez-Salguero et al., 1995; Mimura et al., 1997) or the second (Schmidt et al., 1996)
| 13
exons of AhR gene. As expected, the resulting AhR null mice strains fail to induce Cyp1a1 and
Cyp1b1 gene expression upon ligand challenge and are resistant to TCDD-mediated toxicity. In addition, AhR null mice strains also display a range of physiological defects, including growth retardation, reduced liver size, abnormalities in vascular structure, portal tract fibrosis, and decreased fertility (for a review, see McMillan and Bradfield, 2007b), thus, arguing for endogenous functions of AhR, especially during development.
The liver defect seen in AhR null mice has been linked to patent ductus venous (DV) (Lahvis et al.,
2000; Lahvis et al., 2005), a clinical manifestation characterized by abnormal persistence of an open lumen in the ductus venous after birth. During normal fetal development, oxygen- and nutrient-rich blood is fed into the developing heart and brain via the ductus venous, bypassing the requirement for hepatic filtration through the embryonic liver. However, shortly after birth, the DV is closed, forcing the blood from the portal vein to pass through the liver sinusoids before reaching the other organs such as heart and lung. Persistent opening of the DV during adulthood reduced the blood supply of the liver, leading to impaired postnatal liver growth, potentially via nutrient deprivation.
Thus, it is clear that the expression of AhR in mice is required in postnatal closure of DV, although the molecular mechanism by which this is achieved is not well understood (Lahvis et al., 2000;
Lahvis et al., 2005).
In addition to patent ductus venous, all three AhR knock out (KO) mice strains display reduced fertility, again, implying a role of AhR in development. It appears that the effect of AhR knock out on female reproduction is multi-dimensional, ranging from difficulties in maintaining conceptuses during pregnancy to abnormal placental development as well as reduced litter numbers and poor pup survival (Abbott et al., 1999). Studies by Baba and colleagues showed that AhR null female mice have marked reduced production of mature follicles, which is potentially due to insufficient synthesis of estradiol within the developing follicle (Baba et al., 2005). Thus, AhR appears to be estrogenic in this context, and expression of AhR is required for the proper biosynthesis of estradiol during oocyte maturation. Furthermore, the expression of AhR is up-regulated in the tissues connecting the embryos with the placenta and the uterus vasculature in wild type female mice (Kitajima et al., 2004).
Loss of AhR expression in AhR null mice resulted in placental labyrinth enlargement, which altered
14 |
the dam-to-pup filtration, leading to an increased deposition of teratogenic compounds to the embryonic compartment (Thomae et al., 2004). Finally, impaired mammary gland development has also been reported in the AhR null mice (Hushka et al., 1998), which could contribute to the observed low postnatal pup survival rate in littermates raised by AhR null dams.
Further evidence in support of an endogenous/physiological function of AhR came from studies of invertebrates AhRs, including those in nematode, fruit fly, zebra/blue mussels, and soft-shell clam.
Unlike mammalian AhRs, the invertebrate AhR orthologs, although working as transcription factors, do not bind to any of the known AhR ligands (Butler et al., 2001; Duncan et al., 1998;
Powell-Coffman et al., 1998). Instead, they regulate specific aspects of embryonic development, including GABAergic motor neuron fate specification in C. elegans (Huang et al., 2004), and dendrite arborization neuron diversification as well as antenna and leg development in D. melanogaster (Duncan et al., 1998; Kim et al., 2006a). Phylogenetic analysis suggested that the ability of mammalian and vertebrate AhRs to sense xenobiotics is acquired at later stage of evolution, which is secondary to their role in development. It is possible that the driving force for the evolutionary conservation of AhR not only lies on its role in xenobiotic metabolism both also in normal development.
The third line of evidence pointing to an endogenous and physiological function of AhR came from genome scale microarray studies, in which gene expression profiles of AhR null mice/cells were compared with their wild type counterparts, independent of AhR ligands. It becomes apparent that even without ligand treatment, disruption of a functional AhR expression in cells significantly alters the gene expression (Frericks et al., 2006; Tijet et al., 2006; Yoon et al., 2006), which highlights an intrinsic function of AhR in shaping the normal transcriptome of the cell.
Finally, AhR is also known to “cross-talk” with a broad variety of transcription factors, ranging from nuclear hormone receptors (NRs) to nuclear factor-kappa B (NF-κB) and retinoblastoma tumor suppressor protein (pRb) (reviewed in Barouki et al., 2007; Carlson and Perdew, 2002; Denison et al., 2011). Interaction between AhR and NRs/NF-κB can occur both in the presence and in the absence of exogenous AhR ligands (Ohtake et al., 2009; Tian, 2009 and references therein), thus
| 15
opening up new avenues for investigating the physiological function of AhR. A study by Kim and coworkers suggests that AhR physically interacts with the RelA subunit of NF-κB, leading to an AhR dependent and AhR ligand independent activation of c-myc gene expression, which promotes mammary cell proliferation and contributes to its tumorigenesis (Kim et al., 2000). On the other hand, interaction between AhR and retinoblastoma is ligand dependent, and TCDD dependent AhR/pRb interaction results in cell cycle arrest at G1 phase (Huang and Elferink, 2005; Kolluri et al., 1999;
Marlowe et al., 2004; Puga et al., 2000). Two independent mechanisms have been reported in
AhR/pRb mediated cell cycle arrest. In one study, interaction between AhR and pRb was shown to potentiate cdk2 (cyclin dependent kinase 2) inhibitor gene p27kip1 expression (Kolluri et al., 1999).
As cdk2 promotes G1 to S phase transition, inhibition of cdk2 activity by p27kip1 inhibits cell cycle progression. In the other study, interaction between AhR and pRb also leads to reduced cdk2 activity.
However, in this scenario, AhR forms a repression complex with pRb and E2F, which represses the expression of E2F target genes such as cyclin E (Marlowe et al., 2004; Puga et al., 2000), an obligate partner factor for cdk2 function. Interestingly, this AhR dependent gene repression does not require the transcriptional activity of the receptor (Puga et al., 2000), which is in striking contrast to
AhR dependent xenobiotic metabolism gene induction.
Taken together, these studies strongly support an endogenous and developmental role of AhR beyond simple adaptive xenobiotic metabolism. In the rest of this section, I will use two examples to further illustrate the role of AhR in normal physiology.
1.3.2.1 Cross talk between AhR and ER
Cross-talk between AhR and ER represents one of the best studied examples of transcription factor cross-talk, which involves both XRE dependent and XRE independent mechanisms (Figure 1.5).
However, the exact function of AhR on estrogen receptor signaling appears to be complex, and is likely to be cell context dependent (Boverhof et al., 2008; Boverhof et al., 2006; Cummings et al.,
1996; Gibbons, 1993). On the one hand, interaction between AhR and ER is pro-estrogenic, as mice deficient in AhR exhibited impaired ovarian follicle maturation (Benedict et al., 2000). On the other hand, activation of AhR by TCDD and other AhR agonists inhibits estrogen dependent uterus
16 |
Anti-estrogenic Pro-estrogenic
1) Degradation of ER by E3 ubiquitin ligase pathway 1) Enhancing the transcriptional activity of ER AhR E3 ubiquitin ER ER ligase Co-activator AhR 2) Sequestering transcription coactivators e.g. PR, TFF1 ER ER Co-activator ERE
3) Steric hindrance
e.g. TFF1
ER ER Arnt
AhR 2) Induction of Cyp19 (aromatase) iDRE ERE expression
4) Induction of
inhibitory protein Inhibitory Co-activator
protein Cyp19 Arnt
Co-activator AhR
XRE
Arnt AhR XRE Required for Cyp1a1,Cyp1b1 biosynthesis
5) Metabolising
17β-estradiol & estrone
Figure 1.5 Cross talk between AhR and ER. Activation of AhR by AhR ligands can be both pro- and anti-estrogenic depending on cell context. See main text for details. XRE: Xenobiotic Response Element, ERE: Estrogen Response Element, iDRE: inhibitory Dioxin Response Element, PR: Progesterone Receptor, TFF1: Trefoil factor 1.
development, highlighting an anti-estrogenic role of AhR in this tissue.
At the molecular level, ligand activated AhR has been reported to function as transcription coactivator of ER by directly interacting with ER (Ohtake et al., 2003). Interaction between AhR and
ER enhances the transcriptional potential of ER at estrogen responsive elements (EREs) even in the absence of ER ligands, resulting in increased ER target gene expression. This is perhaps not surprising given that other mammalian PAS proteins such as NCoA1, NCoA2 and NCoA3 have been shown to function exclusively as transcription cofactors (Anzick et al., 1997; Onate et al., 1995;
Voegel et al., 1996). Furthermore, there are also precedents for AhR to function as a ligand dependent transcriptional coactivator (Boutros et al., 2004; Sogawa et al., 2004). A study by
Sogawa and colleagues found that in response to ligand treatment, AhR is recruited to AhRE-II
(AhR response element like sequence) elements at the enhancer region of rat cyp1a2 gene via an unknown mediator protein(s) and upregulates cyp1a2 gene expression. In both of these cases, AhR may simply function as a scaffold protein that facilitates assembling of basal transcriptional machinery necessary for productive transcription.
Direct interaction between AhR and ER could also negatively affect ER target genes by promoting
ER degradation (Harris et al., 1990). In this case, the ligand bound AhR functions as substrate-specific recognition subunit of the CUL4B ubiquitin ligase complex, which polyubiquitinates ER and results in proteasome mediated ER degradation (Ohtake et al., 2007).
Further studies indicate that this AhR mediated protein degradation is not unique to ER, and ligand activated AhR could also target other transcription factors such as androgen receptor or β-catenin for degradation via a similar pathway (Kawajiri et al., 2009; Ohtake et al., 2007).
Cross-talk between AhR and ER may also involve indirect mechanisms. For example, activation of
AhR by AhR agonists upregulates the expression of key xenobiotic metabolizing enzymes such as cytochrome P450 Cyp1a1, Cyp1a2 and Cyp1b1, which promotes the clearance of 17β-estradiol (E2) and estrone (E1), the two main circulating forms of estrogens, and thus reduces ER target genes expression (Lee et al., 2003; Spink et al., 1990). Activation of AhR by AhR ligand could also titrate down the cellular pool of common transcription cofactors in a mechanism similar to that proposed
| 17
for other steroid hormone receptors in the “squelch” model (Meyer et al., 1989). Overexpression of
AhR transactivation domain in both rat hepatoma BP8 and human breast cancer T47D cells reduced E2 dependent ER activity in a reporter gene assay (Reen et al., 2002). Recently, Arnt, the obligate dimerization partner of AhR, has been shown to interact with ER directly, which could potentiate the transcriptional activity of ER (Brunnberg et al., 2003). Thus, activation of AhR may sequester Arnt away from ER, which in turn represses ER dependent target gene activation (Ruegg et al., 2008).
Direct competition between AhR and ER could also occur at the level beyond cofactor binding. It was suggested that several estrogen-responsive genes also carry a XRE like sequence known as iDRE (inhibitory Dioxin Response Element) at proximal promoter regions (Gillesby et al., 1997;
Krishnan et al., 1995; Safe et al., 1998; Wang et al., 2001). Intriguingly, iDREs are often present in close proximity with estrogen response elements or even overlapping with EREs. Thus, activation of
AhR by ligand could also promote binding of AhR/Arnt complex at iDRE, which blocks the E2 induced ER from binding at the adjacent ERE sites through steric hindrance.
The mechanism of AhR mediated interference of ER activity amy also stem from induction of inhibitory proteins, as blocking of protein synthesis with translation inhibitor cycloheximide prevents
TCDD induced inhibition of estrogen-responsive pS2 gene transcription in human ovarian carcinoma BG-1 cells (Rogers and Denison, 2002). However, this mechanism is likely to be cell context dependent and is less likely to be the major mechanism underlying the anti-estrogenic effect of AhR in most cell types.
Finally, a study by Bata and colleagues suggests that activation of AhR by AhR ligand upregulated ovarian P450 Cyp19 gene expression (Baba et al., 2005). The Cyp19 gene encodes an enzyme known as aromatase that catalyzes the final step of estrogen synthesis, and thus provides yet another mechanism for the pro-estrogenic effect of AhR.
18 |
1.3.2.2 AhR and immunity
Numerous studies have suggested a role of AhR (both ligand activated and unliganded) in immunomodulation. Exposure of laboratory rodents to TCDD has long been known to induce a profound immune suppression response and results in thymic involution (Kerkvliet, 1995; Kerkvliet,
2002). In addition, mice exposed to TCDD exhibit increased susceptibility to viral infection (Head and Lawrence, 2009). All of these effects were subsequently confirmed to be AhR dependent, as knock out of AhR in mice renders them complete protection against TCDD induced immune toxicity
(Esser, 2009). Furthermore, transgenic mice expressing a constitutively active form of AhR under the control of a T cell specific promoter recapitulates the thymic defect seen with TCDD exposed mice (Nagai et al., 2006; Nohara et al., 2005), again highlighting an important role of AhR in immunomodulation.
As the thymus is the major organ of T cell development, and age related thymus atrophy is proposed to be one of the major reasons behind increased susceptibility to infection in the elderly, a detailed understanding of the mechanisms underlying TCDD induced thymus involution has generated considerable interest. Studies by Gasiewicz and others suggested that persistent AhR activation reduces the number of T-lymphocytes in the thymus as a result of both AhR dependent impairment of thymus seeding by bone marrow-derived prethymic stem cells (Fine et al., 1990), and a reduction of thymocyte proliferation (Kremer et al., 1994; Laiosa et al., 2003). Furthermore, premature emigration of CD4/CD8 double-negative (DN) progenitor T-cells from the thymus to peripheral lymphoid organs (Temchura et al., 2005), as well as AhR dependent induction of KLF2
(Kruppel-like factor 2) gene expression in developing thymocytes, which inhibits thymopoeisis
(McMillan et al., 2007c), may also contribute to the observed severe thymus atrophy following
TCDD exposure.
In addition to immune suppression and thymic involution discussed above, studies from mice exposure to low dose of TCDD (i.e. AhR over-activation) as well as AhR null mice (i.e. AhR under-activation) have also suggested a role of AhR in hematopoiesis (Casado et al., 2010; Singh et al., 2009). It was proposed that AhR acts as a negative regulator of HSC (hematopoietic stem cell) proliferation, while knock out of AhR in mice leads to spleen enlargement in juvenile and adult mice, | 19
as well as an increased proliferation of HSCs (Singh et al., 2011). In contrast, activation of AhR by
TCDD leads to inappropriate expression of AhR target genes which blocks the differentiation of progenitor cells into different lineages, depletes HSC compartment by stem cell exhaustion, and alters HSC/stroma microenvironment interactions (Casado et al., 2010). Consistent with this notion, accidental exposure to TCDD and related PAHs in humans is associated with increased incidence of leukemia and lymphoma (Bertazzi et al., 1999; Fingerhut et al., 1991; Hooiveld et al., 1998).
In a simplified model, immune defense can be broadly divided into innate immunity and adaptive immunity (for a recent review, see Moser and Leo, 2010). The innate immune response provides the first line of defense against environmental pathogens that have entered the body and is mediated primarily through neutrophils, eosinophils, mast cells, natural killer (NK) cells, macrophages and dendritic cells (DCs). However, the innate immune response is nonspecific, which does not lead to the development of immunological memory. Switching from innate to adaptive immunity results in clonal expansion of T and B lymphocytes, which accelerates pathogen clearance, and promotes the generation of memory cells capable of mounting a faster and stronger attack each time the same pathogen is encountered. Not surprisingly, cells from both innate and adaptive immune systems are vulnerable targets of TCDD toxicity (Kerkvliet, 2009; Stevens et al., 2009; Stockinger et al., 2011). In the rest of this section, I will discuss the current understanding on the mechanism of TCDD mediated immunomodulation with regard to each of these immune cells.
Neutrophils
The effect of TCDD on neutrophil function is best studies in the context of influenza virus infection
(Head and Lawrence, 2009). Following respiratory viral infection, there is a marked increase in neutrophil recruitment and neutrophil dependent interferon-γ (IFN-γ) secretion in the airway and lung interstitium of TCDD treated wild type mice as compared to AhR null mice (Neff-LaFord et al.,
2007; Teske et al., 2005). The AhR dependent excessive neutrophil recruitment is likely to contribute to the increased pathology of TCDD treated mice, such as decreased host response and reduced survival. In addition, elevated neutrophil infiltration has also been observed in the peritoneal cavity and spleen of TCDD treated mice following intraperitoneal sodium caseinate injection or P815 tumor cell challenge, respectively (Ackermann et al., 1989; Choi et al., 2003).
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Dendritic cells
The dendritic cell is one of the two major types of antigen presenting cells (APCs), which migrates to the lymph nodes and activates T cells when charged with antigen, thus linking the innate and adaptive immunity (Moser and Leo, 2010). Interestingly, the expression level of AhR is up-regulated in both bone marrow derived DC and splenic DC following 24 hours of lipopolysaccharide (LPS) or
CpG DNA treatment (Nguyen et al., 2010), thus making DC an ideal candidate for AhR dependent regulation. On the one hand, DCs isolated from mice exposed to TCDD exhibited an increased
T-cell stimulatory ability, characterized by elevated expression of MHC-II (major histocompatibility complex class II), ICAM-1 and CD24 adhesion molecules, and CD40 co-stimulatory receptor (Lee et al., 2007; Vorderstrasse and Kerkvliet, 2001). On the other hand, the number of DCs in the spleen or bone marrow of TCDD treated mice decreased due to premature apoptosis of DCs (Ruby et al.,
2005; Vorderstrasse et al., 2003).
Macrophages
Similar to DCs, macrophages are also APCs that express high levels of AhR following LPS or CpG
DNA stimulation (Kimura et al., 2009). In this case, elevated expression of AhR is thought to be protective against persistent inflammation, as activation of macrophage in AhR null mice leads to increased production of pro-inflammatory cytokines such as IL-6 (interleukin-6), IL-12 and TNF-α
(tumor necrosis factor-α), whereas at the same time, the production of anti-inflammatory cytokine
IL-10 is largely impaired (Kimura et al., 2009). Furthermore, consistent with this difference in inflammatory cytokine productions, AhR null mice were also found to be more sensitive to LPS induced lethal shock than their wild type littermates.
The AhR dependent repression of IL-6 pro-inflammatory cytokine production following LPC stimulation of macrophages is believed to occur via two independent mechanisms (Figure 1.6).
Firstly, AhR is thought to form a repressor complex with Stat1 (signal transducer and activator of transcription 1) and NF-κB which acts directly on the IL-6 promoter and inhibits its transcription in
LPS stimulated macrophages (Kimura et al., 2009). Secondly, interaction between AhR and Sp1 in
LPS stimulated macrophages could also inhibit HDC (histidine decarboxylase) expression and thus reduce the production of histamine, an important signal molecule involved in the inflammatory
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response via promoting secretion of pro-inflammatory cytokines by macrophages (Masuda et al.,
2011).
Th1 and Th2 cells
Both Th1 and Th2 T cells are CD4+ helper T cells that function to aid other white blood cells; however, there are fundamental differences between these two cell types (Moser and Leo, 2010).
The Th1 T lymphocyte secretes mainly IFN-γ that promotes the differentiation of CD8+ CTLs and maximizes the phagocytosis of macrophages, thus providing effective cellular immunity against intracellular pathogens. In contrast, Th2 T lymphocytes produce cytokines such as IL-4 and IL-5, which stimulate B-cells proliferation, leading to an IgE mediated humoral immune response against extracellular pathogens. However, excessive IgE secretion leads to immune hypersensitivity such as asthma, although the production of IgE antibodies is beneficial against extracellular parasites.
Thus, the balance between Th1/Th2 cytokine production determines the direction and outcome of an immune response, including allergy response.
It has been shown that activation of AhR by benzoimidazole derivatives M50354/M50367, as well as classic AhR ligands TCDD, β-NF and 3MC skewed Th1/Th2 cell balance towards Th1 dominance and repressed Th2 cytokines production (Figure 1.6) (Fujimaki et al., 2002; Inouye et al., 2005;
Negishi et al., 2005). This repression in Th2 cell polarization is also coupled with suppressed IgE antibody secretion and reduced peritoneal eosinophil count (Negishi et al., 2005). A similar result was also reported in a mouse model of antigen-induced eosinophilic inflammation, while treatment of VAF347, a known ligand of AhR (Lawrence et al., 2008), blocks lung eosinophilia and serum IgE level (Ettmayer et al., 2006). Furthermore, ectopic expression of a constitutively active form of AhR in anti-CD3/CD28 antibody stimulated naïve T cells recapitulates the AhR dependent immune suppression seen in M50354 treated mice. On the other hand, addition of AhR antagonist resveratrol increases both Th1 and Th2 cell populations, which mirrors the result observed in AhR null mice (Negishi et al., 2005). Mechanistically, M50354 dependent reduction in Th2 polarization is believed to occur via inhibition of GATA-3 expression, and requires the presence of AhR. However, the molecular mechanism underlying M50354 mediated GATA-3 gene repression has not been fully elucidated, although M50354 is known to function as an agonist of AhR (Morales et al., 2008;
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Cellular immunity AhR (M50354) 1) α-CD3 & α-CD28 IFN γ intracellular 2) DNP Ascaris extracts pathogens AhR (M50354) Gata3 AhR (β-NF or 3MC) Humoral immunity CA_AhR IL4, IL5 extracellular pathogens AhR inhibits Stat1 & Stat5 AhR AhR IL6 (or IL21)/TGFβ+FICZ or EAE+FICZ Defence against AhR IL6/TGFβ + AhR antagonist extracellular ligands pathogens & AhR+/+ > AhR -/- & IMDM > RPMI IL17a, IL17f IL17a, IL17f, IL22 Autoimmunity Naive T cell (α-CD3/CD28+IL6/TGFβ or EAE)
Parent-into-F1 GVH CTL response + TCDD AhR PIA + VAF347(VAG539) CD4+ CD25+ AhR Suppression of T IL10 cell response & NOD mice + TCDD or EAE + TCDD AhR ligands α-CD3/CD28 + AhR antagonist Immune Tolerance + + AhR+/+ > AhR -/- & AhRb > AhRd CD4 FoxP3 2) HDC AhR LPS AhR LPS or CpG DNA AhR (Kyn) 1) PIA VAF347(VAG539) Mϕ DC IL6 histamine
Figure 1.6 AhR and immunomodulation. See main text for details. EAE: Experimental Autoimmune Encephalomyelitis, GVH: Graft-vs-Host, NOD: Nonobese Diabetic, PIA: Pancreatic Islet Allotransplantation, IMDM: Iscove's Modified Dulbecco's Medium, RPMI: Roswell Park Memorial Institute medium, DC: Dendritic Cell, Mϕ: Macrophage.
Negishi et al., 2005).
Regulatory T cells (Treg)
The regulatory T cell is a recently identified subpopulation of CD4+ T lymphocytes that is loosely defined by the expression of CD25 surface antigen and/or FoxP3 (forkhead box P3) transcription factor (Moser and Leo, 2010). Increased Treg polarization prevents the development of autoimmune diseases by suppressing the inflammatory response against self-antigens, which is accomplished in part by the production of IL-10 family anti-inflammatory cytokines.
Recent studies have suggested a link between Treg differentiation and AhR signaling pathways, providing a plausible mechanism for TCDD mediated immunosuppression. It appears that activation of AhR promotes Treg polarization (Figure 1.6). Consistently, naïve T cells isolated from either AhR null mice or mice carrying a low affinity AhR receptor (encoded by AhRd allele) are inefficient at generating Treg cells (Kerkvliet et al., 2009; Mezrich et al., 2010; Quintana et al., 2008). Moreover, in contrast to naïve T cells, which have low levels of endogenous AhR (Frericks et al., 2007), the expression of AhR is increased in both CD4+FoxP3+ and CD4+CD25+ Treg cells, further suggesting a role for AhR in these cells (Funatake et al., 2005; Quintana et al., 2008).
It was proposed that the regulatory T cell marker FoxP3 is a direct target of AhR, since activation of
AhR by TCDD enhances AhR binding at the FoxP3 promoter, and increases FoxP3 gene expression (Quintana et al., 2008). In addition, a single dose of TCDD treatment suppresses EAE
(experimental autoimmune encephalomyelitis), a mouse model of multiple sclerosis, in MOG35-55
(myelin oligodendrocyte glycoprotein peptide) immunized mice (Quintana et al., 2008). Chronic
TCDD exposure in nonobese diabetic (NOD) mice also leads to an increased FoxP3+ Treg differentiation in the pancreatic lymph nodes and suppresses autoimmune type 1 diabetes (Kerkvliet et al., 2009). Furthermore, anti-CD3/CD28 antibody stimulated naïve T cells treated with AhR agonist TCDD or β-NF show increased Treg differentiation (Quintana et al., 2008), while co-treatment with AhR antagonists resveratrol or CH223191 inhibits Treg polarization (Mezrich et al.,
2010; Quintana et al., 2008).
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The effect of TCDD and AhR on Treg polarization has also been demonstrated in an in vivo acute parent-into-F1 graft-vs-host (GVH) model (Funatake et al., 2005; Marshall et al., 2008). In this model, T cells isolated from C57BL/6 mice (H-2bb) were injected intravenously into F1 host mice
(derived from crossing C57BL/6 and DBA/2 mice [H-2dd]) of mixed haplotype (H-2b/d). As the T cells from F1 host express both H-2b and H-2d antigens, the cells will not recognize the injected donor cells as foreign. However, alloreactive donor T cells recognize the H-2d antigen expressed on F1 host, leading to a donor T cell mediated CTL response against host cell (Kerkvliet et al., 2002). On the other hand, treatment of the host mice with TCDD promotes CD4+CD25+ Treg cells differentiation from alloreactive donor derived CD4+ T cells, which reduces allograft response via production of IL-10 family anti-inflammatory cytokines and suppression of IL-2 secretion by responder T cells. This process is likely to be AhR dependent as addition of TCDD had no effect on the CTL response when the donor T cells were obtained from AhR null mice (Funatake et al., 2005;
Marshall et al., 2008).
Recently, two independent groups have proposed a third model for AhR dependent CD4+FoxP3+
Treg differentiation that is mediated by dendritic cells and kynurenine, a tryptophan metabolite and a recently identified agonist of AhR (Mezrich et al., 2010; Nguyen et al., 2010). Production of kynurenine in dendritic cells is catalyzed by tryptophan oxidoreductase enzymes known as indoleamine-2, 3-dioxygenases (IDO). Interestingly, the expression of IDO itself is induced by AhR
(Mezrich et al., 2010; Nguyen et al., 2010; Vogel et al., 2008), leading to a feed forward loop for AhR dependent accumulation of kynurenine in dendritic cells. In agreement of these results, elevated levels of kynurenine have been detected in the area of clinical inflammation (Heyes et al., 1997).
Kynurenine dependent activation of AhR in naïve T cells promotes Treg differentiation, which functions to control the level and the extent of inflammation (Mezrich et al., 2010; Nguyen et al.,
2010). The identification of the tryptophan metabolite kynurenine as a modulator of inflammation response is intriguing, and has stimulated a general interest in designing a high affinity and nontoxic
AhR agonist to treat autoimmune disease. For example, the anti-allergy compound Tranilast®, a synthetic derivative of the kynurenine metabolite 3-hydroxyanthranilic acid, was shown to suppress
EAE through generation of Treg via AhR dependent pathways (Kerkvliet, 2009; Platten et al., 2005).
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In addition to Tranilast®, other pharmaceutical compounds that show promise in long term allograft acceptance include anti-allergy drugs VAF347 and VAG539 (Hauben et al., 2008). Both VAF347 and VAG539 are ligands of AhR (Lawrence et al., 2008), and oral administration of VAF347 in mice having undergone pancreatic islet allotransplantation (PIA) promotes Treg differentiation, coinciding with reduced graft rejection (Hauben et al., 2008).
Th17 cells
The Th17 cell is a relatively new member of CD4+ effector T cells which, in contrast to Treg cells, secretes pro-inflammatory cytokines IL-17a, IL-17f, and IL-22, and is involved in autoimmune conditions such as multiple sclerosis and rheumatoid arthritis (Marshall and Kerkvliet, 2010). Owing to their directly opposing roles in autoimmune disease, the outcome of immune response against self-antigens and thus the severity of autoimmune diseases are likely to be determined by the balance between Treg and Th17. It appears that the balance between Treg and Th17 is modulated by the local cytokine milieu. In in vitro in cell culture models, treatment of anti-CD3/CD28 antibody stimulated naïve CD4+ T cells with TGF-β (transforming growth factor β) alone induces Treg differentiation, whereas co-treatment with TGF-β plus IL-6 or IL-21 induces Th17 polarization.
Under Th17-polarizing conditions, activation of AhR by FICZ (6-formylindolo[3,2-b]carbazole), a candidate endogenous ligand of AhR, enhances Th17 polarization and pro-inflammatory cytokine production (Veldhoen et al., 2008), suggesting a possible link between ligand dependent AhR activation and Th17 differentiation (Figure 1.6). Consistent with this result, activation of AhR in vivo by FICZ treatment of mice immunized with MOG35-55 peptide exacerbates the severity of EAE and is associated with the early onset of the disease (Veldhoen et al., 2008). It was proposed that AhR did not participate in the initial stage of Th17 differentiation, but rather, is required for the terminal differentiation of Th17. Consistent with this notion, CD4+ T cells isolated from AhR deficient mice are competent in generating Th17 cells in Th17 conditioned medium. However, the AhR-/- Th17 cells produce significantly less IL-17 and the production of IL-22 is completely silenced in these cells
(Kimura et al., 2008; Veldhoen et al., 2008). Furthermore, treatment of Th17 cells with AhR antagonist CH-223191 blocks AhR dependent IL22 secretion, similar to that seen in AhR-/- Th17 cells (Veldhoen et al., 2009), while on the other hand, reconstitution of AhR in AhR null Th17 cells by
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retroviral transduction restores IL-22 production by mature Th17 cells (Veldhoen et al., 2008). The observation that FICZ, a photooxidation product of tryptophan, is a potent inducer of Th17 differentiation could also provide a logical explanation on the efficiency of different culture media in generating Th17 cells. In line with this hypothesis, it was shown that aromatic amino acid rich medium IMDM (Iscove's Modified Dulbecco's medium), proposed to contain more FICZ type of
“endogenous” AhR ligands, is far more efficient than standard RPMI (Roswell Park Memorial
Institute) medium for optimal Th17 cell differentiations (Veldhoen et al., 2009).
Perhaps one of the most intriguing features of Th17 cells is their marked induction of AhR expression (Kimura et al., 2008; Veldhoen et al., 2008), which is comparable to that seen in liver cells and is several orders of magnitude higher than that observed in other immune cells including
Tregs, again, pointing to a direct role of AhR in Th17 polarization. In fact, studies by Kimura and colleagues suggested that AhR directly interacts with transcription factors Stat1 and Stat5, both of which are negative regulators of Th17 differentiation, and promotes Th17 polarization by interfering with Stat1 and Stat5 functions in a currently unknown mechanism (Kimura et al., 2008).
1.3.3 Evolutionary diversity of AhR-- What have we learnt?
AhR orthologs have been identified from a large number of species, including mammals, fish, amphibians, birds, and invertebrates such as flies and insects (for reveiws, see Hahn, 2002; Okey et al., 2005). Mammals and invertebrates have only a single AhR gene, while fish and birds may have up to three AhR genes. Expression analysis together with in vitro radioactive [3H]TCDD binding assays and reporter gene assays indicated that AhR1 is the predominant form of AhR in birds, while in fish, the AhR2 is the primary receptor involved in the xenobiotic response (Prasch et al., 2003;
Tanguay et al., 1999; Yasui et al., 2007). A third AhR homolog has been suggested for a number of fish and avian species including cartilaginous fish (e.g. spiny dogfish), bony fish (e.g. zebrafish), and chicken (Hahn et al., 2006). These AhR homologs were believed to have arisen from tandem and genome-wide duplication events prior to the separation of fish and tetrapod lineages, which was subsequently lost sometime around the divergence of bird and mammalian lineages (Hahn, 2002;
Hahn et al., 2006; Yasui et al., 2007). In addition, phylogenetic tree analysis of AhR between
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chordates (i.e. vertebrates, fishes, amphibians, birds, mammals) and invertebrates suggested that the ability of AhR to bind exogenous ligands was likely to be acquired at the split of chordate and echinoderm lineages (Hahn et al., 2006).
Recently, Wirgin and colleagues performed a comprehensive survey on Atlantic tomcod AhR2 alleles along the Atlantic coast estuaries, including St. Lawrence, Miramichi, Margaree, Squamscott,
Niantic, Hudson, and Hackensack Rivers (Wirgin et al., 2011). Two predominant AhR2 alleles
(AhR2-1 and AhR2-2) have emerged from this study. These two alleles differ by five polymorphisms, including one nonsynonymous base substitution at exon 11 and a six nucleotide deletion in exon 10.
Interestingly, Atlantic tomcod from Hudson River, which has been pre-exposed to TCDD and other
HAHs, carry almost exclusively the AhR2-1 allele. In contrast, tomcod from nearby estuaries such as Hackensack River, Shinnecock Bay and Niantic River are heterozygous for AhR2-1 and AhR2-2 alleles, while tomcod from more distant estuaries carry exclusively the AhR2-2 allele. In vitro
[3H]TCDD binding assays suggested there is a significant difference in TCDD binding affinity between the two AhR2 alleles, while the binding affinity of AhR2-1 is at least 5 fold less than that of
AhR2-2. Thus, these differences in ligand binding affinity may explain the observed difference in
TCDD tolerance among tomcods along Atlantic coast estuaries, with tomcods from Hudson River expressing almost exclusively AhR2-1 allele have highest resistance to TCDD and other HAHs
(Wirgin et al., 2011).
Intra-species variations in TCDD sensitivity have also been observed in vertebrate species, which could again be attributed to the difference in AhR alleles. For example, the Han/Wistar (H/W) rats are 1000 fold more resistant to TCDD induced toxicity than the Long-Evans (L-E) rats (Pohjanvirta et al., 1988; Unkila et al., 1994). Comparison between AhR proteins from H/W rats and L-E rats suggests that there is virtually no difference between two AhRs in terms of TCDD binding, nor is there any marked differences in their ability to heterodimerize with Arnt or bind to XRE in vitro
(Pohjanvirta et al., 1999). However, genetic characterization of AhR locus between TCDD-resistant
H/W rats and TCDD-sensitive L-E rats revealed two single nucleotide mutations in the AhR of H/W rats (Pohjanvirta et al., 1998). One of these mutations is found at exon/intron junction near exon 10, which disrupts normal splicing of the rat AhR gene, resulting in two short forms of AhR with 38 or 43
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amino acids truncation at the C-terminal transactivation domain, respectively (Pohjanvirta et al.,
1998). Interestingly, although this genetic variation in AhR alleles leads to a dramatic difference in
TCDD sensitivity, comparable TCDD induced Cyp1a1 and AhRR induction was observed in both
H/W and L-E rats (Korkalainen et al., 2004).
Similarly, extensive polymorphisms have been identified in mice AhR. At least four phenotypic haplotypes of AhR exist in mice, namely AhRd, AhRb1, AhRb2, and AhRb3 (Poland et al., 1994;
Thomas et al., 2002). All AhR isoforms encoded by the AhRb alleles (i.e. AhRb1, AhRb2, and AhRb3) bind TCDD with high affinity. In contrast, the binding affinity of AhR encoded by AhRd allele is approximately 10 fold lower than that of AhRb1 alleles. Sequence analysis revealed two alternations between the AhRb1 allele of C57BL/6 mice and the DBA/2 mice AhRd allele, including a single SNP
(single nucleotide polymorphisms) at exon 9 and a T to C mutation at the first letter of the stop codon, leading to a 43-amino acid extension at the C-terminus of C57BL/6 AhR (Okey et al., 1989).
A subsequent mutagenesis study suggested that the key determinant for the observed difference in the sensitivity of TCDD between AhRb1 allele and AhRd allele resides on the SNP at exon9, which is substituted from alanine in C57BL/6 AhR to valine in DBA/2 AhR (Ema et al., 1994). It is proposed that the side chain of alanine 375 in murine AhR points directly into the ligand binding pocket of the receptor. Substitution of alanine (-CH3) with more bulky amino acid valine (-CH(CH3)2) at this position increases the size of the amino acid side chain, which blocks ligands from access to the
LBD, and thus reduces the affinity of AhR to its ligands (Ema et al., 1994). Consistent with this notion, ectopic expression of mutant AhR with even bulkier amino acids isoleucine
(CH(CH3)CH2CH3) or phenylalanine (-CH2-benzene ring) at position 375 failed to induce any
AhR-mediated transcriptional activity in AhR null hepatocytes (Murray et al., 2005).
The common tern (Sterna hirundo) AhR also possesses alanine at codon 381, which is positionally equivalent to A375 in the mouse AhR. However, in contrast to chicken AhR, which has a slightly bulkier amino acid serine (-CH3OH) at the equivalent position, the tern AhR is about 80- to 250-fold less sensitive in TCDD mediated toxicity, and ~7 fold weaker in its ability to bind TCDD (Karchner et al., 2006). Interestingly, mutating of A381 in tern AhR to serine significantly increases the TCDD binding affinity of tern AhR to a comparable level of chicken AhR. Thus, substitution of this crucial
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alanine amino acid with a slightly bigger but more polar amino acid may in fact increase the binding affinity of AhR to TCDD, potentially by stabilizing the ligand-receptor interaction through hydrogen bonding.
Similar to DBA/2 mice, human AhR also carries valine at the equivalent position of murine AhR
A375, and thus has much weaker binding affinity to TCDD than mouse AhRb1 allele (Ema et al.,
1994). Due to their difference in TCDD sensitivity, “humanized AhR” knock-in mice were developed as a model system to study the toxicity of TCDD in human (Moriguchi et al., 2003). As expected, the humanized AhR mice are less responsive to TCDD than C57BL/6 and DBA/2 mice in general, consistent with reduced affinity of hAhR to TCDD, although homozygous hAhR fetuses developed severe kidney defects following maternal exposure of TCDD (Moriguchi et al., 2003). Microarray comparison was also performed with 6 hour TCDD treated primary hepatocytes freshly isolated from hAhR knock-in mice or C57BL/6 control mice (Flaveny et al., 2010). In general, fewer genes were regulated by hAhR than mAhRb1 proteins with only 15% of mouse AhRb1 regulated genes also regulated by hAhR (Flaveny et al., 2010), again highlighting the difference between hAhR and mAhR in TCDD sensitivity. In addition to the difference in the ligand binding domain, differences in the C terminal transactivation domain may also contribute to increased TCDD tolerance in AhR humanized mice as human AhR shares only 58% of amino acid identity with mouse AhR. This difference in CTD between human and mice AhR may result in differential recruitment of transcription co-activators, leading to different sets of target gene expression following activation by xenobiotics (Flaveny et al., 2008).
A number of genetic variations in the human AhR locus have also been identified; however, most of these polymorphisms do not interfere with receptor function, although in vitro [3H]TCDD binding assays revealed approximately 10-fold variation among human AhR variants in their ability to bind
TCDD (reviewed in Harper et al., 2002). In 2008, the international 1000genomes project
(http://www.1000genomes.org/) was launched with the aim of sequencing 2500 individuals from all ethnic populations to identify the majority of genetic variants that have frequencies of at least 1% in the populations. At this stage, there are more than 100 SNPs documented for hAhR cDNA alone, including 30 synonymous and nonsynonymous SNPs in the coding region and 75 SNPs in the
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untranslated region (UTR). Extending the search to include the entire AhR locus identified hundreds of SNPs. There is no doubt that with the timely completion of this project in the near future, more of the AhR polymorphisms will be revealed. It will thus be interesting to study these AhR polymorphisms in the context of diseases such as cancer progression and autoimmunity.
Perhaps one of the best studied hAhR polymorphisms is the CTD polymorphism at codon 554, which encodes for either arginine or lysine with relative frequencies of 0.57 and 0.43, respectively, in a Japanese population study (n=277) (Kawajiri et al., 1995). Subsequent studies by Wong and colleagues extended this study to include other ethnic groups, and showed that the hAhR L554 allele is also prevalent in the African population (allele frequency = 0.53) and Canadian Chinese populations (allele frequency = 0.32), but is very rare in German Caucasians (allele frequency =
0.07) (Wong et al., 2001a). Interestingly, although the initial report by Kawajiri and the subsequent report by Cauchi failed to establish a link between the hAhR codon 554 variant and the incidence of lung cancer in either Japanese or French populations (Cauchi et al., 2001; Kawajiri et al., 1995), studies by Berwick and coworkers suggested that having lysine at the 554 position of hAhR rather than arginine significantly and adversely affect the survival rate of soft tissue sarcoma patients from a mixed background (Berwick et al., 2004). However, the molecular basis of this association is unknown, and in vitro EMSA assay indicated that both hAhR variants can be readily transformed to a DNA binding species in the presence of TCDD (Wong et al., 2001b).
In addition to the intra-species variations, inter-species variation in TCDD toxicity has also been reported among mammalian species. Guinea pig is perhaps one of the most TCDD-sensitive mammals with LD50 (Lethal Dose) for TCDD at about 1µg/kg. In contrast, hamsters are approximately 1000-fold more resistant to TCDD than guinea pigs (reviewed in Hahn, 2002). As with the intra-species variation observed in L-E and H/W rats, the large degree of variation between guinea pig and hamster in TCDD sensitivity is likely to reside in the composition of their C-terminal transactivation domains of AhR rather than their ligand binding domain, as both AhRs dimerize with
Arnt and bind to XRE in vitro with roughly equal affinities, but they differ significantly in the sequence of their C-terminal glutamine (Q)-rich subdomains (Korkalainen et al., 2001).
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1.3.4 AhR ligands
In the canonical pathway, activation of AhR requires AhR agonists. One of the most potent agonists of AhR is TCDD, which activates AhR in the picomolar (pM) range (Fisher et al., 1989; Israel and
Whitlock, 1983). Other high affinity AhR agonists commonly used in AhR research include
β-Naphthoflavone (β-NF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3’,4,4’,5-pentachlorobiphenyl
(pentaCB), 3-methylcholanthrene (3MC), and benzo[α]pyrene (B[α]P) (Figure 1.7). Structure activity relationship (SAR) analysis using the above mentioned AhR ligands together with other high affinity
HAH/PAH type of AhR ligands suggested that the ligand binding pocket of AhR is promiscuous, which can accommodate a large number of planar and hydrophobic compounds with maximal van der Waals dimensions of 14 Å x 12 Å x 5 Å (Waller and McKinney, 1995). In addition, analysis of a large group of polychlorinated dibenzo-p-dioxin (PCDD) type of AhR ligands suggests that the general requirements for a high affinity ligand is a concentrated negative electrostatic potential at the extremes of the long axis and a depleted charge at the center of the molecule above and below the aromatic ring (Fraschini et al., 1996).
In addition to HAHs and PAHs, synthetic compounds that fall outside aromatic classification such as isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439) have also been shown activate AhR (Lee et al., 1996). Furthermore, cellular metabolites and dietary plant compounds including heme metabolites, tryptophan metabolites, lipoxin A4, prostaglandin G2, and indirubin have been shown to induce AhR activation in a fashion that is similar to classical AhR agonists (reviewed in Denison and Nagy, 2003; Nguyen and Bradfield, 2008). However, with exception of indirubin and FICZ (Adachi et al., 2001; Oberg et al., 2005), most of these ligands are low affinity ligands, which are likely to be rapidly metabolized under normal physiological conditions.
Recently, kynurenine, the first breakdown product in the tryptophan metabolism pathway, has been shown to function as an endogenous agonist of AhR and play an important roles in the tumor microenvironment (Opitz et al., 2011). The average concentration of kynurenine in tumour tissue from mice bearing human glioma xenografts is around 37 ± 13 μM, which is well beyond the estimated dissociation constant (Kd) of kynurenine for mouse AhR (~4μM). Unlike the kynurenine produced in dendritic cells (section 1.3.2.2), production of kynurenine in tumor cells is thought to | 31
acheived via another tryptophan oxidoreductase enzyme known as tryptophan-2,3-dioxygenase
(TDO). Knock down of TDO in U87 glioma cells reduces kynurenine production, which coincides with decreased AhR target gene expression, again, arguing for an endogenous role of kynurenine in
AhR activation. It is possible that the endogenous tumor-derived kynurenine activates AhR in both autocrine and paracrine fashions by enhancing tumor growth and mobility (autocrine regulation) and decreasing the infiltration of anti-tumor immune cells (paracrine regulation) (Opitz et al., 2011).
However, this effect may be cell type specific, as a previous study suggested that in both human hepatoma HepaG2 and primary human hepatocytes, kynurenic acid (KA), a kynurenine derivative further down the tryptophan metabolism pathway, rather than kynurenine itself, is responsible for most of the observed agonist activity of AhR (DiNatale et al., 2010).
Interestingly, although all AhR agonists lead to AhR transformation, the kinetics of AhR activation is different among different AhR agonists. For example, the PAH type of ligand β-NF is able to induce an oscillatory recruitment of AhR and Arnt complex at Cyp1a1 promoters within a 150mins time course (Hestermann and Brown, 2003; Wihlen et al., 2009); however, a similar cyclic pattern of AhR recruitment was only observed for TCDD and 3MC in a much longer time course (up to 4.5 hours)
(Matthews et al., 2005; Pansoy et al., 2010).
Despite the difference in their chemical characteristics, all of the above mentioned AhR ligands function as AhR agonists and induce AhR target gene expression via the canonical AhR signaling pathway. In addition, other compounds have also been shown to interact with AhR, but instead, they function as either AhR antagonists or selective AhR modulators (SAhRMs).
The PAH compound α-Naphthoflavone (α-NF) (Blank et al., 1987) is one of the first characterized
AhR antagonists, which inhibits TCDD dependent AhR activation as well as AhR target gene induction at low concentration (<1μM). It was thought that α-NF bound to AhR via the same ligand binding pocket as AhR agonists. But unlike agonists, α-NF induces a different kind of conformational change that inhibitss AhR transformation. As a result, binding of AhR antagonists to the receptor blocks agonists from accessing the ligand binding pocket, and inhibits AhR activation. Strikingly, the ability of α-NF to inhibit AhR activity is concentration dependent. At higher concentration (i.e.
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AhR agonists
HAHs
TCDD PentaCB TCDF
PAHs
3MC B(α)P βNF
Endogenous
Kynurenine Indirubin FICZ
Atypical
YH439 Lipoxin A4 AhR antagonists
Resveratrol MNF
GNF351 αNF CH-223191 SAhRMs
Anti- estrogenic 6-MCDF DIM
Anti- inflammatory Way-169916 SGA360
Figure 1.7 Structures of AhR ligands. AhR ligands are planar and hydrophobic compounds with maximal dimensions of 14 Å x 12 Å x 5 Å. Functionally, AhR ligands can be divided into three classes, including agonists, antagonists and selective AhR modulators (SAhRMs). See main text for detail.
1-10μM), α-NF also exhibits weak agonist activity (Santostefano et al., 1993), which seems to be a common feature for most AhR antagonists (Murray et al., 2010a; Smith et al., 2011).
In addition to α-NF, other compounds such as 2-Methyl-2H-pyrazole-3-carboxylic Acid
(2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) (Kim et al., 2006b), ellipticines (Gasiewicz et al.,
1996), resveratrol (Ciolino and Yeh, 1999), curcumin (Ciolino et al., 1998), certain anthraquinones
(Fukuda et al., 2009), vegetable constituents (Amakura et al., 2003), and flavone substitutes (Henry et al., 1999; Lee and Safe, 2000) have all been shown to function as AhR antagonists that represses agonist dependent AhR activation. Interestingly, a recent study suggests that the AhR antagonist
CH-223191 is also selective and preferentially inhibits HAH type of AhR agonists, but has little antagonistic effect towards PAHs, flavonoids and indirubin (Zhao et al., 2010).
Structure activity relationship analysis of the ellipticine type of AhR antagonists suggested that potent AhR antagonists often have an electron-rich center near a terminal position along the lateral-axis (Gasiewicz et al., 1996). Similarly, potent flavone type of AhR antagonists also have an electron withdrawing group such as nitro (-NO2), azide (-N3), or thiocyanate (-SCN) at carbon 4’ position (Henry et al., 1999). Recently, a study by Henry and Gasiewicz suggested that the strong electronegative substituents in the flavone type of AhR antagonist form hydrogen binding with positively charged basic amino acid residues in the AhR LBD, restraining the orientation of antagonists in a way that prevents AhR transformation. In support of this hypothesis, mutation of arginine 355 to isoleucine in murine AhR changed 3’-methoxy-4’-nitroflavone (MNF) from a potent
AhR antagonist to agonist (Henry and Gasiewicz, 2008).
Selective AhR modulators represent the third class of AhR ligands. The concept of SAhRM was initially introduced by studying the cross talk between AhR and ER (Safe et al., 1999). It has long been known that AhR exhibits both pro- and anti-estrogenic effect, with agonist (e.g. TCDD) activated AhR either potentiating or inhibiting ER activity depending on the cellular context (section
1.3.2.1). It was reasoned that compounds that do not induce the typical AhR battery genes but retain their anti-estrogenic activity may represent an exciting new class of drugs for treatment of ER associated disease such as breast and ovarian cancers (Safe et al., 2000). To this end, two main
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classes of SAhRMs were generated, namely, alternate-substituted (1,3,6,8- or 2,4,6,8-) alkyl PCDFs
(polychlorinated dibenzofurans) and ring-substituted DIMs (diindolylmethanes) (Safe and Wormke,
2003). Studies by Safe and colleagues suggested that the prototypical SAhRM 6-MCDF
(6-methyl-1,3,8-trichlorodibenzofuran) inhibits tumor growth in a carcinogen-induced mammary tumor model, potentially by limiting the cellular level of ER via AhR mediated ER degradation
(McDougal et al., 2001). A subsequent study also suggested that mice fed with 6-MCDF can be protected from prostate cancer and pelvic lymph node metastasis in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model (Fritz et al., 2009).
A recent study with AhR DNA binding mutant knock in mice suggested that ligand activated AhR could also cross talk with the NF-κB signaling pathway and repress NF-κB regulated acute-phase gene expressions in an XRE independent manner (Patel et al., 2009). Thus, the term “SAhRM” has also been used to refer to a subset of AhR ligands that is unable to induce classical AhR battery gene expression but retain their ability to repress cytokine-mediated inflammatory gene expression.
Examples of such SAhRMs include 4-[1-allyl-7-(trifluoromethyl)-1H-indazol-3-yl] benzene-1
(Way-169916), 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360), and
3’,4’-Dimethoxy-α-naphthoflavone (DiMNF), which repress IL-1β induced SAA (serum amyloid associated), CRP (C-reactive protein), and HP (haptoglobin) gene expression in human hepatoma
Huh7 cells (Murray et al., 2011; Murray et al., 2010b; Murray et al., 2010c). However, several questions still remain unanswered. The molecular mechanism of AhR dependent inflammatory gene repression is unclear at this stage, although a study by Patel and colleagues suggests that activation of AhR reduces the occupancy of C/EBPβ (CCAAT/enhancer-binding protein β) and the
RelA subunit of NF-κB at SAA-2 and SAA-3 promoters, without direct contact with DNA (Patel et al.,
2009). Furthermore, the structure determinant of this ligand selectivity is unknown, and the effects of other AhR antagonists on the anti-inflammatory effect of AhR have not been systematically studied.
Finally, as opposed to SAhRMs, the AhR antagonist N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2
-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) was shown to function as a “complete” antagonist, which inhibited both agonist induced AhR target gene expression and SAhRM mediated acute-phase inflammatory gene repression (Smith et al., 2011). However, the word “complete”
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should be use with caution, as the complex nature of AhR signaling pathway has not been fully explored and the effect of GNF351 on the ER signaling pathway has not been assessed.
1.3.5 The LBD of AhR
A number of groups have attempted to localize the region in AhR required for ligand binding
(Burbach et al., 1992; Fukunaga et al., 1995; Whitelaw et al., 1993). Initially, the minimal ligand binding region was mapped to amino acids 230-421 of mAhR, which overlaps with the proposed
PAS.B domain of mAhR (Whitelaw et al., 1993). However, further domain deletions coupled with in vitro photoaffinity ligand (2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin) binding assay has refined this region to a smaller region, encompassing amino acids 230-397 of mAhR (Fukunaga et al., 1995). In addition, deletion of amino acids between 288 and 421 in mAhR converts mAhR from a ligand regulated receptor to a constitutively active receptor, further supporting a role of AhR PAS.B domain in ligand binding (McGuire et al., 2001).
Despite many attempts by others and our own research, structure of the ligand binding domain of
AhR has not been solved experimentally. This is presumably due to the absolute requirement for mammalian chaperone proteins for the correct folding of AhR LBD, which is missing in bacterial expression systems. Thus, in the absence of a high resolution experimentally determined structure, a number of AhR LBD homology models have been built to study the molecular detail of AhR/ligand interaction, as well as the structural requirement for high affinity AhR ligands and the mechanism of ligand induced conformational change of AhR (Jogalekar et al., 2010; Motto et al., 2011; Procopio et al., 2002). Furthermore, homology models of AhR LBD have been successfully used in in silico nature compound library screening to identify novel AhR ligands (Bisson et al., 2009).
The first homology model of AhR LBD was developed by Tramontano’s group about 10 years ago
(Procopio et al., 2002). At the time this model was built, PAS structures had only been solved experimentally for 3 proteins, namely, the bacterial photoactive yellow protein PYP, the human potassium channel HERG, and the bacterial O2 sensing FixL. As the HERG protein lacks the ability to bind ligand, and the PYP bound its ligand via covalent bonds, which is distinct from the
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non-covalent interactions between AhR LBD and its ligand, the PAS domain (aka. heme binding domain) of FixL was chosen as the template for AhR LBD modeling in Tramontano’s study. However, it should be pointed out that this model was not an optimum model, as the homology between mAhR
LBD and heme binding domain of FixL is low, making the alignment extremely difficult.
Base on the studies from Tramontano’s group, the modeled mAhR LBD revealed a classical PAS fold with 5 stranded antiparallel β-sheet, a central α-helix core and a α-helical connector spanning the two halves of the β-scaffold, while the ligand binding pocket was proposed to sit in the middle of the molecule, sandwiched by the β-sheet and α-helical connector at either side (Figure 1.8).
Interestingly, cross comparison between the modeled mAhR LBD with the PAS domain of HERG,
PYP, and the heme binding domain of FixL suggested that the orientation of the α-helical connectors determine the size of the ligand bind pockets, such that in FixL, the α-helical connector is linked to the β-sheet backbone via a flexible linker, which opens up the space between the α-helical connector and the β-sheet, allowing access of large ligands, whereas in HERG, which does not bind ligand, the α-helical connector is anchored to the β-sheet in a more compact structure that would restrict the access of ligand. In addition, the amino acid side chains pointing towards the ligand binding pocket also appeared to be tailored to suit the different requirements for ligand binding. For example, the amino acids facing toward ligand binding pocket in FixL tends to have smaller side chains such as glycine (G224 and G251), whereas the modeled mAhR LBD carries the bulkier amino acids L347 and A375 at equivalent positions, which may explain the decreased ligand binding cavity in mAhR LBD compared with the heme binding domain of FixL.
In FixL, amino acids R206, T210 and D212 lie at the entrance of the ligand binding pocket and were proposed to interact via hydrogen bonding in the ligand unbound state. Interaction between ligand and R206 disrupts hydrogen bonding, resulting in re-orientation of the D212 side chain (i.e. conformation change) that signals to switch off the downstream kinase activity (Gong et al., 1998).
Remarkably, these three amino acids are highly conserved in mAhR (R333, T337 and E339), but not in other PAS proteins (Procopio et al., 2002). Thus, a similar mode of regulation could also apply to AhR, which may illuminate the mechanism of ligand induced AhR transformation. Consistent with this notion, mutation of R333 and E339 to leucine reduced the [3H]TCDD binding as well as ligand
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A) B)
Eα
Hβ Cα
Fα Dα Iβ Bβ
Gβ Aβ Helicalconnector
C) D)
Figure 1.8 Homology models of mAhR LBD. A) Topology and nomenclature of mAhR LBD. Similar to other PAS domains, the LBD of mAhR is characterized by having a 5 stranded antiparallel β-sheet (Aβ, Aβ , Gβ, Hβ, and Iβ) with a central α-helix core (Cα-Eα) and a α-helical connector (Fα) spanning the two halves of the β-scaffold . B) Cartoon representation of NMR structure of HIF2α PAS.B domain (1P97) commonly used as the template of mAhR LBD homology modelling. C) Cartoon representation of modelled mAhR LBD bound by TCDD (blue stick). In this model, the ligand binding cavity (cyan surface) is delimited by the β-sheet and α-helical connector. D) A closer look of mAhR PAS.B region involved in ligand binding. The TCDD “finger print” amino acids are shown as sticks, and the molecular surface that delineates the ligand binding cavity is highlighted (cyan), which is proposed to be ~ 496 Å3 in size Color scheme: green for carbon, red for oxygen, blue for nitrogen, and yellow for sulfur. Part C and D adapted from Pandini et al, 2009.
dependent XRE binding (Henry and Gasiewicz, 2008).
According to this homology model of mAhR LBD, other amino acids important in AhR/ligand interaction include R282, C327, T343, F345, A375, and Q377. Amino acid A375 has long been known as a determinant amino acid involved in ligand binding (section 1.3.3). Mutation of A375 to a bulkier and non-polar amino acid such as valine blocks ligand from accessing the ligand binding pocket, as well as disrupts the H-bond formed between A375 and adjacent histidine residue at position 285, thus reducing ligand binding (Ema et al., 1994; Murray et al., 2005; Pandini et al., 2007;
Xing et al., 2012). In addition, mutagenesis studies have also established roles of amino acids C327,
F345, and Q377 in ligand binding (Pandini et al., 2007; Pandini et al., 2009). On the other hand, mutation of R282 to alanine has no effect on ligand induced XRE binding and a very limited impact on the ability of the mutant AhR to bind TCDD (Pandini et al., 2007), suggesting that this amino acid is less likely to play a key role in ligand/AhR interaction. Finally, the contribution of amino acid T343 in ligand/AhR interaction has never been verified experimentally. Interestingly, the side chains of amino acids adjacent to R282 and T343, namely the T283, H285 and F345, were all proposed to project towards the ligand binding cavity in a more recent mAhR LBD homology model (see below), and function to stabilize the AhR/ligand interaction (Pandini et al., 2009). Thus, amino acids R282 and T343 might be misassigned as boundary amino acids in Tramontano’s model due to the low sequence homology between the mAhR LBD and the FixL.
With more PAS structures being solved experimentally, the initial mAhR LBD structure developed by
Tramontano’s group was refined by Pandini and co-workers using the NMR structure of HIF-2α and
Arnt PAS.B domains, either separately or combined (Pandini et al., 2007; Pandini et al., 2009). This refined mAhR LBD model revealed a large ligand binding cavity of approximately 496 Å3, delimited by β-sheet and α-helical connector (Figure 1.8). To further test the role of the α-helical connector in ligand/AhR interaction, amino acid I332, lying in the middle of the α-helical connector, was mutated to a helical incompatible amino acid proline. Replacement of I332 with proline completely disrupts
α-helical connector formation, leading to a significant reduction in the size of the ligand binding pocket by ~20% (Pandini et al., 2007). In addition, the resulting mAhR mutant becomes completely inert, showing no [3H]TCDD binding above background in in vitro TCDD binding assays and no
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ligand induced AhR transformation in EMSA assay with radioactively labeled XRE oligonucleotide probes.
In contrast to the Pandini’s model, the mAhR LBD model developed by Bisson and colleagues revealed a much more compact ligand binding pocket, encompassing an area of only 202 Å3
(Bisson et al., 2009), which is almost half of the size of that identified in Pandini’s model. However, despite this dramatic difference in the size of ligand binding pocket, both studies have highlighted a group of 5 amino acids at the α-helical connector (C327 and M334), as well as βA (H285) and βI
(A375 and Q377) of mAhR as critical boundary amino acids involved in AhR/ligand interaction. This is further supported by a recent study from Bradfield’s group suggesting that the two adjacent
H-bond networks formed between amino acids H285/T283/A375 and Q377/S359, respectively, define the ligand binding specificity of the AhR (Xing et al., 2012).
In addition to mouse AhR, homology LBD models have been developed for other high affinity mammalian AhRs including rat, hamster, rabbit, guinea pig, beluga whale and seal AhRs based on the same NMR structure of HIF-2α PAS.B (Pandini et al., 2009). Cross comparison between these
LBD models and mAhR LBD identified “TCDD binding fingerprint” required for high affinity
AhR/TCDD interaction, including 10 highly conserved boundary amino acids (i.e. those with side chains facing internally, towards the ligand binding pocket), and 3 borderline amino acids (i.e. those with side chains orientated to project either towards or away from ligand binding pocket in different
AhR sequences) (Pandini et al., 2009). Some of the proposed fingerprint amino acids such as T283,
H285, C327, M334, F345, A375 and Q377 have previously been implicated in AhR/ligand interaction, by either projecting into the ligand binding cavity, and thus defining the size of ligand binding pocket or forming hydrogen bonding with ligand to stabilize AhR/ligand interaction (Bisson et al., 2009; Pandini et al., 2007; Procopio et al., 2002). However, cross species modeling also allowed identification of additional amino acids (i.e. F289, P291, Y316, F318, I319, and H320) involved in
TCDD/AhR interaction, providing further insight into the molecular detail of AhR/ligand interactions.
The contribution of F318, I319, and H320 to AhR/ligand interaction has been confirmed by an independent laboratory (Goryo et al., 2007). It was proposed that interaction between AhR and
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TCDD is mediated partially via π-π stacking forces. Owing to its aromatic nature, F318 may stabilize
AhR/ligand interaction by interacting with the aromatic ring systems of TCDD. Consistent with this notion, the AhR orthologs in D. melanogaster and C. elegans, which do not bind any of the known
AhR ligands, have leucine in place of phenylalanine at position 318 of mAhR. Furthermore, mutation of F318 to non-polar amino acids such as alanine or leucine reduced TCDD dependent AhR activity in reporter gene assay, whereas substitution of F318 with aromatic amino acids tyrosine or tryptophan has no effect on AhR activity (Goryo et al., 2007). Similarly, the imidazole ring of histidine at adjacent amino acid 320 could also play a role in electrostatic stabilization of AhR/ligand interaction (Pandini et al., 2009). In contrast, I319, the last amino acid of three at the same patch of central α-helix core was proposed to determine the size of ligand binding pocket. This is further supported by in silico modeling, in which mutation of I319 to bulkier amino acid tyrosine, the equivalent amino acid in HIF-2α, reduced the size of the ligand binding cavity (Pandini et al., 2009).
Interestingly, the I319 of mAhR correspond to V325 in low affinity tern AhR and I324 in high affinity chicken AhR. Mutation of V325 to isoleucine alone in tern AhR dramatically increased its affinity to
[3H]TCDD to a comparable level to chicken AhR in in vitro TCDD binding assays (Karchner et al.,
2006).
The role of F289 in AhR/ligand interaction has also been supported by a molecular dynamics simulation study, looking at the interplay between mAhR and a set of 18 AhR ligands (Jogalekar et al., 2010). Intriguingly, molecular docking studies suggested that upon ligand binding, the side chain of F289 rotates to re-orient itself, such that the aromatic ring of F289 is now positioned parallel to the aromatic ring of AhR ligands, and thus strengthens AhR/ligand interaction via π-π stacking interaction, in a similar fashion as F318. However, ligand induced rearrangement of F289 has not been observed with other mAhR LBD homology models (Motto et al., 2011; Pandini et al., 2009).
In addition to the above mentioned “TCDD binding fingerprint” amino acids, L302, L309 and L347 were also identified as highly conserved boundary amino acids among mammalian AhRs. These amino acids were excluded from the Pandini study initially as they lie relatively far from the core of ligand binding cavity according to the homology models (Pandini et al., 2009). However, recent evidence suggested that in the ligand binding state, the ligand binding pocket of mAhR expands
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(Motto et al., 2011), bringing distant amino acids L302, L309 and L347 close to the ligands. This conclusion was drawn based on new mAhR LBD homology models developed (Motto et al., 2011) using the holo X-ray structures of HIF-2α PAS.B co-crystallized with artificial ligands THS-044,
THS-017 and THS-020 (Key et al., 2009; Scheuermann et al., 2009). Accordingly, ligand binding at the LBD of AhR leads to an “induced fit” effect, which increase the size of the ligand binding cavity to
~800 Å3, approximately twice as much as that proposed in Pandini’s model and about 4 times larger than that proposed by Bisson, bringing more amino acids to the forefront of the ligand binding cavity.
Thus, mutation of L302, L309 and L347 to alanine significantly reduced [3H]TCDD binding as well as
TCDD dependent DNA binding (Motto et al., 2011). Consistently, the size of AhR LBD proposed by the “induced fit” model is in a good agreement with maximal dimensions the proposed for AhR ligands (section 1.3.4). According to the SAR analysis, the maximal van der Waals dimensions of
AhR ligands is 14 Å x 12 Å x 5 Å (Waller and McKinney, 1995), which gives a maximal volume of
840 Å3, a volume that is much larger than the size of the proposed ligand binding pocket in the apo models of LBD (varies from 202Å3 to 496Å3), but is well compatible with the proposed holo model of mAhR LBD (i.e. ~800 Å3).
Finally, homology models have also been built for the LBD of human AhR, again, using the NMR structure of HIF-2α PAS.B as template (Salzano et al., 2011). In contrast with mAhR LBD, which has only a single ligand binding pocket, four ligand binding cavities ranging from 431.7Å3 to 781.4Å3 were identified for human AhR. Out of the four putative candidate ligand binding pockets, TCDD can be docked into two of them, one of which also overlaps with the ligand binding cavity identified in mouse and other mammalian species, while the other appears to be novel for human AhR.
Furthermore, AhR ligands such as quercetin, indirubin, pentaCB and B[α]P could also be docked into a third ligand binding cavity in addition to the one bound by TCDD (Salzano et al., 2011). As a result, this difference in the ligand binding cavity choice may explain some of the observed differences in AhR activity among different AhR ligands. It should be noted, however, that additional ligand binding cavities have yet to be reported for AhR from other species. Thus, mutagenesis-based experimental approaches are required to verify the roles of additional ligand binding pockets in AhR/ligand interaction.
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1.4 Aims and Approaches
As discussed above, it is clear that AhR is multi-functional, both as a master modulator in adaptive xenobiotic response, and as a key regulatory protein in development and immune response.
However, experiments designed to separate these two functions are extremely challenging. The work described in this thesis sought to tackle this problem from the perspective of studying the mechanisms of AhR regulation during both xenobiotic and physiological activation. More specifically, three aspects of AhR regulation were explored in this thesis, including:
1) Rational site-directed mutagenesis on the LBD of mouse AhR to study the ligand binding
specificity of AhR.
2) Random mutagenesis screening of the N-terminal PAS regions of AhR and Arnt to reveal amino
acids key for dimerization between AhR and Arnt and amino acids in Arnt which may be specific
to the AhR/Arnt heterodimers.
3) Microarray analysis on mouse AhR null and reconstituted AhR expressing hepatocytes to study
the target gene specificity of AhR following activation by xenobiotic or suspension culture
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Chapter 2: Results
2.1 Discovery that the dimerization specificity of bHLH/PAS factor Arnt resides in its N-terminal PAS domain (Paper II)
The bHLH/PAS family transcription factors are key regulatory proteins important in development, stress response, oxygen homeostasis and neurogenesis (Figure 1.1). Target gene specificity for bHLH/PAS proteins depends in part on their partner protein choices, where dimerization with common partner factor Arnt is an essential step towards forming active DNA binding complexes.
To gain a better understanding of the molecular basis of bHLH/PAS protein interactions, a novel
Reverse Bacterial two Hybrid (RevB2H) screen was developed (Figure 1a, Paper II). This system differs from the conventional bacterial two hybrid technique (Dove et al., 1997) in that the original
HIS3-aadA reporter cassette was replaced by coliphage 186cI C-terminal domain encoding cDNA, allowing positive selection for loss of protein interaction in Escherichia coli (E. coli) strain KS1033
(Pinkett et al., 2006). In Paper II, I showed that the RevB2H method is simple, robust and efficient, and reliably able to identify genuine loss of interaction mutations with less than 50% false positive rate (Supplementary Table 1, Paper II).
Using the RevB2H screen, 22 evolutionarily conserved amino acids in the N-terminal PAS and linked α-helical cap region of Arnt were identified as key amino acids involved in AhR/Arnt heterodimerization (Figure 2a and 2e, Paper II). The reduced dimerization between AhR and Arnt mutants in bacteria was not simply a result of reduced mutant protein expression or global protein misfolding as demonstrated by quantitative fluorescence-based western blotting and circular dichroism (CD) spectroscopy analysis (Figure 2 and Supplementary Figure S2, Paper II).
Since all bHLH/PAS proteins function as an obligate dimer to regulate gene expression, reporter gene assays were used as an indirect measurement for dimerization. Consistent with the two hybrid
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results, the majority of Arnt mutations that disrupted AhR/Arnt heterodimerization in bacteria also significantly reduced full length AhR/Arnt interaction in mammalian cells (Figure 3, Paper II).
Furthermore, screening of Arnt mutants with other bHLH/PAS proteins Sim1, HIF-1α, Sim2s, NPAS4 as well as Arnt itself suggested that Arnt uses the same face of the N-terminal PAS domain for both homo- and heterodimerization. However, dimerization selectivity appears to reside in a few key amino acids within the dimerization interface as several identified mutations were selective only for
Arnt/AhR interaction, and mutations at these positions had little or no effect on Arnt interaction with other partner proteins (Figure 4, Paper II). Strikingly, mutation of Arnt E163 to lysine, which is detrimental for AhR/Arnt heterodimerization (Figure 2, and 3, Paper II), increased Arnt homodimerization by approximately 2 fold in reporter gene assays using a E-box driven reporter construct (Figure 4g, Paper II).
A similar mutagenesis screen was also performed for the N-terminal PAS region of mouse AhR using the newly developed RevB2H methodology. Data indicated that the equivalent region is used in AhR when dimerizing with Arnt (Figure 5, Paper II), suggesting a novel model for AhR/Arnt heterodimerization via N-terminal PAS domain that is different from the β-scaffold surfaces used for dimerization between the C-terminal PAS domains of HIF-2α and Arnt, and commonly used for PAS domain interactions (Figure 6, Paper II).
2.2 Discovery that Xenobiotic and Suspension Activation of AhR regulates both overlapping and distinct sets of gene expression (Paper III)
The AhR is traditionally defined as a signal regulated transcription factor, which becomes active in the presence of xenobiotic ligands and promotes their elimination by induction of xenobiotic metabolizing enzymes. While this is certainly true, recent studies have also suggested alternative mechanisms for AhR activation independent of xenobiotics (Section 1.3.1). For example, switching adherent cell to suspension cultures induces AhR target genes Cyp1a1 and Cyp1b1 expression in a number of cells culture systems (Cho et al., 2004; Ikuta et al., 2004; Monk et al., 2001; Sadek and
Allen-Hoffmann, 1994a; Sadek and Allen-Hoffmann, 1994b). However, whether this also represents
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a genus mode for AhR activation is currently unknown.
To answer this question, genome wide microarray analysis was performed on both mouse AhR null and AhR reconstituted hepatocytes following 8 hours ligand (i.e. YH439) or suspension treatment, and compared with non-treated controls (Figure 1, Paper III). Microarray results indicated that a panel of classic AhR target genes, including Cyp1a1, Cyp1b1, Nqo1, Tiparp Aldh3a1 and Ugt1a7c were upregulated by both YH439 and suspension culture in an AhR dependent manner (i.e. comparing null vs. reconstituted cell lines), which is consistent with suspension being a genus mode for AhR activation.
In addition, the mechanism of suspension mediated AhR activation was also explored in Paper III. It appeared that AhR is recruited to the XRE of representative AhR target genes by both xenobiotics and switching adherent culture to suspension (Figure 4 and 7b, Paper III), suggesting a common mode of activation between these two mechanisms.
Activation of AhR by xenobiotics or suspension appears to lead to both overlapping and distinct target gene regulation (Figure 2, Paper III). According to the microarray results, AhR target genes can be broadly divided into three Classes, including those I) regulated predominately by xenobiotics,
II) regulated predominately by suspension culture, and III) regulated by both xenobiotics and suspension culture. Interestingly, most of the classic xenobiotic metabolizing type of AhR target genes belong to the third class that were regulated by both xenobiotic and suspension culture activation of AhR.
To validate the microarray results, selected AhR target genes from each class were analyzed by qRT-PCR in an independent hepatoma Hepa-1c1c7 cell line. In general, the results obtained from qRT-PCR were in good agreement with the microarray data (Table 1, Paper III). Furthermore, the temporal expression patterns of AhR target genes were also analyzed by qRT-PCR. Strikingly, the temporal expression patterns among different AhR target genes appeared to differ significantly.
Activation of AhR by both ligand and suspension culture led to sustained activation of Cyp1a1,
Cyp1b1, Nqo1, and Por (P450 (cytochrome) oxidoreductase), while on the other hand, the induction
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of Serpinb2, Spint1, and GTRAP3-18 genes was more transient, declining back towards baseline levels after 8 hours treatment (Figure 3, Paper III).
Finally, sequence analysis coupled with ChIP analysis and reporter gene assay identified a functional XRE within the Tiparp locus, which differs from the conventional xenobiotic response element by having a tandem repeat of 4 XRE core sequence arranged in a concatemer configuration (Figure 7a, Paper III). This XRE concatemer site is likely to coordinately regulate both the protein coding Tiparp gene and expression of its cis anti-sense ncRNA Tiparp-as1 (Figure 6 and
7, Paper III), providing the first experimentally validated example to link the AhR signaling pathway with ncRNA expression.
2.3 Discovery that the mode of interaction between AhR and atypical ligand YH439 is distinct from that of HAH/PAH ligands (Paper I)
As previously described, AhR is a signal regulated transcription factor, which provides adaptive responses to environmental pollutants such as TCDD and other structurally related HAHs/PAHs
(Section 1.3.1). In addition, compounds such as YH439 that fall outside the aromatic classification has also been shown to activate AhR, presumably by functioning as an AhR agonists (Section
1.3.4).
Work by Fiona Whelan suggested that mutation of H285 to tyrosine at the LBD of mouse AhR prevents its activation by HAH/PAH ligands TCDD, 3MC B[α]P (Figure 1.7), by shear stressed fetal calf serum (FCS) or suspension culture, but not by atypical ligand YH439 (Figure 4, Paper I).
Furthermore, the mAhR A375I mutation, which has been proposed to prevent ligand from accessing the ligand binding pocket by steric hindrance (Murray et al., 2005 and Section 1.3.5), was also partially activated by YH439 in transient transfection reporter gene assays (Figure 6c, Paper I). In order to validate these results, stable wild type, and mutant H285Y or A375I mAhR expressing cell lines were generated from mouse AhR null hepatocytes using lentiviral-mediated infection. While
YH439 induces the endogenous AhR target gene Cyp1a1 expression in all three cell lines, addition
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of TCDD or switching adherent cells to suspension culture had no effect on the expression levels of
Cyp1a1 in either of the mutant cell lines (Figure 6b, Paper I), pointing to a novel mode of AhR activation by YH439.
In addition, receptor transformation and DNA binding was assessed by in vitro EMSA using an XRE containing oligonucleotide derived from mouse Cyp1a1 promoter. Consistent with the results of reporter gene assays (Figure 4, Paper I) and qRT-PCR analysis (Figure 6, Paper I), the mAhR
H285Y and A375I mutants only formed a DNA binding complex in the presence of YH439, but not with HAH ligand TCDD, while wt mAhR bound to DNA in the response to both ligands (Figure 7,
Paper I). Collectively, these results point to a novel mode of AhR activation by YH439 that does not require full access the ligand binding cavity. In further support of this notion, HAH-type AhR antagonist 3’4’DMF was able to inhibit TCDD, but not YH439, activated XRE driven luciferase gene in MCF7 breast cancer cells or human embryonic kidney HEK 293T stably AhR expressing cells
(Figure 8, Paper I).
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Chapter 3: Discussion
As previously discussed, proteins are modular in nature (section 1.1). However, the origin of this modularity is still controversial (Lorenz et al., 2011; Wagner et al., 2007). It appears that modular design enhances evolvability and is associated with increased complexity (Figure 3.1). This is perhaps not surprising given that from an evolutionary point of view, building a new protein by rewiring existing components is much more cost effective than starting everything from scratch. On the other hand, evolution could also positively select for modularity under fluctuating environments
(Kashtan et al., 2007). It is likely that modularity emerges with the evolutionary process, such that the time-varying more complex goal is achieved via rearrangement of existing modules, each of which implements an elementary goal (Kashtan et al., 2007).
At the protein level, the modularity concept can best be illustrated by using transcription factor as an example. Pioneering studies by Fields and Song showed that all transcription activators have an uniform molecular design, consisting of at least two relatively autonomous units, commonly referred to as DNA binding domain (DBD) and transactivation domain (TAD) (Fields and Song, 1989). More sophisticated transcription factors may include additional modules such as signal sensing domains
(SSD). One such example is given by the bHLH/PAS family transcription factors, which possess three distinct classes of modules, including the obligate DBD and TAD modules, as well as a pair of degenerate PAS modules (Figure 1.1).
The PAS module is a classic signal recognition module that senses changes in cell microenvironment and modulates the activity of linked effector domain (section 1.2.2). In the most recent release of Pfam database (Version 26.0, November 2011 Build), there are at least 1390 unique domain organizations involving the PAS module, suggesting that the PAS module is extremely prevalent and is extensively embedded in protein design. The modular nature of the PAS domain is further supported by a series of elegant experiments conducted by Moglich and colleagues (Moglich et al., 2009a; Moglich et al., 2010). In one experiments, the authors replaced the heme-binding PAS domain of Bradyrhizobium japonicum FixL, which confers oxygen sensitivity
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on its histidine kinase activity, with the blue-light sensing PAS like LOV (light, oxygen, voltage) domain of Bacillus subtitis YtvA. Substitution of FixL oxygen sensing PAS module with the LOV domain of YtvA had no effect on the kinase activity of FixL, but altered its mode of activation from an oxygen sensing kinase to a blue-light regulated kinase (Moglich et al., 2009a). Furthermore, the
PAS domain can also be assembled in a tandem configuration as demonstrated by Moglich et al., in which chimeric FixL protein with N-terminal fusion of LOV domain is capable of sensing both blue-light and oxygen levels in a synergistic manner, owing to its dual-input PAS modules (Moglich et al., 2010).
In addition to modular protein design, the modularity concept has also gained popularity at the level beyond the individual protein (Wagner et al., 2007). Numerous studies suggest that networks of protein-protein interactions also have a modular nature (reviewed in Fraser, 2005). This could also be illustrated by using bHLH/PAS proteins as an example. Paper II of this thesis describes the molecular characterization of dimerization among bHLH/PAS proteins using a novel Reverse
Bacterial Two-Hybrid (RevB2H) screen approach that selects for protein loss of interaction. Using this system, 22 key amino acids in the N-terminal PAS domain and the linked α-helical cap region of
Arnt that are involved in AhR/Arnt heterodimerization were identified. Intriguingly, the majority of these Arnt mutants also displayed a marked reduced binding affinity towards other class I bHLH/PAS proteins such as HIF-1α, Sim1, Sim2s, or NPAS4, as well as disrupting Arnt homodimerization (Paper II). Taken together, these results are consistent with the notion that dimerization between bHLH/PAS proteins is modular, where the common partner factor Arnt uses the same dimerization interface for both homodimerization and heterodimerization with other class I bHLH/PAS factors.
Furthermore, it was proposed that modularity only works on a robust system (Kitano, 2004; Rorick and Wagner, 2011). Conceptually, this is relatively easy to rationalize as in order to build a complex protein, each underlying module must be robust enough to cope with perturbations, introduced either as a result of intrinsic genetic drift or by domain joining and rearrangement. This is again illustrated by PAS proteins.
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Complexity Evolvability
Modularity
Degeneracy Robustness
Figure 3.1 Relationship between modularity and other essential trails of biology, including evolvability, complexity, degeneracy, and robustness. Modularity is a central trail for evolvability, which promote degeneracy, enhances complexity, and is associated with robustness. See main text for details.
A NOTE: This figure/table/image has been removed to comply with copyright regulations. It is included in the print copy of the thesis held by the University of Adelaide Library.
Figure 3.2 Degeneracy is a trade-off for robustness. Suppose that there are two ways to represent letter “I”, either by a rectangle or a set of degenerate circles. Although more components are required to generate the same letter by circles than rectangle, having degenerate design confers flexibility that can be easily “rewired” to form new letters (i.e. O and E). Figure modified from Dovrolis and Streelman , 2010.
The PAS sequences have been identified from all kingdom of the life with now over 10,000 sequences documented in the Pfam Database (Version 26.0, November 2011 Build). Sequence alignment shows that the sequence homologies among different PAS proteins at primary amino acid levels are extremely low, generally less than 20%, yet all PAS sequences are folded in a well-defined three dimensional structure, consisting of a five-stranded anti-parallel β barrel flanked by a central α-helix core and a α-helical connector (Figure 1.3). This high level of structural conservation suggests an intrinsic structural robustness of the PAS module, such that the key structural determinants rely only on a small number of key amino acids within the modules. This is further supported by an elegant experiment performed by Philip and colleagues, in which the authors took a systematic alanine scanning mutagenesis approach on the entire 125 amino acid residues of prototypical PAS protein PYP, and measured 6 functional and structural parameters of the resulting mutants, including p-coumaric acid (pCA) binding, isomerization and protonation, thermodynamic stability against unfolding, and protein production levels (Philip et al., 2010). It appears that all PYP mutants retain a significant thermodynamic stability against unfolding, and only one mutant caused complete loss of function, consistent with the notion that the PAS domain is extremely robust against perturbations.
In addition to robustness, the other key feature that stems from modularity is degeneracy (Figure
3.1). Degeneracy refers to a situation where a specific goal can be attained by different mechanisms that function independent of each other (Whitacre, 2010), and represents another common trail in protein designs. As previously noted, the bHLH/PAS proteins possess a pair of degenerate PAS domains. At first, it may seem contradictory as in theory, a robust system should evolve towards the elimination of degeneracy. This is because energetically, it is non-economic to maintain a parallel
“backup” system when the default system is perfectly functional. On the other hand, degeneracy can also be viewed as a trade-off for robustness, which provides the necessary buffering spaces against unpredictable lethal perturbations (Dovrolis and Streelman, 2010). In fact, both modularity and degeneracy enhance evolvability. As illustrated in Figure 3.2, systems built with degenerate modules are more versatile than “monolithic” design, although it comes at a cost of increased energy expenditure.
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Given the discussion above, it is clear that modularity operates at every level of biology. However, how specificity is achieved under a highly modular system is less well understood. It becomes apparent that the bHLH/PAS family transcription factors represents a good example to study protein modularity. All bHLH/PAS proteins have a modular molecular design, comprising of three distinct modules (Figure 1.1). Furthermore, all bHLH/PAS proteins function as obligate dimers in a modular fashion, with the N terminal PAS domains determining the dimerization specificity (Pongratz et al.,
1998 and Paper II), and all bHLH/PAS dimers recognize closely related DNA sequences to exert their transcriptional activities (section 1.2.1). On the other hand, different bHLH/PAS proteins regulate different sets of target gene expression, demonstrating that even in a highly modular system, mechanisms must exist to ensure target gene specificity.
There are at least four mechanisms that may promote protein specialization. First of all, there is no doubt that the functionalities of closely related proteins are ultimately determined by their primary amino acid sequences. However, due to the robust nature of protein design (see above), the real determinant for this specificity is likely to reside on only a few key amino acids within the modular domains, rather than the entire domain sequences. For example, AhR interacts with both Arnt and
Arnt2 at a similar affinity (Dougherty and Pollenz, 2008). However, in contrast to AhR/Arnt dimers, interaction between AhR and Arnt2 is non-productive upon xenobiotic challenge (Dougherty and
Pollenz, 2008; Hankinson, 2008; Sekine et al., 2006). A parallel study suggests that the lack of AhR functionality in vivo is in part due to a single amino acid change at the C-terminal PAS domain of
Arnt2, which is substituted from an evolutionally conserved histidine in Arnt to proline in Arnt2
(Sekine et al., 2006). Another example to illustrate the role of key amino acids in discriminating protein activities is given by AhR polymorphism studies. It has been shown that the high affinity and low affinity mouse AhRs encoded by AhRb and AhRd alleles differ by ~10 fold in their sensitivity to
TCDD, resulting in a single amino acid substitution at codon 375 of mouse AhR (Ema et al., 1994;
Okey et al., 1989). However, having a relatively bulky amino acid valine at the entry site of the ligand binding cavity prevents ligand from accessing the cavity, resulting in a significantly reduced AhR activity in response to ligand treatment (section 1.3.3).
The contribution of key amino acids in protein specialization was also demonstrated in Paper II.
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Although all bHLH/PAS proteins share a uniform molecular design (Figure 1.1), there is a marked difference in terms of binding affinity between Arnt and its Class I partner proteins, leading to competitive interaction in their co-existence (Woods and Whitelaw, 2002). Paper II of this thesis provides evidence that the binding between Arnt and other class I bHLH/PAS proteins such as AhR,
HIF-1α, Sims and NPAS4 is determined in part by a small number of evolutionally conserved key amino acids in the N terminal PAS domain of Arnt. Mutation of Arnt at amino acid E163 or S190 (i.e.
E163K and S190P) results in significantly reduced AhR/Arnt heterodimerization as well as concurrent loss of AhR function in cells. In contrast, neither of these mutations affects Arnt heterodimerization with other class I PAS proteins HIF-1α, Sim1, Sim2s, and NPAS4. Similarly, the
Arnt D217G mutant also appears to discriminate between partner proteins, substitution of Arnt D217 to glycine reduces both AhR/Arnt and HIF-1α/Arnt interactions, with little effect on AhR and Sim1,
Sim2s or NPAS4 dimerization. In addition to heterodimerization, the Arnt E163K mutant also appears to strengthen Arnt homodimerization and upregulates E-box driven luciferase reporter gene expression.
The target gene specificity of the ligand activated bHLH/PAS protein AhR could also be determined by the chemical/physical properties of the ligands it encounters. For example, both HAH and PAH ligands activate AhR; however, activation of AhR by chemically more stable HAHs leads to persistent activation of AhR, and is proposed to contribute to AhR toxicity (section 1.3.1). Ligand specific regulation of AhR was further explored in Paper I. It appears that both TCDD and YH439 activate AhR, while mutation of histidine at codon 285 of murine AhR to tyrosine renders it non-responsive to TCDD, although this mutant form of AhR is still responsive to YH439 to the same extent as wild type AhR protein in reporter gene assays (Paper I). This result suggests that the binding mode between AhR and atypical ligand YH439 is distinct from that of the well characterized
HAH ligands, which tolerates tyrosine at position 285 of mAhR, presumably via binding to distinct ligand binding cavities within the LBD of mAhR. Consistent with this notion, a recent modeling study identified 4 putative ligand binding cavities in the LBD of human AhR (Salzano et al., 2011 and section 1.3.5). In the same study, the prototypical AhR ligand TCDD was docked into two of the four ligand binding cavities, while the atypical ligand hemin was docked to the third cavity that is distinct from those bound by TCDD. Moreover, in Paper I, I showed that the PAH type AhR antagonist
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3',4'-dimethoxyflavone (3',4'-DMF) selectively blocks activation of AhR by HAH ligand TCDD but not
YH439, further supporting the notion that TCDD and YH439 lead to non-identical modes of AhR activation. Interestingly, ligand-selective inhibition of AhR mediated gene regulation has also been reported in a more recent study using another AhR antagonist CH223191, which preferentially antagonizes HAH type of AhR ligands, with little inhibiting effect towards PAHs, flavonoids, and indirubin (Zhao et al., 2010 and Section 1.3.4). It could be possible that different AhR ligands induce different conformational changes of AhR, resulting in the regulation of different sets of target gene expression. Consistent with this hypothesis, a limited proteolysis study showed that different degrees of proteolysis can be observed when in vitro translated AhR was bound with agonists and antagonists (Henry and Gasiewicz, 2003).
In addition to being regulated by xenobiotics, switching adherent cell to suspension cultures or disrupting cell-cell contacts can also activate AhR, the latter being proposed to mimic an endogenous activation mechanism of AhR (section 1.3.1). It is possible that the target gene specificity of AhR is also regulated by the route of activation. In agreement with this hypothesis, activation of AhR by xenobiotic or suspension culture drives both overlapping and distinct sets of target gene expression, although both modes of AhR activation results in recruitment of AhR at target XRE sites (Paper III). Mechanistically, route-of-activation dependent target gene regulation could be linked to post translational modifications (PTMs) of AhR, with different combination of
PTMs potentially recruiting different set of transcription co-activators. Preliminary data from our laboratory has suggested that AhR is extensively modified even in the latent state, while activation of AhR by xenobiotic or suspension culture significantly changes the PTM pattern of AhR (Ph.D thesis, Fiona Whelan, the University of Adelaide, 2009).
PTM dependent differential gene regulation has previously been demonstrated with other sequence specific transcription factors such as tumour suppressor protein p53 and nuclear retinoic acid receptors (RARs). Depending on the potency of the DNA-damaging agents, p53 is acetylated at either K320 or C-terminus lysine residues (K370/K372/K373), leading to different sets of target gene expression with opposing effects on cell survival (Knights et al., 2006). Similarly, retinoic acid receptors can be phosphorylated at different serine residues in response to retinoic acid treatment
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or exogenous signals such as growth factors, insulin, stress or cytokines, which drives diverse transcription machinery to either potentiate or repress target gene transcription (for a review, see
Rochette-Egly and Germain, 2009). Furthermore, the C-terminal domain (CTD) of RNA polymerase II (RNAPII) is also shown to be extensively post-translationally modified, where the stage and the level of transcription (i.e. active, paused, or poised) is determined by the PTM patterns of its CTD (reviewed in Brookes and Pombo, 2009).
Finally, target gene specificities can also be regulated by the differential tissue distribution of bHLH/PAS proteins. Although this idea has not been explicitly explored in this thesis, previous studies suggested that most of bHLH/PAS proteins in the Arnt network (Supplementary Figure S1,
Paper II) display tissue restricted expression patterns. For example, NPAS4 is expressed exclusively in the brain (Hester et al., 2007), while expression of the Sim proteins is restricted predominantly to the muscles, kidney, and brain (Ema et al., 1996; Fan et al., 1996). Interestingly, although Arnt is ubiquitously expressed, the expression level is generally low in the brain, while the
Arnt2 homologue is expressed predominantly in the kidney and brain, and displays a somewhat reciprocal expression pattern with Arnt (Aitola and Pelto-Huikko, 2003; Hirose et al., 1996; Jain et al.,
1998). It has been proposed that Arnt2 is the endogenous binding partner for the neuronal bHLH/PAS proteins Sim1 and NPAS4, and knock out of Arnt2 in mice phenocopies the effect seen in Sim1 null mice (Hosoya et al., 2001; Keith et al., 2001; Michaud et al., 2000; Ooe et al., 2004).
Thus, the reciprocal expression pattern of Arnt1 and Arnt2 may provide another layer of complexity in terms of gene regulation.
Future perspectives
The aryl hydrocarbon receptor was first identified by Poland and colleagues over 30 years ago
(Poland et al., 1976). Since its first discovery, a tremendous amount of research has been dedicated to understanding both the biology and the toxicology of the AhR, which has led to a rich body of literature on the function of AhR. Despite significant advances being made towards the mechanism of AhR activation and the molecular outcomes following AhR activation, a number of central questions still remain unanswered.
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It is apparent that AhR exerts the majority of its functions by engaging with AhR ligands; however, an experimentally derived three dimensional structure of the AhR ligand binding domain is still missing. Part of the problem that prevents physical characterization of AhR LBD is the absolute requirement for mammalian chaperone proteins for the correct folding of LBD, missing in bacterial based expression systems. This could potentially be overcome by mutating surface exposed hydrophobic amino acids to hydrophilic amino acids, an approach that has been successfully employed to crystallize the LBD domain of glucocorticoid receptor (Bledsoe et al., 2002), in combination with knowledge gained from studies of related PAS structures, such as the crystal structure of HIF-2α/Arnt PAS.B heterodimers (Scheuermann et al., 2009) and the recently determined crystal structure of Clock and BMAL (Huang et al., 2012). Alternatively, one could also attempt to express and purify the AhR LBD domain from insect cells using baculovirus based vector, a methodology successfully adapted by a number of laboratories to express recombinant MAP kinase in crystallization trials (Lee et al., 2006).
The second long lasting question that puzzled AhR researchers is the endogenous functions of AhR that are strongly implied from the AhR knock out studies. In addition, increasing evidence emerged in recent years to suggest a developmental role of AhR, especially in the immunology field (section
1.3.2.2). A detailed understanding on the endogenous function of AhR independent of adaptive xenobiotic response is lacking. This is partially due to our limited understanding on the activation mechanisms of AhR independent of xenobiotic ligands. It is clear from Paper III that loss of cell contact by suspension culture activates AhR. However, the mechanism underlying suspension dependent AhR activation is unknown, although suspension dependent AhR activation could occur via either a series of signaling cascade such as phosphorylation, or the production of endogenous ligand. To this end, numerous studies have suggested the existence of endogenous AhR ligands
(section 1.3.4). It appeared that the endogenous AhR ligands, if any, are likely to be tissue specific and are much more labile than xenobiotic ligands. One way to identify candidate endogenous AhR ligands is by utilizing AhR reconstituted AhR null hepatocytes, which showed over 250 fold difference in the basal expression level of Cyp1a1 gene when wild type or the LBD mutant (i.e.
A375I) AhR is expressed (Figure 6, Paper I, and data not shown). This marked difference in the basal expression of Cyp1a1 is likely due to the presence of endogenous ligands, which could
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potentially be co-purified together with the recombinant AhR proteins by affinity chromatography, followed by mass spectrometry identification, a strategy similar to that used for identifying physiologically relevant endogenous ligands of PPARα (Peroxisome proliferator-activated receptor
α) (Chakravarthy et al., 2009).
Finally, as illustrated in Paper I and Paper III, activation of AhR by different ligands (e.g. YH439 and
TCDD) or by different mechanisms (e.g. ligand and suspension) results in different modes of AhR activation. We propose different PTM codes could be imposed to the AhR following different means of AhR activation, which in turn regulates different sets of target gene expression. Thus, the AhR research could also benefite from a detailed deciphering of the PTM patterns of AhR both in the latent status and following activation. As elucidated earlier, a detailed understanding on the molecular mechanism underlying AhR activation will greatly advance our current understanding on the functions of AhR both as a receptor in adaptive xenobiotic response and as key regulatory protein in normal cell physiology and development.
Concluding remarks
Regulation of eukaryotic gene transcription is a central theme in biology. It has been estimated that up to 10% of the mammalian coding capacity is devoted to the synthesis of transcription regulators
(Alberts, 2002). Uncontrolled gene expression often leads to disrupted cell function and is associated with disease states such as cancer. Thus, maintaining a robust, versatile and adequate transcriptional control is essential for the normal physiology of the cell. In this thesis, I have explored the mechanisms underlying differential transcriptional control among closely related transcription factors of the bHLH/PAS family. The data present in this thesis will further contribute to our understanding of how eukaryotic genes are regulated at the transcriptional level.
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Paper I
Appendix 1
Amino acid substitutions in the aryl hydrocarbon receptor ligand binding domain reveal YH439 as an atypical AhR activator
Whelan F1,2, Hao N1,2, Furness SG3, Whitelaw ML1,2, Chapman-Smith A1,2
1School of Molecular and Biomedical Science (Biochemistry), and 2Australian Research Council
Special Research Centre for the Molecular Genetics of Development,
The University of Adelaide, South Australia, Australia
3Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia
Molecular Pharmacology 2010; 77(6):1037-1046.
Appendix 1 Paper I
A Whelan, F., Hao, N., Furness S.G., Whitelaw, M.L. & Chapman-Smith, A. (2010) Amino acid substitutions in the aryl hydrocarbon receptor ligand binding domain reveal YH439 as an atypical AhR activator. Molecular Pharmacology, v. 77 (6), pp. 1037-1046
A NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library. A It is also available online to authorised users at: A http://dx.doi.org/10.1124/mol.109.062927 A
Paper II
Appendix 2
Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR
Hao N1,2, Whitelaw ML1,2, Shearwin KE1, Dodd IB1, Chapman-Smith A1,2
1School of Molecular and Biomedical Science (Biochemistry), and 2Australian Research Council
Special Research Centre for the Molecular Genetics of Development,
The University of Adelaide, South Australia, Australia
Nucleic Acids Research 2011; 39(9):3695-3709.
Appendix 2 Paper II
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Appendix 2 Paper II Supplementary Material
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Paper III
Appendix 3
Xenobiotics and Loss of Cell Adhesion Drives Distinct Transcriptional Outcomes by Aryl Hydrocarbon Receptor Signaling
Hao N1,2, Lee KL3, Furness SG4, Poellinger L3,5, Whitelaw ML1,2
1School of Molecular and Biomedical Science (Biochemistry), and 2Australian Research Council
Special Research Centre for the Molecular Genetics of Development,
The University of Adelaide, South Australia, Australia
3Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
4Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia
5Departments of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
Molecular Pharmacology 2012; submitted paper
Appendix 3 Paper III
A Hao, N., Lee, K.L., Furness, S.G., Bosdotter, C., Poellinger L., & Whitelaw, M.L. (2012) Xenobiotics and Loss of Cell Adhesion Drives Distinct Transcriptional Outcomes by Aryl Hydrocarbon Receptor Signaling. Molecular Pharmacology, v. 82 (6), pp. 1082-1093
A NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library. A It is also available online to authorised users at: A http://dx.doi.org/10.1124/mol.112.078873 A