Cn¡.n tcrnRrsrNc MncUANISMS oF RnculATIoN

oE HyPOXIA.INDUCIBLE FACTORS

By David Lando

Department of Molecular BioSciences (Biochemistry)

University of Adelaide

Submitted for degree of Doctor of Philosophy (PhD)

September 2002

TABLE OF CONTENTS

THESIS SUMMARY

MAINREFERENCES

DECLARATION

ACKNOWLEDGEMENTS

ABBREVIATIONS

INTRODUCTION

CeIIuLnn,tND SYSTEM WIDE RESPONSES TO OXYGEN DEPRTVATION

ERYTHROPOIETIN INDUCTION BY HYPOXIA

CLONTNG OF HYPOXIA-INDUCIBLE FACTOR-1

HIF-2a and HIF-3aSTRUCTURE AND FUNCTION OF bHLH-PAS TNNNSCRPTION FACTORS

BIoLoGICAL ROLE OF HIF PROTEINS

HYPoXIA, HIF AND DISEASE

REGULATION OF HIF PROTEINS BY HYPOXTA

Oxygen sensor - many candidatesHIF protein stabilityControl of HIFø stability by proline hydroxylationHIF prolyl hydroxylasesHIF transactivation

ALTERNATTVE MECHANISMS OF HIF-1CT ACTIVATION

DISCUSSION

DIFFERENTIALDNA BINDING ACTIVITYoFHIF PROTETNS- IMPLICATIONS FORHIF BIOLOGY

REGULATION oF HIF PROTEIN FUNCTION BY ASPARAGINE HYDROXYLATION

S tr uctur al imp li c atio ns

Oxygen sensingSubstrate specificityOther mechanisms of aclivationOther possible CAD modificationsTherapeutic benefitsConcluding remarlcs

REFERENCES

ilIV

V

VI

VII

I

AIMS OF' PRESENT INVESTIGATION 2t

RESTJLTS 22

DISCOVERY THAT DNA BINDING ACTTVITY OT HIF-2ct IS ENHANCED BY REF-I (PAPERS I & ID 22

DISCOVERY THAT HIF TRANSCRIPTIONAL ACTIVITY IS REGULATED BY ASPARAGINE HYDROXYLATION

(PAPER III) 24

CHARACTERISATION OF FIH-I AS HIF ASPARAGINE HYDROXYLASE (PAPER IV) 26

1

2J

45

6

10

1l12

13

I41516

20

29

293031JJ

343536J/

38

39

APPENDIX IAPPENDIX IIAPPENDIX III

Papers I-IV

SupplementaryData for Paper III

Comments on Methodology

Tnnsrs SUIUMARY

All humans have a constant and absolute requirement for oxygen, which is necessary to

produce energy for normal cell growth and survival. Mammalian cells adapt to low oxygen

stress (hlpoxia) through a transcriptional response pathway mediated by the Hlpoxia-

inducible factor protein, HIF. HIF is a heterodimer consisting of one of three alpha

subunits (HIF-1o, HIF-2c or HIF-3cr) and abeta subunit called Amt. The activation of HIF

by hypoxia is a multistep process involving increases in both protein stabilisation and

transcriptional potency of the alpha subunits. Protein stabilisation is a result of inhibition

of ubiquitin dependent degradation while increased transactivation is a result of

recruitment of transcriptional coactivators. Once activated the HIF alpha subunits

accumulate in the nucleus where they heterodimerise with Arnt to bind hypoxia response

elements (HRE) located in genes involved in helping cells adapt to low oxygen stress. To

better understand the mechanisms by which HIF is regulated I have undertaken in my PhD

a study to investigate both the DNA binding and transcriptional capaci|y of the HIF-lcx, and

HIF-2cr subunits.

In my thesis I report that the DNA binding of HIF-2cx, to a HRE, but not HIF-lcx,, is

dependent on redox reducing conditions. In-vitro DNA binding and mammalian two-

hybrid assays demonstrate that a unique cysteine residue located in the DNA binding basic

region of HIF-2a is a target for the reducing activity of redox factor ReÊl. Our data are the

first to establish a discriminating control mechanism for differential regulation of HIFs that

targets the DNA binding potential of HIF-2cr.

In a separate investigation we show that the induction of the hypoxia sensitive HIFa

carboxy-terminal transactivation domain (CAD) occurs through abrogation of

hydroxylation of a conserved asparagine residue. Hypoxia and chemical mimetics of

h¡poxia, such as iron chelators and analogs of 2-oxoglutarate, prevented hydroxylation of

the asparagine allowing the CAD to interact with the transcriptional coactivator protein

p300. We then demonstrate that the protein factor inhibiting HIF-I (FIH-l), previously

shown to interact with HF, is an asparaginyl hydroxylase enzvrrre capable of hydroxylating

the asparagine in the HIF CAD. Like other known hydroxylases FIH-I is an iron and 2-

II

oxoglutarate-dependent enzyme that uses molecular oxygen to modify its substrate, thus

comprising a critical regulatory component of the oxygen sensing and HIF response

pathway.

ilI

MaTx REnERENCES

This thesis is based on the following publications (see appendix I) refened to in the text by

their Roman numerals.

Paper I. Lando, D., Pongtatz,I., Poellinger, L., and 'Whitelaw, M.L. (2000) A redox

mechanism controls differential DNA binding activities of hypoxia-inducible

Paper II. Lando, D., Peet, D.J., Pongratz, I., and V/hitelaw, M.L. (2002) Mammalian

two-hybrid assay showing redox control of HIF-like factor. Methods in

Enzymology 353,3-10.

Paper III. Lando, D., Peet, D.J., Whelan, D.4., Gorman, J.J., and Whitelaw, M.L. (2002)

Asparagine hydroxylation of HIF transactivation domain: A hypoxic switch.

Science 295,858 861.

Paper IV. Lando, D., Peet, D.J., 'Whelan, D.4., Gorman, J.J., Whitelaw, M.L., and

Bruick, R.K. (2002) FIH-I is an asparaginyl hydroxylase enzyme that regulates

the transcriptional activity of hl,poxia-inducible factor. Genes & Dev. 16,1466-

t471.

All publications have been reproduced with the permission of the copyright holders

IV

AcxxowLEDGEMENTS

After 4 years work, there are many whom I give my thanks, they include

My supervisor Murray Whitelaw for his support, encouragement and wealth of ideas.

Thanks for showing me that science can be an enjoyable experience. I have no doubt that

the skills and knowledge that that I have gained during my studies will be invaluable in the

years to come. It has truly been a privilege to be a student in your lab.

My surrogate supervisor Dan Peet. Thanks Dan for being there during the years and

offering your words of advice, it was much appreciated.

To my fellow Whitelaw lab members past and present Michael, Anthony, Seb, Robyn,

Susi, Cameron, Anne, Gerwin, Christine, Alix, Sarah, Jodi, Marika and Ben.

To all that attended Lorne conferences (1999-2001). Definitely the best scientific (party)

meeting in the world.

All at the Department of Molecular BioSciences and old Department of Biochemistry for

their practical help and support.

My Mum, Dad, sister Vanessa and family for giving me support.

Finally, I would like to thank Christine for her love, devotion and above all patience and

understanding.

That's all folks...

VI

AnnnEVrATroNS

Ala

Asn

ATP

Arnt

bHLH basic helixJoop-helix

CAD

alanine

asparagme

adenosine hiphosphate

aryl hydrocarbon nuclear

translocator

carboxy-terminal transactivation

domain

CREB binding protein

cysteine-histidine

carbon monoxide

cyclic AMP-response element

binding

dimethyloxalyl glycine

deoxyribonucleic acid

2,2'-dipyridyl

dioxin receptor

desferrioxamine

dithiothreitol

epidermal growth factor

erythropoietin

endothelial PAS

hypoxia-inducible factor

HIF like factor

HIF prolyl-4-hydroxylase

heat shock protein-9O

hypoxia response element

HIF related factor

MALDI-TOF matrix assisted laser desorption

ionisation-time of flight

MAPK mitogen activated protein kinase

MS mass spectrometry

MS-MS tandemMS

mRNA messenger ribonucleic acid

NAD amino-terminal transactivation

domain

nicotinamide adenine dinucleotide

phosphate

nitric oxide

NADPH

ODD oxygen-dependent de gradation

domain

organ ofzuckerkandl

CBP

CH

CO

CREB

EGF

Epo

EPAS

HIF

HLF

HPH

HSP-90

HRE

HRF

PAS

Per

PEST

Pro

Ref-1

RLL

ROS

SRC-1

Sim

TIF2

Trl

VEGF

VHL

NO

OZ

DMOG

DNA

DP

DR

Dsfx

DTT

Per-Arnt-Sim

period

proline-glutamic acid-serine-

threonine

proline

redox factor-1

arginine-dileucine

reactive oxygen species

steroid rsceptor coactivator- I

srngle minded

transcription intermediary factor-2

trachealess

vascular endothelial growth factor

von Hippel-Lindau

FIH-I factor inhibiting HIF-1

iPAS inhibitoryPAS

vII

INrnoDUcTroN

To sustain life humans have an absolute and continual requirement for oxygen. The

detrimental consequences of removing oxygen by burning a candle on a mouse in a bell jar

were first noted by English scientist Joseph Priestley back in the late 18th century (Gibbs

1965). The ability of oxygen to readily accept electrons enables it to participate in

reduction-oxidation reactions and thus serve as the terminal electron acceptor in

mitochondrial oxidative phosphorylation, the main ATP energy producing source for all

higher organisms (Bunn and Poyton, 7996).

For nearly two centuries after Priestley's observations little progress was made on

understanding the way the human body adapts to changes in oxygen availability. With the

emergence of molecular biology it is now only recently that we have begun to unravel how

our bodies cope with changes in oxygen levels and thus respond to periods of oxygen

deprivation, termed hypoxia. Here I will give an overview of the current concepts of how

oxygen homeostasis in humans is maintained, focussing primarily on the way oxygen

availability regulates the activity of Hypoxia-inducible factor (HIF) proteins, a group of

oxygen regulated transcription factors which I have investigated in my thesis.

Cellular and system wide responses to oxygen deprivation

The development of complex cardiovascular, respiratory and hemopoietic systems in

humans provides a means to efficiently capture and deliver oxygen from the environment

to every cell of the body. 'While a sufficient supply of oxygen is essential for energy

production, excess oxygen can be detrimental. For example, hyperoxia can result in the

generation of reactive oxygen species such as superoxide and hydrogen peroxide, which

are toxic to cells by virtue of their ability to damage DNA and proteins (Storz and Imlay,

1999). Therefore, to maximise oxygen use and minimise the impact of oxygen free

radicals, cells endeavor to tightly regulate and maintain oxygen concentrations within a

narrow physiological range. To achieve this humans regulate oxygen consumption and

levels by a combination of both cellular and system wide processes. For example, certain

1

hypoxic responses such as the switch to anaerobic metabolism are inherent to all cells.

When oxygen is limiting cells abandon oxidative phosphorylation and rely on glycolysis as

the primary means of ATP production. To achieve this switch cells up-regulate the

expression of a select set of genes such as those encoding glycolytic enzymes and glucose

transporters that function more efficiently when oxygen is limiting (Webster and Murphy,

1988).

In conjunction with intracellular mechanisms, further hypoxic responses seem to monitor

global oxygen levels and affect system wide changes in tissue oxygen availability. For

instance, in response to oxygen deprivation type I cells of the carotid body produce and

release the neurotransmitter dopamine which signals to the brain to increase breathing and

thus oxygen uptake by the respiratory system (Czyzyk-Krzeska et a1., 1992). Another

example is the hypoxic induction of the hormone erythropoietin (Epo) by the kidney,

which stimulates red blood cell production to help increase the oxygen carrying capacity of

the blood (Jelkmann, 1992). Tissues and cells experiencing reduced oxygen supply like

those associated with wound healing increase the levels of the angiogenic cytokine

vascular endothelial growth factor (VEGF). VEGF then acts on endothelial cells to

stimulate the proliferation of new blood vessels that in turn help maintain an adequate

supply ofoxygen (Ferrara, 1999).

Erythropoietin induction by hypoxia

For many years Epo was the model system used to study the molecular mechanisms

associated with the induction of hypoxia responsive genes. In response to hypoxia Epo

levels can be increased by as much as 1000 fold (Jelkmann,1992). The cloning of the Epo

gene in 1985 (Jacobs et al., 1985; Lin et al., 1985), greatly facilitated the study of Epo

induction by hypoxia. It was shown that two human hepatoma cell lines, HepG2 and

Hep3B, were able to produce significant amounts of Epo upon hypoxia stimulation and

more importantly, this induction required ongoing transcription (Goldberg et al., l99l,

Schuster et a1., 1989). The finding that Epo levels were regulated at the level of messenger

RNA synthesis strongly implied that hypoxia was most likely targeting a transcription

factor, for which increased activity during hypoxia was resulting in increased Epo mRNA

production. This was supported by evidence that hypoxic induction of Epo mRNA was

2

blocked by the translational inhibitor cyclohexamide, indicating that the hypoxic response

required the de-novo synthesis of a protein (Goldberg et al., 1988). V/ith this in mind

experiments with transgenic mice then went on to identify regulator regions of the Epo

gene important for this hypoxic induction (Semenza et al., 1990; Semenza et al., 1991).

Definitive studies in cell culture systems then mapped the hypoxic responsive region to a

50 nucleotide sequence located at the 3' end of the gene (Beck et al., I99l; Pugh et al.,

l99l; Semenza et al., 1.991; Semenza and Vy'ang, 1992).

Cloning of Hypoxia-Inducible Factor-L

In a series of experiments Semenza and co-workers then went on to characterise and

identify the factors mediating the hypoxic response on the Epo gene. Utilising a

combination of reporter gene, DNase I footprinting and electrophoretic mobility shift

assays they demonstrated that the 50 nucleotide Epo enhancer bound a nuclear factor,

which was induced by hypoxia (Semenza and Wang,1992). They called this nuclear factor

Hypoxia-Inducible Factor-1 (HIF-1). Then by systematically mutating the enhancer they

mapped the DNA binding of HIF-I to an 8-base pair DNA sequence 5'-TACGTGCT

which they termed the Epo Hypoxia Response Element (HRE) (Wang and Semenza,

I993a). Finally, utilising a biochemical approach they purified this nuclear factor and

found it was composed of a heterodimer for which they designated the subunits HIF-lcr

and HIF-IB (Wang et al., I995a). The cloning of HIF-1c¿ and HIF-1p genes revealed that

both subunits belonged to the basic-helix-loop-helix (bHLH), Per-Arnt-Sim (PAS)

homology domain family of transcription factors (Wang et al., 1995a). HIF-1P subunit was

found to be identical to the previously described bHLH-PAS protein aryl hydrocarbon

receptor nuclear translocator (Arnt), which is known to heterodimerise with a number of

other bHLH-PAS proteins to form transcriptionally active complexes (Crews, 1998).

Subsequent biochemical analysis of the induction of HIF-1 by hypoxia revealed that HIF-1

was being primarily regulated by enhanced protein stabilisation and transcriptional activity

of the HIF-1ct subunit. In contrast, the Arnt subunit was found to be constitutively

expressed and its transcriptional potency was not affected by hypoxia (Huang et a1., 1996;

Pugh et al., 199'7; Kallio et al., l99l). Therefore the HIF-1c¿ subunit represented the

hypoxia responsive component of the HIF-1 complex.

3

Erythropoiesis andlron Metabolism

ErythropoietinTransferrinTransferrin receptor

Ventilation

Tryrosine hydroxylase Vascular Biology

Energy Metabolism

Glucose transporter-1,-2Aldolase A & CEnolase 1

Hexokinase -1 & -2

Proliferation & Survival

Phosphoglycerate kinase-1Pyruvate kinaseLactate dehydrogenaseGlyceraldehyde-3-phosphate-dehydrogenase

HYPOX¡A

Figure 1. Hypoxia-inducible factor (HlF) target genes and their roles in oxygen homeo-

stasis. Hypoxia activates the HIF complex which binds to hypoxia response elements contain-

ing the core recognition sequence 5'RCGTG found in numerous genes involved in a variety of

cellular and system wide responses to low oxygen stress.

Vascular endothelial growth factorHeme oxygenase-1Nitric oxide synthase-2Endothelin-1Plasminogen activator inhibitor-1

lnsulin-like growth faclor-2 (lGF-2)IGF binding protein-1 , -2 & -3cyclin G2p21NIP3

The importance of the discovery and cloning of the HIF-1 complex was underscored by the

finding that the DNA binding activity of HIF-I and the activation of Epo response element

driven reporter genes occurred in a number of non Epo producing cell lines (Beck et al.,

1993; Maxwell et al., 1993; 'Wang and Semenza,1993c). These results suggested that HIF-

1 may represent a factor responsible not only for Epo induction, but the induction of other

hypoxia responsive genes. It had been shown that other hypoxia mediated responses such

as angiogenesis are due to the transcriptional up-regulation of specific target genes such as

VEGF (Goldberg and Schne\der, 1994). Subsequent studies carried out by numerous

groups over the next few years then went on to identify functional HIF-1 binding sites in

several genes encoding proteins known to be important for the adaptive response to low

oxygen stress (see Figure 1) (Guillemin and Krasnow, 1997; Semenza, 1999b). Thus, the

investigation into the hypoxic induction of the Epo gene had uncovered a common

pathway for the activation of a broad spectrum of hypoxia responsive genes. The

importance of the HIF complex as a global regulator of oxygen homeostasis was further

substantiated by the finding that the HIF subunits were well conserved among many

multicellular organisms ranging from the simple round worm C. elegans (Jiang et al.,

2001), through Drosophila ( Nambu et al., 1996) and complex mammals, such as mice and

humans (Wang et al., I995a).

HIF-2ct and HIF-3u

The complexity associated with the regulation of hypoxia responsive genes was further

extended with the discovery and cloning of related HIF-lcr like proteins. Referred to as

HIF-2o (also known as endothelial PAS (EPAS), HIF-Like factor (HLF), and HlF-related

factor (HRF)) (Tian et al., I99l; Ema et al.,199'7 Flamme et al., 1997) and HIF-3o (Gu et

a1., 1998), these closely related proteins share approximately 487o and 407o am\to acid

sequence homology with HIF-Iü, respectively. Analysis of HIF-2a and HIF-3cr have

shown that both proteins share similar biochemical properties with HIF-lct. For example,

both proteins are able to heterodimerise with Arnt with similar affinities, both bind the

same consensus hypoxia response element (HRE) found in a large number of hypoxia

responsive genes, and in cell culture assays, both activate HRE driven reporter genes when

exposed to hypoxic stress (Ema et al., l99l; Gu et al., 1998; Tian et a1,.,1997).

4

ABbHLH PAS TAD TAD*

HIF-1a

HIF-2cr, Hypoxia

HIF-3a

Arnt General partner factor

Dioxin Receptor Xenobiotic metabolism

Clock Circadian cycles

trachealess Tubular formation

SimNeurogenesis

Sim

RED

Figure 2. A general schematic representation of some basic helix-loop-helix (bHLH)/ Per-Arnt-Sim

(PAS) transcription factor proteins. The two hydrophobic repeat regions within the PAS domain are

denoted A and B, TAD and RED denote transactivation and repression domains respectively, *

denotes the C terminal transactivation domain. Drosophila proteins are in italics.

IA B

AB

AB

AB

AB

AB

AB

AB

Structure and function of bHLH-PAS transcription factors

The bHLH-PAS family of proteins comprise a continually growing class of transcriptional

regulators, which control a diverse range of physiological and developmental events in

both vertebrates and invertebrates (Figure 2). For example, the mammalian Dioxin

Receptor (DR) responds to xenobiotic compounds such as dioxin by activating target

genes associated with cellular detoxification (Poellinger, 1995), whereas the Drosophila

Single-Minded (Sim) (Nambu et al., 1995) and Trachealess (Trl) (Wilk et al., 1996)

proteins are involved in regulating neurogenesis and tracheal development respectively.

As outlined previously HIF-lol2oJ3o" and Arnt aÍe members of the bHLH-PAS

transcription factor family. The domain structure and organisation of bHLH-PAS members

is astonishingly similar (Figure 2). The bHLH motif is located near the amino terminus,

closely followed by the PAS domain, while the carboxy-terminus of the proteins contain

transcriptional activation domains or repression domains which interact with

transcriptional regulator complexes to control transcription of target genes. The bHLH

motif is found in a large number of other transcription factors including the oncogenic and

myogenic protein complexes Myc/Max and MyoD/BzA, respectively. The HLH region is

responsible for promoting protein dimerisation while the basic region binds DNA (Kadesh,

1993). PAS is an acronym derived from the names of the first three proteins Per, Arnt and

Sim, in which this protein motif was formally defined (Nambu et al., 1991). The PAS

domain in bHLH-PAS proteins is typically 250-300 amino acids in length and is

subdivided into two well conserved repeat regions designated PAS A and PAS B,

separated by a poorly conserved spacer region.. The two repeats that are 40 to 50 amino

acids in length contain a high content of hydrophobic amino acids (Crews et al., 1988;

, Nambu et al., 1991). The role of the PAS domain is to help mediate protein-protein

interactions and act as a second dimerisation interface in conjunction with the HLH motif

(Huang et al., 1993; Lindebro et a1.,1995). In the case of the DR the PAS B region is also

known to bind ligands such as dioxins and related polycyclic aromatic hydrocarbons. The

binding of ligands to The PAS B region activates the latent cytoplasmic form of the dioxin

receptor by unmasking a nuclear localisation signal resulting in the translocation of the DR

to the nucleus, where it forms a transcriptionally active complex with Amt (Lees &'Whitelaw

1999 ; Poellinger, 1 995).

5

The PAS domain is now considered to be an ancient protein motif as it has been identified

in well over 100 other proteins ranging from Bacteria, Archaea and Eucarya (reviewed by

Taylor and Zhulin, 1999). These proteins include histidine and serine/threonine kinases,

chemoreceptors and photoreceptors which have been shown to perform a diverse range of

sensory functions associated with monitoring changes to light, redox, small ligands, energy

usage and even oxygen levels. The two component FixLÆixJ system in the bacterium

Rhizobium meliloti is the best characterised PAS oxygen sensor. FixLÆixJ system is able

to sense changes in oxygen concentration and couple the level of oxygen to the rate of

nitrogen fixation (David et al., 1988). The mechanism of oxygen sensing is attributed to a

heme moiety associated with the PAS domain in FixL (Gong et a1., 1998). 'When

associated with oxygen the PAS domain undergoes a conformational change and inhibits

the histidine kinase activity of FixL. However, in the oxygen free state the histidine kinase

is derepressed and can catalyse a series of events that activates the FixJ transcription

factor, which then turns on genes associated with nitrogen fixation. Therefore, with the

FixLÆixJ system in mind it would be inviting to speculate that the PAS domain of the

hypoxia responsive HIFcr. subunit is somehow also involved in sensing oxygen levels.

However, the PAS domain of the ct subunit has not been shown to associate with a heme

moiety and as will be discussed in more depth later has not been shown to be directly

involved in oxygen sensing.

Biological role of HIF proteins

Northern analysis has shown that both HIF-lcr and HIF-2cr rnRNA is found widely

expressed in many tissues (Tian et al., 1997). However, at a cellular level the expression

profile of both HIF-lcr, and HIF-2a is quite different. In-situ analysis of various tissue

samples and examination of a number of culture cell lines has shown that HIF-lcr, is

expressed in almost all cell types, whereas, HIF-2cr shows a more restricted expression

pattem and is found highly expressed in lung alveolar cells, endothelial cells and the

carotid body (Compemolle et al., 2002; Ema et al.,1997; Flamme et al., l99l; Tian et al.,

1991; Wiesener et al., 1998). The expression analysis of HIF-3cr, rnRNA has shown HIF-3cx,

to be primarily located in adult thymus, lung, brain, heart and kidney (Gu et al., 1998).

6

To help decipher the biological role of these factors a number of groups have now made

targeted disruptions of the HIF-1c and HIF-2o. genes in mice and found that both HIF-1c¿

and HIF-2a are critical for embryo development via quite distinct pathways. Null mice for

HIF-1o die at embryo day 11 due to gross neural tube defects, cadiovascular

malformations and complete lack of cephalic vascularisation (Iyer et al., 1998; Ryan et al.,

1998). In the HIF-1o-/- embryos vascularisation is initiated properly but fails to progress

during embryo development between days 88.75 and 89.'75, which correspondingly

coincides with the appearance of HIF-1o expression in wild type embryos. The improper

progression of vasculogenesis was shown to be a result of increased mesenchymal cell

death, leading to defective support structures for the growing vessels (Kotch et al., t999).

In a different study designed to investigate the developmental role of HIF-lcr in avascular

tissue, Schipani et al. (2001) created a targeted disruption of HIF-lcr. in mouse chondrocyte

cells of the cartilaginous growth plate. During development the cartilaginous growth plate

is responsible for forming cartilage and bone in the growing embryo. Disruption of HIF-lcr

in chondrocytes resulted in perinatal lethality usually within 2 hours of birth. Analysis of

the bone structure of the null animals revealed gross abnormalities in cartilage formation

associated with the stemum and rib cage, which resulted in the trachea collapsing at birth

and the animals dying from asphyxiation. Further analysis then revealed that the cartilage

abnormalities were a result of massive chondrocyte cell death in the cartilaginous growth

plate, which was shown to be due to a decrease in the expression of the cell growth arrest

factor p57kip2 (Schipani et al., 2001). Thus, loss of HIF-1o in chondrocyte cells led to an

inability of the growth plate to initiate proper growth arrest and then differentiation. Taken

together these gene knockout studies of HIF-1c¿ in mice suggest that during embryo

development HIF-lcr may play an important role in helping to maintain normal cell

survival signals during periods of hypoxic stress.

Most embryos null for the HIF-2cr gene die mid-gestation at embryo day 812.5-16,

suffering from pronounced bradycardia (Compernolle et a1.,2002; Tian et al., 1998) and

gross defects in vascular remodeling (Peng et al., 2000), while those that survive to birth

die within a few hours due to respiratory distress syndrome (Compernolle et al., 2002).

Bradycardia or reduced cardiac output in the null HIF-2o mice was shown to be due to a

reduction in the level of catecholamines (Tian et al., 1998). In wild type mice HIF-2cr is

highly expressed in the organ of zuckerkandl (OZ) during embryonic days E13.5-15.5. The

7

OZ \s the principle source of fetal catecholamines and catecholamines are essential for the

normal functioning of the heart. Interestingly, the defects in vascular biology observed in

the HIF-2cr, null embryos were not associated with formation of blood vessels but attributed

to either improperly fused vessels and/or poorly assembled vessel networks (Peng et al.,

2000). Therefore, unlike the HIF-lcr null embryos where vascularisation is impeded

leading to reduced blood vessel formation, HIF-2o¿ null embryos can form blood vessels

but the vessel remodelling is disorganised leading to poor oxygen delivery. The HIF-2cr

null mice that developed respiratory distress syndrome were shown to produce insufficient

surfactant in the alveolar type 2 cells of the developing lung (Compernolle et al., 2002).

Surfactant consists of a mixture of phospholipids and surfactant-associated proteins and

their role in the lung is to help lower surface tension between the air-water interface and

prevent aveolar collapse. Further analysis then revealed that low surfactant production in

the HIF-2o null mice was due to low levels of VEGF expression. If VEGF was delivered

in-uterine this protected preterm HIF-2cr, null mice against respiratory distress syndrome

(Compernolle et al., 2OO2), suggesting that HIF-2cr induced VEGF expression is an

essential component of normal lung function. Currently, the difference in the major

phenotypes obtained for the three independent HIF-2cr, knockout studies are unclear. It is

unlikely that the differences are due to construct targeting design, because all three groups

disrupted exon 2 containing the DNA binding motif. It is possible that the discrepancy is a

consequence of the use of different mouse strains and possibly also subtle differences in

the ES cell lines used by the three groups.

The biological role of HIF-3cr remains largely unknown because no gene knockout studies

in mice have been conducted. However, an alternative spliced transcript of HIF-3c¿ termed

inhibitory PAS (iPAS) protein, which lacks the C terminal region of HIF-3cr, to consist of

an incomplete PAS B region and complete loss of transactivation domain (Makino et al.,

2002), has been shown to be a negative regulator of HIF activity (Makino et al., 2001). The

inhibitory effect of iPAS has been shown to be due to the ability of iPAS to sequester HIF-

lcr away from its regular dimerisation partner Arnt and the subsequent iPAS/HIF-1a

heterodimer is then unable to bind to HRE of target genes. Then in a mouse model of

coûlea induced angiogenesis it was shown that overexpression of iPAS was able to block

HIF-lcr mediated induction of VEGF and subsequent neovascularisation of the

surrounding comea. (Makino et al., 2001).

I

Despite strong amino acid sequence and biochemical similarities, target disruption

analyses of HIF-lcr and HIF-2cr in mice have clearly shown that both HIF-1c¿ and HIF-2cr

perform critically distinct functions during embryo development. How and by what

mechanism do HIF-1ct and HIF-2c¿ mediate these different phenotypes? It is possible that

some of the phenotypes observed are due to differences in expression pattems for both

HIF-lcr, and HIF-2cr. This may be plausible for some of the phenotype observed in the HIF-

1c¿ knockout mice since expression of HIF-2cr is known to be restricted. However, this is

difficult to reconcile for some of the HIF-2c¿ knockout studies since HIF-lcr, is found more

widely expressed (Ema et al.,l99l; Flamme et al., 19971' Tian et a1.,1997; 'Wiesener et al.,

1998). Another possible reason for the differences in knock-out studies is that HIF-1o and

HIF-2o may in certain circumstances activate different target genes. Interestingly, in two

studies it was demonstrated that HIF-2ø was able to activate reporter gene constructs

containing the endothelial cell specific promoter and enhancer regions from the Tie-Z

(Tian et al., I99l) and Flk-l (Kappel et al., 1999) genes. Both Tie-2 and Flk-1 genes

encode for different vascular endothelial growth factor receptor tyrosine kinases which are

specifically found in endothelial cells (reviewed by Mustonen and Alitalo, 1995). In

contrast, HIF-1o was unable to activate the T\e-2 and Flk-l reporter gene constructs in

similar experiments. Therefore, these observations raise the possibility that HIF-2cr may

have some distinct target genes, and may specifically regulate Tie-2 and Flk-1 expression

within endothelial cells.

Differences in biological function of HIF-loc and HIF-2cr, may also be due to alternatively

spliced isoforms. While no altematively spliced isoforms have been found for HIF-2ø, a

number of altematively spliced variants of HIF-lcr have been discovered in mouse

(Wenger et al., 1998), rat (Drutel et al., 2000) and humans (Gothie et al., 2000).

Interestingly, one of the human isoforms (HIF-1a736) has been shown to lack exon 14,

which gives rise to a shorter form of HIF-lcr, lacking the potent carboxyl terminal

transactivation domain. While this shorter isoform was shown to be regulated by hypoxia

and bound Amt and a HRE in a similar manner as full length HIF-lcr, the ability to

transactivate target genes such as VEGF was, however, substantially reduced (Gothie et al.,

2000). Thus, like the iPAS isoform of HIF-3c¿, the HIF-lcr.73u isoform may represent a

negative regulator of HIF-1cr, activity in certain cellular situations.

9

To help gain a better understanding of the mechanisms contributing to the biological

functions of HIF-lcr, and HIF-2c¿ further studies will need to focus on,

i) deciphering the target genes regulated by both factors,

ii) investigating if their exists alternative mechanism of activation for both HIF-lcr and

HIF-2o and

iii) determining the functional role of the various spliced isoforms for HIF-1o.

Hypoxia, HIF and Disease

Humans have developed a complex respiratory and circulatory system to ensure adequate

oxygen supply to all the cells in the adult organism. Unfortunately in many disease states

these oxygen delivery systems fail and a lack of oxygen then becomes a major component

of the pathophysiology of these diseases, which include stroke, heart disease, diabetic

retinopathy and pulmonary hypertension (reviewed by Semenza, 2000). In each case the

disruption of oxygen supply has a drastic affect on cell, tissue and organ viability. For

example, in the heart condition atherosclerosis where plaque build up in the arteries leads

to oxygen and nutrient deprivation to the surrounding myocardial tissue the end result is

usually an infarction (heart attack). It is known that both HIF-lcr, and VEGF expression are

induced by heart attack as a mechanism to increase oxygen delivery via enhanced

angiogenesis (Lee et a1.,2000). It is also well known that the level of the VEGF response

is directly related to the severity of the heart attack (ie. greater response leads to better

survival outcomes) (Schultz et a1., 1999). Therefore therapeutic strategies aimed at

increasing HIF-1a activity and the VEGF mediated angiogenic response may provide

benefits to heart attack victims.

On the other hand cancer cells adapt to low oxygen conditions by exploiting the above

same oxygen delivery systems to promote tumor growth, invasion and metastasis. For

primary tumors to develop beyond a few cubic millimeters, where the diffusion of oxygen

and nutrients become limited, they must establish a reliable blood supply. Therefore

increased expression of VEGF has been shown to be essential for the development of

angiogenesis in many solid tumors (reviewed by Hanahan and Folkman, 1996).In addition

to increased angiogenesis, tumor cells adapt their metabolism to reduced oxygen

10

availability by increasing the expression of genes associated with the switch to glycolysis

which help to maintain ATP energy supplies (Rodriguez-Enriquez and Moreno-Sanchez,

1998). With the above observation that many known HIF target genes are found up-

regulated in many cancers, it was not unexpected to find that both HIF-lor and HIF-2cr,

subunits are found overexpressed in many malignant tumors (Talks et al., 2000; Zagzag et

al., 2000; Zhong et al., 1999). To further support the notion of HIFs aiding tumor

progression it was found that disruption of the HIF-1c¿ subunit can retard tumor growth

(Ryan et a1., 1998). Therefore, strategies aimed at blocking the activity of HIFs may

provide therapeutic benefits to cancer patients

In the US nearly two thirds of all deaths can be in some way attributed to the above

mentioned diseases states (Greenlee et al., 2000). A better understanding of the role of

hypoxia and the contribution of the HIFs in disease progression may lead to better

strategies and therapies to combat these disease outcomes. It is envisaged that further

research should focus on two general areas that aim to understand, in particular disease

states,

i) biological processes and target genes regulated by the various HIF complexes,

and

iÐ the mechanisms by which the activity of the various HIF complexes are

regulated.

Regulation of HIF proteins by Hypoxia

One of the major challenges facing the HIF research field has been to understand the

molecular mechanism by which cells are able to sense oxygen levels and transduce the

physiological signal of reduced oxygen levels to changes in HIF activity. As mentioned

earlier the HIFcr subunits represent the hypoxia responsive component of the HIF complex.

It has been reported that oxygen levels affect the protein stability, subcellular localisation,

DNA binding capacity and transcriptional potency of the HIFcr, subunits (reviewed by

Semenza, 1999b). While the HIFo subunits maybe subject to numerous levels of regulation

by oxygen it has been the the analysis of HIF-lcr. and HIF-2c¿ protein stability and

11

transactivation potency that have provided the greatest insights into cellular oxygen

sensing.

Oxygen sensor - many candidates

Insights into the molecular mechanisms of oxygen sensing in mammalian cells, like the

cloning of HIF, initially came from studies investigating the hypoxic induction of the Epo

gene. In these early studies it was shown that the Epo gene could be transcriptionally

induced with transition metals such as cobalt chloride and nickel chloride, while carbon

monoxide blocked hypoxia induced Epo production (Goldberg et al., 1988). The

conclusion drawn from these initial studies was that oxygen sensing involved a

hemoprotein whose activity could be affected by replacement of iron (cobalt chloride) or

by binding of carbon monoxide. With the discovery of HIF, initial investigations showed

that cobalt chloride and the iron chelator desferrioxamine (Dsfx) were potent inducers of

HIF activity (V/ang and Semenza,l993b) and other hypoxia (HIF) responsive genes (Zhu

and Bunn, 1999), while the kinase inhibitor 2-aminopurine blocked HIF activity (Wang

and Semenza,l993a). Together these observations supported the concept of a hemoprotein

oxygen sensor that was linked to a phosphorylation signalling cascade. However, over time

a bewildering collection of oxygen sensing models in addition to the hemoprotein model

have been proposed (for comprehensive review see Semenza, 1999a). One model

suggested the sensor may constitute a iron-sulfur cluster. While an initial report suggested

HIF-1c¿ may bind non heme iron, the results could not be repeated and the report was

retracted. Experimental data derived from the use of redox sensitive fluorescent

compounds that measure reactive oxygen species (ROS) also proposed that the oxygen

signalling pathway may involve changes in the level of ROS, either by a heme containing

NADPH oxidoreductase (Fandrey et al., 1997) or by the mitochondrial electron transport

chain (Chandel et al., 2000). However, the NADPH oxidoreductase and mitochondrial

electron transport chain models were in contradiction as they proposed a decrease and

increase in ROS generation during hypoxic stress, respectively. Even though the above

models were complex and at times contradictory, they did have the common threads of

requirements of iron and oxygen in the generation of the oxygen-hypoxia signal,

suggesting that these two elements might be critical components of the oxygen sensor. In

certain cell types oxygen sensitive ion channels exist (Seta et a1.,2002), but the ubiquitous

12

expression of HIF meant that a more general hypoxia sensing mechanism must exist. To

decipher a universal mechanism of hypoxia sensing, effort over the next few years

switched from trying to identify the sensor to trying to better understand the parameters by

which the HIFo subunits were regulated by low oxygen levels.

HIF protein stability

Biochemical analysis of HIF-lcr revealed that HIF-Iø was subject to rapid turnover at

normoxia whereas hypoxia, cobalt chloride or iron chelators blocked degradation leading

to the accumulation of the HIF-lcr protein (Huang et al., t996; Kallio et al., 1997).

Treatment with proteasomal inhibitors and mutation of the ubiquitin activating enzyme E1

revealed that HIF-Iû, was being degraded by the ubiquitin proteasome pathway under

normoxic conditions (Salceda and Caro, 1997). To search for regions that conveyed

ubiquitin mediated degradation, various portions of HIF-1ct were fused to the stable Gal4

DNA binding domain and stability of the constructs were analysed by western blot and

pulse chase assays. Using this approach it was found that the instability of HIF-lcr, mapped

to a region of approximately 200 amino acids (residues 401-603) located C-terminal to the

PAS domain (Huang et al., 1998). This region was subsequently called the oxygen-

dependent degradation domain (ODD) and removal of the entire ODD rendered HIF-1o

stable at normoxia. Likewise, analysis of HIF-2cr. revealed that HIF-2u, was also subject to

proteasomal degradation at normoxia (Wiesener et al., 1998) via a similar ODD like region

(Ema et al., 1999). Hypoxia, cobalt chloride or iron chelating agents were also shown to

block degradation of HIF-2c¿ (Wiesener et al., 1998), suggesting a similar mechanism was

responsible for degrading both HIFcr subunits.

Germline mutations of the von Hippel-Lindau (VHL) protein lead to the development of a

wide variety of tumors including clear cell carcinomas of the kidney and vascular tumouts

of the central nervous system and retina. A hallmark of VHL disease is the high degree of

vascularisation, which is due to constitutive expression of a large number of hypoxia

inducible genes such as VEGF (reviewed by Kaelin and Maher, 1998). Since VEGF is a

known target for the HIF proteins these observations raised the question of whether VHL

disease and HIF were somehow related. Using various cell lines deficient in VHL Maxwell

et al., (1999) demonstrated that VHLJ cells expressed high levels of HIF-lcr. and HIF-2a

13

protein at normoxia. Moreover, the protein levels of HIF-1c¿ and HIF-2cr could not be

further induced by hypoxia or Dsfx treatment. Reintroduction of VHL back into these

VHL deficient cell lines resulted in a reduction of normoxic HIF-lcr and HIF-2oc protein

that was now able to be induced by hypoxia or Dsfx treatment (Maxwell et al., 1999).

Collectively these observations suggested that VHL may be an important regulator of

HIFq, stability. To support this the VHL protein was shown to form a complex with

elongins B and C and cullin 2 and this subsequent complex contained E3 ubiquitin-protein

ligase activity (Lisztwan et al., 1999). Further analysis then demonstrated that VHL could

physically interact with (Maxwell et al., 1999) and ubiquitylate (Cockman et al., 2000;

Kamura et al., 2000; Ohh et a1., 2000; Tanimoto et al., 2000) the HIFø subunits via the

ODD. Together these experiments defined the VHL complex as a ubiquitin ligase capable

of ubiquitinating the HIFcr, subunits targeting them for destruction by the proteasome.

Control of HIFa stability by proline hydroxylation

V/ith the identification of the VHL ligase complex the next obvious question was what

signal primed the ODD for VHL mediated degradation? The ODD contained a number of

proline, glutamic acid, serine and threonine (PEST)-like sequences, which have previously

been shown to be important signal sequences for recruiting E3 ubiquitin ligases

(Rechsteiner and Rogers, 1996). However, deletion analysis (Huang et al., 1998) and

misense mutations of some of the residues (Sutter et al., 2000) of the the PEST sequences

revealed that they were not critical for HIF-1cr stabilisation. Previous reports had suggested

that phosphorylation may play an important role in the activation of HIF-lcr (Salceda and

Caro, 1997; Wang et al., 1995b), and it had been observed that both HIF-lcr, (Richard et al.,

1999) and HIF-2cr. (Conrad et al., f999) were phosphoproteins. However, the replacement

of potential phosphoacceptor residues in the ODD was shown to have no effect on HIF-lcr

activity (Srinivas et al., 1999). Taken together these observations suggest that the

proteasomal mediated degradation of HIFo subunits may occur via a novel mechanism.

Protein interaction and ubiquitylation assays narrowed down the major VHL binding

region to a20 amino acid stretch within the ODD of HIF-1cr and HIF-2o (Cockman et al.,

2000; Tanimoto et al., 2000 Yu et al., 2001a). Treatment with hypoxia or cobalt chloride

was able to induce the dissociation of VHL from HIF-1cr, suggesting that some cellular

L4

Flgure 3. Sequence alignment of the proline sites (*) targeted for

hydroxylation in the oxygen-dependant degradation domain of various

HlFa subunits. The core motif is shaded.

component in normoxic cells may be responsible for promoting VHL association (Yu et

aI.,200la). To support this proposal synthetic peptides made to the minimal VHL binding

motif of HIF-lcr were unable to interact with VHL unless pretreated with normoxic cell

extracts (Ivan et al., 2001; Jaakkola et al., 200I; Yu et al., 2001b). Further experiments

utilising this minimal binding motif then went on to discover that enzymatic hydroxylation

of proline (Pro) residue (HIF-1o Pro564, HIF-2cr Pro530) was critical for VHL binding to

HIFcr subunits (Ivan et a1.,2001: Jaakkola et a1., 20OI; Yu et al., 2001b). A second proline

hydroxylation site (Pro402) was also found in the N-terminus of the ODD of HIF-lcr,

(Masson et al., 2001). As expected the proline residues targeted for hydroxylation are well

conserved in HIFcr, members (Figure 3). Hypoxia or iron antagonists (Dsfx and cobalt

chloride) blocked hydroxylation of these proline residues and subsequent VHL binding

(Ivan et a1.,200I; Jaakkola et al., 200I; Masson et al., 2O0I; Yu et al.,2001b) providing

the long sort after link between oxygen availability and iron in the regulation of HIFcr

protein stability.

HIF prolyl hydroxylases

Synthetic peptides of the minimal VHL binding motif of HIF-lct synthesised with a 4-

hydroxyproline at position 564 were able to bind VHL whereas a wild type non Pro-OH

peptide did not bind VHL (Ivan et al., 2OOl; Jaakkola et al., 200I Yu et al., 2001b).

Structural analysis demonstrated the tight binding of VHL to hydroxylated peptide was

due to the a 4-hydroxyproline residue forming critical hydrogen bonds with residues in

VHL (Hon et al., 2002; Min et al., 2002). Together this suggested that VHL can

specifically recognise a 4-hydroxyproline and the HIF modifying enzyme responsible for

hydroxylating the proline residues in the ODD was likely a prolyl-4-hydroxylase eîzyme.

The best characterised prolyl-4-hydroxylases are those that modify proline residues in

collagen (Kivirikko and Myllyharju, 1998). In addition to oxygen and iron the collagen

prolyl-4-hydroxylases also require the cofactor 2-oxoglutarate. Using an inhibitor of 2-

oxoglutarate it was demonstrated that the HIF modifying enzyme like the collagen prolyl

hydroxylase required 2-oxoglutarate to efficiently hydroxylate HIF-1o (Jaakkola et al.,

15

PAS

NORMOXIA

o22-oxoglutarate

cozsuccinate

,91

X

HYPOXIA

Stabilised

CAD

HlFcr

HPH(Fe2*)

P

ñ +Nuclear accumulation

heterodimerise with ArntC-term i nal transactivation

IProteasomal Degradati on

Figure 4. Protein stabilisation of Hypoxia lnducible Factors (HlF) by oxygen depen'

dent prolyl hydroxylation. ln oxygenated conditions (normoxia) HIF Prolyl Hydroxylase

(HPH) hydroxylate proline residues(P-OH) within the oxygen-dependent degradation

domain (ODD) of HlFcr. von Hippel Lindau ( VHL) protein ligase recognises the hydroxylated

proline and targets HlFo for destruction by the proteasome. Under hypoxic conditions the

HPH is inactive and HlFcr escapes destruction to activate target genes. bHLH (basic helix-

loop-helix), PAS (Per-Arnt-Sim), CAD (Cterminal transactivation domain).

2001), providing further evidence that the HIF modifying enzyme was a prolyl-4-

hydroxylase.

The prolyl hydroxylases that modify collagen were not able to modify the proline residues

within the ODD (Jaakkola et al., 2001). Subsequent database searches in combination with

biochemical assays were then employed to identify and clone three mammalian HIF-

prolyl-4-hydroxylases (HPH-l, HPH-2 and HPH-3) capable of hydroxylating the key

proline residues within the ODD (Bruick and McKnight, 2001; Epstein et a1., 2001). The

enzymatic reactions carried out by the HPHs reveal that the proline hydroxylation reaction

requires oxygen (in the form of dioxygen Or), iron (Fe'*) and the cofactor 2-oxoglutarate

(Figure 4). The reaction is inherently dependent on ambient oxygen because the oxygen

atom used to form the proline hydroxyl group is derived from molecular oxygen. The

cofactor 2-oxoglutarate is required because it undergoes decarboxylation to succinate and

accepts the remaining oxygen atom. Finally, experiments using recombinant HPH showed

that the rate of hydroxylation of Pro564 varied with changes in oxygen concentration,

providing evidence that the HPHs were sensitive to oxygen levels and may represent an

oxygen sensor (Epstein et al., 2001). Therefore, the rapid turnover of the HIFa protein

subunits involves oxygen-dependent prolyl-4-hydroxylation by the HPHs, this

modification then serves as a signal for VHL binding and polyubiqutination which targets

the HIFa subunits for proteasomal degradation. The importance of the HPH-HIFcI-VHL

pathway in the oxygen response is further confirmed by the finding that components of

this pathway are functionally conserved in C.elegans (Epstein et al., 2001) and Drosophila

(Bruick and McKnight, 2001).

HIF transactivation

In addition to protein stability the transcriptional potency of the HIFo subunits are also

known to be regulated by changes in oxygen levels. When HIF-1a was initially cloned it

was shown that truncation of the carboxy-terminus downstream of the PAS B region

resulted in a dramatic loss of hypoxia inducible activation of a HRE containing reporter

gene (Jiang et al., 1996a). This observation suggested that HIF-1o may contain amino acid

regions important for transactivation of target genes. To support this the transcriptional

coactivator protein CREB-binding protein (CBP)/p300 which contains acetyltransferase

16

activity was shown to interact with HIF-lcr via its cysteine-histidine rich-l (CHl) domain

(Arany et a1., 1996).

To search for regions that conveyed transcriptional activity various portions of HIF-1a

were fused to the DNA binding domain of Ga14 and the transcriptional activity of the

various chimeric constructs were analysed using reporter gene assays. Using this approach

it was found that HIF-lc¿ contained two transactivation regions, termed the amino-terminal

transactivation domain (NAD, residues 53I-515) and the carboxy-terminal transactivation

domain (CAD, residues 786-826) (Jiang et al., 1991; Pugh et al., 1997). Treatment with

hypoxia, cobalt chloride and Dsfrx all enhanced the activity of the minimal NAD. Since

the NAD overlaps one of the proline hydroxylation sites (Pro564) within the ODD its

increase in transcriptional activity at hypoxia is difficult to dissect from the influence of

protein stability (Pugh et al., 1997). Unlike the NAD, the potent activity of the CAD did

not change with hypoxia treatment. However, if amino acid residues C terminal to the

minimal CAD (652-785) were included a substantial decrease in activity was observed at

normoxia and more interestingly, full activity of the CAD could be restored if cells were

treated with hypoxia, cobalt chloride or Dsfrx (Jiang et al., 1997; Pugh et a1.,1997). Unlike

the NAD, the increase in transcriptional activity of the CAD was not attributed to changes

in protein stability (Pugh et a1., 1997).Instead, hypoxia and iron antagonists were shown to

promote the recruitment of coactivator proteins such as CBP/p300 (Carrero et al., 2000;

Kallio et al., 1998; Kung et a1., 2000), steroid receptor coactivator-l (SRC-l), and

transcription intermidiary factor 2 (TIF2) (Carrero et al., 2000) to the CAD region. Further

deletion analysis narrowed down the minimal region of inhibition to a 9 amino acid stretch

(716-784) located adjacent to the CAD (Carrero et al., 2000; Kallio et al., 1998).

Substitution of an arginine dileucine (RLL) sequence within this region to alanine residues

greatly enhanced activity of the CAD at normoxia (O'Rourke et a1., 1999).Interestingly,

the CAD contains a number of di-leucine repeat sequences and in other transcription

factors di-leucine rich regions have been shown to be important for mediating coactivator

binding (Torchia et al., 1997). Likewise, analysis of HIF-2o revealed that HIF-2cr also

contained two transactivation domains with similar organisation to the NAD and CAD

found in HIF-1o (Ema et al., 1999; O'Rourke et al., 1999). As with HIF-lcr, the CAD of

HIF-2cr, was inducible by hypoxia and iron antagonists via a similar inhibitory region.

While the transactivation capability of HIF-3o is less characterised sequence alignment

T7

HIFcr

HypoxiaDP, Dfrx,Go

O2CAD

Degradation byproteasome

Stabilisation &nuclear accumulation

ÞHypoxia

DP, Dfrx,Goo

IArnt

lnduction of hypoxia genes

Figure 5. Two step hypoxic activation of Hypoxia lnducible Factors (HlF), ln oxygenat-

ed conditions HIF prolyl hydroxylase (HPH) hydroxylates (oH) the oxygen-dependent degra-

dation domain (ODD) of HlFo and targets HlFa for destruction by ubiquitin proteasome path-

way. Hypoxia and iron antagonists block HPH and HlFo escapes destruction to dimerise with

Arnt and bind hypoxia response elements (HRE) in target genes. Hypoxia and iron antago-

nists also stimulate the recruitment of coactivators (CBP/P300) to the C-terminal

transactivation domain (CAD) to increase induction of hypoxia responsive genes. von Hippel

Lindaul protein (VHL), cobalt chloride (Co), desferrioxamine (Dfrx), 2'-2-dipyridyl (DP).

Id

ODDPAS

HRE

cBP/P300

suggests that HIF-3o lacks an inducible CAD region (Gu et al., 1998). Therefore the

transactivation activity of HIF-1a and HIF-2o CADs is negatively regulated by oxygen

independent of protein stability, suggesting that along with ODD exists a second oxygen

sensing region near the carboxy terminus (Figure 5).

How hypoxia and iron antagonists stimulated CAD coactivator recruitment was initially

not clear. In contrast to in-vivo experiments where the recruitment of CBP/p300 via its

CH1 domain was inhibited by normoxic conditions (Kung et al., 2000) in-vitro studies

showed the CHl domain interaction with HIF-1c¿CAD can occur efficiently at normoxia

(Arany et al., 1996; Kung et al., 2000). These observations strongly suggested that either a

cellular factor or a cellular post-translational modification or both maybe targeting the

CAD for repression at normoxia with hypoxia and iron antagonists blocking these effects.

With the discovery that prolyl hydroxylation is responsible for regulating the stability of

the HIFcr, subunits one may have hypothesised that prolyl hydroxylation may also have

been responsible for regulating HIF-lcr and HIF-2cr CAD activity. While the CAD domain

contains a number of proline residues, none of them conforms to the consensus proline

hydroxylation motif found in the ODD. Also, no evidence has been reported to date to

suggest that either of the three HPH enzymes is responsible for regulating CAD activity.

While an exact mechanism of CAD regulation was not immediately forthcoming, a

number of studies suggested the regulation of CAD activity may have been influenced by

either post-translational modifications targeting the CAD and or repressive protein factors

binding the CAD region. For example, changes in cellular redox conditions were suggested

to influence HIF-1c¿ transcriptional activity (Huang et al., 1996). The redox factor-l (Ref-

1) protein was proposed to regulate HIF-lct and HIF-2cr transcriptional activity by reducing

a specific cysteine residue within the CAD domain to enhance coactivator binding (Carrero

et a1., 2000; Ema et al., 1999). However, substitution of the cysteine to a serine residue

(Ema et a1., 1999) which was previously shown to mimic the reduced sulfhydryl group

produced by Ref-l (Xanthoudakis and Curran, 1992) did not stimulate CAD activity or

coactivator binding, but instead, abolished activity and binding all together. Indeed another

report has now suggested that this cysteine residue is not a target for reduction by a

reducing factor, instead this study concluded the cysteine may represent an important

18

residue in the molecular interface linking the CAD to the CHI domain of CBP/p300 (Gu et

al,, 2001).

As well as redox modification phosphorylation of HIF has also been suggested to be

important in regulating transcriptional activity (reviewed by Minet et al., 2001). In

particular the p38 mitogen activated protein kinase (p38-MAPK) (Hirota and Semenza,

2001; Minet et a1.,2000; Sodhi et al., 2000) and diacylglycerol kinase (Aragones et al.,

2001), have been linked to hypoxic regulation of HIF-lcx, transcriptional activity. Likewise

the phosphorylation state of a threonine residue in the CAD (HIF-2o Thr844, HIF-lc¿

Thr796) was recently suggested to be important for binding p300 (Gradin et a1.,2002).

However, the phosphorylation of this residue was not confirmed by mass spectrometry or

amino acid analysis, thus the presence of this phosphorylation awaits further confirmation.

The gas molecules nitric oxide (NO) and carbon monoxide (CO) have also been shown to

inhibit the induction of HIF target genes such as VEGF (Liu et al., 1998). Further analysis

has suggested that production of NO or addition of CO to cells inhibits the transcriptional

potency of the CAD of HIF-lcr (Huang et al., 1999). Together these observations have led

to the proposal that a heme containing protein may be responsible for sensing oxygen

levels. In this model the NO and CO might bind to the heme moiety and block the

normoxic function of the putative oxygen sensor leading to the activation of the CAD.

Recently a protein called factor inhibiting HIF-I (FIH-l) was identified as a negative

regulator of HIF-lcr, transactivation (Mahon et a1., 200I). FIH-I was shown to interact with

HIF-1c¿, VHL and histone deacetylases to suppress activity of the CAD. While no

mechanism of hypoxic regulation was found in this study it was speculated that the

mechanism of inhibition may involve the oxygen-dependent regulation of either the

association of FIH-I with VHL, or the recruitment of repressive factors of transcription

such as histone deacetylases to the VHL FIH-I complex. It will now be interesting to

determine if FIH-I can interact with and regulate in a similar manner the activity of the

HIF-2cr, CAD.

In summary, when this thesis commenced it had been established that under oxygenated

conditions the interaction of coactivator proteins with the CAD of HIF-1u and HIF-2q was

t9

prevented and by some unidentified process hypoxia and iron antagonists could relieve the

repressive effects on the CAD, thus allowing coactivator protein binding to result in an

increase in CAD activity.

Alternative mechanisms of HIF-la activation

As well as being regulated by hypoxia, the activity of HIF-1o has also been shown to be

influenced by heat (Katschinski et al., 2002) and various growth factors and cytokines such

as insulin (Zelzer et al., 1998), insulin-like growth factors (Feldser ef. a1.,1999), interleukin

1 (Thornton et al., 2000), platelet derived growth factor, thrombin and angiotensin II

(Richard et al., 2000). Invariably these alternative stimuli have been shown to increase

HIF-lcr protein levels under normoxic conditions. In the case of heat treatment it was

demonstrated that this process was dependent on a phosphatase resistant form of HIF-lcr

that associated with heat shock protein-9O (HSP-90) (Katschinski et al., 2002). HSP-90 has

previously been shown to help stabilise HIF-1o by averting a VHl-independent

degradation pathway (Isaacs et al., 20OZ), however, the mechanism by which HSP-90

association helps stabilise HIF-1o is poorly characterised. On the other hand it has been

reported that insulin (Treins et al., 2002) and heregulin (Laughner et al., 2001) can

stimulate HIF-1c¿ through a phosphatidylinositol 3-kinase dependent pathway that targets

the translation of HIF-1o¿ protein. In both cases the rate of synthesis of HIF-1a was

demonstrated to be increased after growth factor stimulation and directly correlated with

an increase in the induction of HIF target genes such as VEGF (Laughner et al., 2001;

Treins et a1., 2002).It will now be interesting to establish whether such an affect on HIF-

1a synthesis is a common feature of other growth factors and cytokines and whether HIF-

2a and HIF-3 ct are also subject to similar control mechanisms.

20

Arus oF PnESENT INvnSTIGATIoN

At the outset of this thesis the main questions facing the HIF research field were twofold.

Firstly, mouse gene knockout studies of the HIF-1c¿ and HIF-2o members had revealed

both factors regulated quite distinct biological programs during mouse development. These

observations raised the question of how different biological programs could be initiated by

two highly similar proteins. Secondly, relatively little was known about how oxygen levels

were sensed by cells and moreover, how a lack of oxygen resulted in the activation of the

HIF proteins. While several lines of evidence had suggested this may involve a

combination of redox and phosphorylation cascades targeting the HIFc¿ molecules, no

exact mechanism of oxygen sensing and signalling had been confirmed at the outset of this

study. With the above observations in mind the general focus and aims of this thesis have

been to;

(i) Determine if the DNA binding activity of HIF-1o and HIF-2cr is differentially

regulated by the redox factor Ref-1.

(ii) Investigate by the use of mass spectrometry whether the hypoxia-inducible C-

terminal transactivation domain of HIF proteins is a target for a oxygen regulated

post-translational modification.

2t

REsuLTs

Discovery that DNA binding activity of HIF-2' is enhanced by Ref-1

(Papers I & II)

Despite preliminary reports of HIF-1ø and HIF-2cr, sharing strong similarities in their

activation mechanisms, targeted disruption of each gene had indicated that both are

essential for embryo development via quite distinct biological pathways. At the outset of

this thesis it was not known how such specificity of action was achieved. The combined

studies outlined in Papers I and II describe work demonstrating the DNA binding potential

of HIF-2o¿ (referred to in Papers I and II as HIF-like factor (Fil-F)), but not HIF-lcr, can be

regulated by Ref-1 protein.

In paper I, an investigation of the DNA binding properties of bacterially expressed HIF-1c¿

and HIF-2cr proteins containing the amino terminus bHLFilPASA regions had surprisingly

found that the DNA binding activity of HIF-2cr, on HRE was substantially weaker than

HIF-1c¿ (Paper I Figure Ð. If the concentration of reducing agent (DTT) in the DNA

binding assay was increased from 0.1mM to 10 mM the DNA binding activity of HIF-2cr

was restored to a similar level as HIF-1o, suggesting that HIF-2cr DNA binding activity

may be sensitive to redox control. By comparing the basic DNA binding domains of both

HIF-lcr, and HIF-2c¿ we had observed that the HIF-2c¿ basic region contained a cysteine

residue, whereas, in HIF-1c¿ this residue was a serine (Paper I Figure 3). In other basic

DNA binding domain containing transcription factors such as Fos-Jun it had been

demonstrated that a cysteine within the basic region was often subject to redox control, and

that a change in redox status affected DNA binding activity (reviewed by Morel and

Barouki, 1999). More importantly, this cysteine was shown to be commonly reduced by

Ref-1 protein. V/ith this in mind we conducted in Paper I (Figures 4 e.7) a number of

experiments to test if the DNA binding properties of HIF-2o were subjected to redox

control by Ref-l. The gel mobility shift experiments revealed that the DNA binding

activity of HIF-2a could be enhanced when bacterially expressed fragments of Ref-l

protein containing the redox domain were included in the assay. No change in DNA

22

binding activity was observed when HIF-lcrwas treated with the same Ref-1 protein. We

therefore concluded that HIF-Ic¿ was resistant to redox control because it contained a

serine at this critical position in the basic region and not a cysteine as found in HIF-2cr.. To

test this further we then made a serine-cysteine point mutant of this critical residue in HIF-

lcr and found that this point mutant bound the HRE with weaker affinity than wild type

HIF-1c¿. 'When we performed the gel shift assays with the Ref-l protein the DNA binding

activity of the serine to cysteine point mutant was restored to wild type levels. From these

experiments we concluded that the reason HIF-2c¿ was susceptible to redox control was

because HIF-2a contained a cysteine residue in its basic region and not a serine as found in

HIF-1c¿. Utilising a mammalian two-hybrid approach we then demonstrated that Ref-1 was

able to specifically interact with the N terminal bHLFVPASA region of HIF-2o, but did not

interact with HIF-lcr bHLH/PASA region. If we mutated the cysteine in the basic region of

HIF-2cr to a serine this interaction was abolished (Figure 2 Paper II). Combined with the

gel mobility shift assays we concluded that the unique cysteine residue in the basic DNA

binding region of HIF-2cr was a target for the reducing activity of Ref-l.

The gel mobility shift experiments had shown that the DNA binding activity of HIF-2cr

was dependent on a reducing activity that could be supplied by Ref-l. We therefore

predicted that in a cell culture system the ability of HIF-2c¿ to activate a HRE driven

reporter gene might be dependent on sufficient Ref-l protein levels. 'When we tested this

(Figure 8 Paper I) using an inducible antisense Ref-1 system to regulate the levels of

endogenous Ref-1 protein, we did observe a reduction in the ability of HIF-2cr to activate a

HRE driven reporter when Ref-1 levels were reduced. However, when we repeated the

experiment with HIF-1o we unexpectedly observed a reduction in HRE reporter activity

when Ref-l levels were reduced. Since we had shown that the amino terminal DNA

binding region of HIF-lcr, was not subject to Ref-l regulation, we sought to investigate if

the C terminal region of HIF-lcr that contains the transactivation domain could be

influenced by Ref-l. To test this we made a chimeric protein that consisted of the bHLH

DNA binding amino terminus of the DR, which is not influenced by Ref-1, fused to the

carboxy terminus of HIF-1ct. When we tested this chimera for activity we found its activity

was decreased when the level of Ref-1 was decreased by expressing antisense Ref-1

(Figure 9 Paper I). We therefore concluded that while the DNA binding activity of HIF-lcr

is not influenced by Ref-l, the C-terminus of the protein which harbours the

23

Table 1. Tryptic peptide fragments generated when 6xHis.myc.HIF2sCAD

protein is digested with trypsin as outlined in Paper III

Peptide Location

(amino acid)

Pl (1-16)

P2 (17-27)

P3 (28-34)

P4 (3s-4s)

Ps (46-s2)

P6 (s3-74)

P7 (7s-81)

P8 (82-e7)

Pe (e8-r16)

P10 (117-r20)

PIr (t2t-r26)

Sequence

MGGSHHHHHHGGSEQK

LISEEDLHMMR

GLGQPLR

HLPPPQPPSTR

SSGENAK

TGFPPQCYAS QFQDYGPPGAQK

VSGVASR

LLGPSFEPYLLPELTR

YD CE\.TNVPVPG S STLLQ GR

DLLR

ALDQAT

Mass (average)

daltons

1759.831 1

1373.6064

739.8670

1226.3901

691.6910

2444.6439

674.7s01

1845.1513

209r.3t23

5t5.6062

618.6688

transactivation domain can be stimulated by Ref-l. While we did not investigate if Ref-l

could influence the C terminus of HIF-2c¿, a report published during this work showed that

the transcriptional activity of HIF-2o could be stimulated by Ref-l (Ema et a1., 1999).

Sebsequent work by others also found that Ref-1 could increase the activity of the HIF

CAD (Canero et al., 2000). Finally, to assess if Ref-l is absolutely required for in-vivo

HIF-2cr. DNA binding activity, further experiments utilising Ref-1/-knockout cells or RNA

interference (RNAi) will need to be undertaken.

Discovery that HIF transcriptional activity is regulated by asparagine

hydroxylation (Paper III)

In addition to regulating protein stability, oxygen has also been shown to affect the

transcriptional activity of both HIF-lcr, and HIF-2o via a region termed the CAD. Work

outlined in Paper III describes the discovery that HIF-1a and HIF-2c¿ transcriptional

potency is regulated by hydroxylation of a conserved asparagine residue within the CAD.

Previous work had established that the transcriptional activity of HIF-1cr and HIF-2cr was

dependent on the binding of coactivator proteins such as CBP/p300 to the CAD. In

normoxia the binding of coactivators is inhibited whereas hypoxia or iron antagonists

stimulated the recruitment of a coactivator complex to the CAD region (Carrero et al.,

2000 Kallio et al., 1998; Kung et a1., 2000). As outlined earlier a number of studies

investigating this mechanism have suggested that phosphorylation and redox modifications

may be important in regulating coactivator binding. In accordance with some of these

observations we had shown in Paper I that the redox protein Ref-1 could influence the

activity of the C-terminus of HIF-1 . However, like many previous studies our observations

in Paper I raised further questions, such as whether there was an essential requirement for

Ref-l in CAD activity. Therefore, to better decipher the mechanism of regulation of CAD

activity by oxygen we decided to investigate the posttranslational state of the CAD at

normoxia and after treatment with hypoxia or DP. Our strategy was to make a stable cell

line in 293T cells that expressed the last 100 amino acid residues of HIF-2cr, which

contained the hypoxia inducible CAD (Figure 1 Paper III). To map potential

posttranslational modifications, tryptic peptide fragments from purified samples of the

CAD protein (Table 1) were analysed by matrix-assisted laser desorption ionisation time-

24

Table 2. Peptide ions derived from MALDI MS for normoxia and DP treated

6xHis.myc.HIF2crCAD protein samples.

a) peaks correspond to sequences/fragments from 6xHis.myc.HIF2otCAD as determined by

MS/I\4S

b) fragment YDCE\TNVPVPGSSTLLQGR hydroxylated (+16 daltons) at asparagine 851

c) incompletely cleaved peptide fragment consisting ofPT &, P8 of 6xHis.myc.HIF2aCAD

d) peptide fragments from contaminant 603 ribosomal protein L27 A as determined by MS/MS

Peak Mass

average

Relative

intensity

Mass

average

Relative peak

intensity

Peak

a1 740.48 0.23450.3255lu 740.28

0.23120.1736 2 798.082 '797.29

0.0987¡dJ 932.693d 932.56 0.1288

4a 969.81 0.10084d 969.48 0,1073

5 1079.36 0.06695 1079.44 0.0990

6d l1 I l.5l 0.46s41111.s7 0.76796d

7 1168.45 0.20530.2292,| 1168.55

1216.49 0.17230.4745 88 t216.55

0.05880. I 080 9a 1232.49gd 1232.57

0.03120.0419 10d 1422.08l0d 1422.s8

0.0281ll 1490.6411 1490.70 0.0393

0.1 136t2 1585.59t2 1585.71 0.l44ll13 1699.66 0.023813 1699.79 0.0409

l4u 1844.82 0.2464l4u 1844.98 0.2848

l5 1901.81 0.0593l5 1901.98 0.0717

l6u 2090.89 0.34182091.02 0. I 355l6^

2t06.86 0.02270.1751 170nb 2107.00

0.28440.2619 l8 2162.9118 2163.06

0.42500.2394 lgn 2444.00lgu 2444.08

0.079720ut 2501.0420u' 2501.1 5 0.0488

2l 2666.16 0.01812t 2666.21 0.0181

of-flight (MALDI-TOF) mass spectrometry (MS). From a maximum of eleven tryptic

peptide fragments we were able to identify five HIF-2cICAD fragments in the MALDI plot

that covered approximately 787o of the 100 amino acid CAD protein sequence (Table 2).

We found that one peptide fragment (846-YDCEVNVPVPGSSTLLQGR-864) from

normoxic treated CAD existed as an extra peak with a mass of 16 daltons greater then the

predicted mass (Figure 2A Paper III). The +16 dalton fragment was absent from the

sample derived from cells treated with the iron chelator,2,2'-d\pyridyl (DP), and greatly

reduced in sample from cells treated with hypoxia. From these observations we reasoned

an amino acid residue in the CAD was being oxidised (ie. contained an additional oxygen

atom) at normoxia to produce a hydroxylated residue and hypoxia or iron chelators could

inhibit this hydroxylation event. We then went on to map the hydroxylation (+16 daltons)

to asparagine residue 851 using tandem mass spectrometry (Figure 2B Paper III). Unlike

the ODD, we did not find hydroxylation of any proline residues in the CAD protein.

To assess the importance of the asparagine residue we mutated the critical asparagine

which is well conserved among HIFo¿ species in both the HIF-1cr, (Asn803) and HIF-2cr

(Asn851). Using Gal DNA-binding domain CAD fusion proteins we tested abilities of the

Asn to Ala mutants to transactivate a reporter gene (Figure 3B Paper III). For both HIFc¿

subunits substitution of the critical asparagine to an alanine resulted in potent induction of

the reporter gene at normoxia, which was not further enhanced with either hypoxia or DP

treatment. In agreement with the constitutive reporter gene activity exhibited by the HIF-

1c¿ mutant (Asn803Ala), we further demonstrated that it displayed strong binding during

normoxia to the CHl domain of p300, whereas the wild type CAD displayed much weaker

binding (Figure 3C Paper III). To verify that the modification of the asparagine residue in

the CAD was due to a hydroxylase we tested a known inhibitor of asparaginyl hydroxylase

enzymes, dimethyloxalylglycine (DMOG). Treatment of normoxic cells with DMOG

resulted in the activation of GalHIFc¿CAD fusion proteins (Figure 3B Paper III) and also

promoted interaction of CAD with p300 (Figure 3C Paper III) to a similar extent as seen

with hypoxia treatment, confirming that hydroxylation of the key asparagine residue was

regulating CAD activity.

To ascertain the significance of asparagine hydroxylation to activity of the HIF full length

proteins, we then analysed activities of a series of modified HIFo¿ proteins with single

25

amino acid substitutions, where the critical proline of the ODD, or asparagine of the CAD,

or both were replaced with an alanine (Figure 4 Paper III). From these experiments we

found that simply stabilising the HIFcr proteins by introducing a proline to alanine

substitution in ODD was not sufficient enough to achieve full HIF activity. However, if

both critical proline and asparagine residues were mutated near full activity was achieved,

suggesting that abrogation of asparagine hydroxylation in the CAD is critical for complete

induction of HIFø subunits. Finally, the results we obtained in Paper III lead us to

conclude that the hydroxylation status of the critical asparagine residue is an important

control mechanism that defines the activity of HIFcr CADs. In support of our findings in

Paper III a manuscript was published shortly after our paper presenting data consistent

with the proposal that the CAD of HIF-1o is regulated by a hydroxylation mediated

association with CBP/p300 (Sang et al., 2002).

Characterisation of FIH-I as HIF asparagine hydroxylase (Paper IV)

In paper III we discovered that the activity of HIF-1c¿ and HIF-2cr CADs is regulated by an

oxygen dependent hydroxylation of a conserved asparagine residue, however, we did not

identify the cellular enzyme responsible for carrying out this modification. The work

conducted in Paper IV identifies the protein FIH-I as an asparagine hydroxylase enzyme

capable of modifying the key asparagine residue in the HIFo CADs.

The HIF prolyl hydroxylase (HPH) enzymes that hydroxylate key proline residues within

the ODD are members of the dioxygenase family of hydroxylase enzymes (Bruick and

McKnight, 20011, Epstein et al., 2001). Experiments in Paper III had shown that the

endogenous asparagine hydroxylase activity that modifies the CAD could be blocked with

inhibitors of 2-oxoglutarate (ie. DMOG) and chelators of iron (ie. DP). Like the HPHs

these observations suggested the putative HIF asparagine hydroxylase enzyme may belong

to the family of dioxygenase enzymes that utilise oxygen, iron and 2-oxoglutarate to

hydroxylate target amino acid residues within polypeptides. The structures for some of the

catalytic domains of these dioxygenase enzymes have been solved and reveal a conserved

enzymatic core with key residues for iron binding (Schofield and Zhang, 1999). Using

these key conserved residues as bait we conducted a Genbank data base search to identify

new candidate hydroxylase enzymes. From this data base search we found that a recently

26

cloned factor called FIH-I contained a predicted hydroxylase enzymatic core with

conserved iron binding residues (Figure 1 Paper IV). Previously, FIH-1 was shown to

suppress HIF-1c¿ transcriptional activity by interacting with the CAD, however, the

mechanism of suppression was not identified (Mahon et al., 2001). 'We therefore

speculated that FIH-I may represent an asparaginyl hydroxylase enzyme that suppresses

CAD activity by hydroxylating the key asparagine residue that we previously identified in

Paper III to be important for regulating coactivator binding.

To investigate if FIH-1 could hydroxylate the asparagine residue within the CAD we

performed in-vitro hydroxylation reactions with recombinant FIH-I and HIF-2oCAD

(Figure 4 Paper IV). Using both MALDI-TOF MS and MS/I\{S sequencing we found that

FIH-I could hydroxylate the critical asparagine residue 851 in HIF2c¿CAD and like other

known hydroxylase enzymes of the dioxygenase family, FIH-I also required the cofactor

2-oxoglutarate as DMOG inhibited hydroxylation. No hydroxylation of other amino acid

residues was found suggesting FIH-1 enzyme activity was specific for asparagine residue

851. To support the in-vitro hydroxylation assays it was found that FIH-I inhibition of

CAD activity in cells also requires the cofactor 2-oxoglutarate (Figure 28 Paper IV).

Further experiments then demonstrated that FIH-1 hydroxylation of the CAD could inhibit

the binding of the CH1 domain of p300 (Figure 3 Paper IV). The addition of inhibitors of

iron (cobalt chloride), 2-oxoglutarate (DMOG), or mutation of a key residue for iron

binding (Asp201Ala) all prevents FIH-I from inhibiting CH1 binding. Previous work had

found that the activity of the HPHs was inhibited by hypoxic conditions (Bruick and

McKnight, 200I; Epstein et al., 2001). Interestingly, in reporter gene assays FIH-I was

still able to reduce the activity of the GaIHIF-lcrCAD fusion protein under hypoxic (0.5Vo

Or) conditions (Figure 5 Paper IV). This observation raises the possibility that activity of

FIH-I may not be solely dependent on oxygen availability, but instead may also be

regulated by other factors such as redox and phosphorylation.

Together the results in Paper IV lead us to conclude that FIH-I is an iron and 2-

oxoglutarate dependent dioxygenase enzyme that can hydroxylate the critical asparagine

residue in the CAD to regulate the recruitment of coactivator complexes. In agreement

with our findings a subsequent manuscript has now presented supporting data showing that

27

the FIH-1 protein can hydroxylate the key asparagine residue in the HIF-Iø CAD to

regulate p300 binding (Hewitson et al., 2002).

28

DISCUSSION

Differential DNA binding activity of HIF proteins - implications for HIF

biology

An ever increasing number of transcription factors including activating protein-l (AP-l),

p53, nuclear factor rcB (NF-rcB) and Myb have been shown to have their DNA binding

activities regulated by redox mechanisms (reviewed by Morel and Barouki, 1999). We

presented data in Paper I & II that demonstrated that the DNA binding activity of HIF-2c¿,

but not HIF-lcr, can be influenced by the redox protein Ref-l via a critical cysteine residue

located within the basic DNA binding region of HIF-2cr. Targeted disruption of HIF-lcr,

(Iyer et a1., 1998; Ryan et al., 1998) and HIF-2cc (Peng et al., 2000; Tian et al., 1998)

proteins has shown that these subunits of the HIF complex perform quite distinct

biological functions during mouse development. Therefore how might the reduction-

oxidation of the critical cysteine residue in the basic region of HIF-2c¿ influence HIF-Za

biological function? There are two mechanistic possibilities that may be considered.

Firstly, the reduction of the cysteine residue by redox proteins such as Ref-l may simply

serve as a switch to regulate the ability of HIF-2o to bind to HIF response elements located

in HIF target genes. In this situation the DNA binding activity of HIF-2o will be dependent

on the reduction of this cysteine by Ref-l. Secondly, the oxidised state of the cysteine may

allow HIF-2c to recognise an altogether different DNA response element to the classical

HRE found in HIF target genes. In this situation the oxidised form of HIF-2a may provide

a mechanism for HIF-2o to activate a different subset of target genes to HIF-1cr,, since HIF-

lcr does not contain the redox sensitive cysteine. A similar type of switch has been

implicated to operate in pair domain proteins, which contain two DNA binding regions

(the PAI and RED domain) that recognise different DNA sequences (Tell et al., 1998).

When a specific cysteine residue in the PAI domain is oxidised this abrogates PAI DNA

binding activity, without affecting the DNA binding activity of the RED domain.

Depending on the redox conditions the pair domain proteins are then able to switch DNA

binding specificity to activate different target gene promoters (Tell et al., 1998). Therefore,

29

like the pair domain proteins, the DNA binding specificity of HIF-2o may also be subject

to complex redox control by Ref-1 protein.

We believe that subtle changes in redox conditions may play an important role in

regulating the critical cysteine residue in the basic region of HIF-2ø, affecting the relative

activity of HIF-2cr protein. Therefore what determines that the cysteine in the basic region

of HIF-2o be subject to redox control? The answer is most likely due to the observation

that the cysteine residue is flanked by a number of basic residues (Figure 3 Paper I),

creating an environment that lowers the pK" of the cysteine thiol rendering it susceptible to

oxidation (Choi HJ, 1998; Snyder et al., 1981). While the exact oxidised form of the

cysteine in the basic region of HIF-2a is not known it has been suggested that oxidised

cysteines may exist as either an intramolecular disulphide (S-S), mixed disulphide with

glutathione (S-SG-S), a cysteine sulfenic acid (S-OH, S-hydroxylation), or a S-

nitrosylation (S-NO) all of which are reversible modifications (Stamler and Hausladen,

1998). Determining these cysteine states and subsequent redox switches is inherently

troublesome because the modifications are difficult to produce in a pure enough form for

detailed structural, chemical and biological analysis. However, a recent report by Kim et

al., (2002) has detailed the development of a number of strategies and methodologies to

overcome some of these problems. Using the bacterial transcription factor protein OxyR

the authors made highly pure and distinct oxidised forms of a previously identified redox

sensitive cysteine residue in the OxyR protein. OxyR is a bacterial protein that is a key

transcriptional regulator of the oxidative response in E.coli (Zheng et al., 1998). Using

these highly pure forms of OxyR it was demonstrated that various oxidised states of the

critical cysteine residue imparted quite distinct biological activities ranging from differing

DNA binding and transcriptional activities (Kim et al., 2002). Therefore using a similar

approach it may now be possible to investigate the nature of the oxidised form of the

critical cysteine in the basic region of HIF-2o and determine the mechanism of reduction

by the Ref-1 protein.

Regulation of HIF protein function by asparagine hydroxylation

One of the main challenges of HIF research over the past few years has been to understand

the mechanism by which the HIFo subunits are regulated by low oxygen stress. It was well

30

CAD

Normoxia

o2

Hypoxia

HypoxiacAD DP,Dfrx,Go

I qHlFcrHlFa

Degradation

Arnt

Stabilised

þ

-+ --..

\T

HypoxiaDP,Dfrx,Co

lnduction of hypoxiagenes

Figure 6. Regulation of Hypoxia lnducible Factors (HlF) by oxygen dependent hydrox'

ylation. ln oxygenated conditions (normoxia) the asparagine and HIF prolyl hydroxylases FIH-1

and HPH hydroxylate (on) HlFo on specific asparagine (Asn) and proline (Pro) residues blocking

transactivation and targeting HlFcr for destruction by ubiquitin proteasome pathway. Hypoxia and

iron antagonists block both HPH and FIH-1 activity and HlFa escapes destruction and recruits

coactivators (CBP/P300) to induce hypoxia target genes. Oxygen-dependent degradation domain

(ODD), Cterminal activation domain (CAD), von Hippel Lindaul protein (VHL), cobalt chloride (Co),

desferrioxam ine (Df rx), 2' -2-dipy ridyl (D P ).

established at the outset of this thesis that oxygen availability affects HIFcr subunits at two

key induction steps; notably protein stability and transcriptional potency. A number of

research groups have now demonstrated that the rapid protein turnovet of HIFo subunits at

normoxia involves proline hydroxylation, which is carried out by a family of three HPH

enzymes. Proline hydroxylation promotes binding of the VHL ubiquitin ligase to HIFcr,

ubiquitylation then targets HIFc¿ for destruction by the ubiquitin proteasome pathway

(reviewed by Semenza, 2001). The work described in Papers III and IV of this thesis has

now discovered that the transcriptional potency of the HIF-1o and HIF-2cr, subunits is

controlled by asparagine hydroxylation. In oxygenated conditions the CAD of HIF-lcr, and

HIF-2o are hydroxylated on a critical asparagine residue by FIH-I, an asparagine

hydroxylase enzyme. The hydroxylation of the asparagine residue prevents binding of

p300 and most likely other coactivator proteins, thereby inhibiting the transcriptional

potency of the CAD. Thus, hydroxylation of the asparagine and proline residues in the

CAD and ODD represent two key regulatory events that modulate HIF activity in response

to changes in oxygen availability (Figure 6).

Structural implications

Very recently the solution structure of the CH1 domain of p300 bound to the CAD of HIF-

lc¿ was solved (Dames et a1., 2002; Freedman et a1., 2002).Interestingly, it was found that

the CAD of HIF-1c¿ was intrinsically disordered and had no structure unless it was

complexed with the CHI domain of p300. Analysis of the bound complex revealed that the

CAD remains relatively extended, wrapping itself around the globular structure of the CHl

domain in a hand grasp or vice like manner. The critical asparagine residue (Asn803) that

we found to be hydroxylated in the CAD of HIF-1o is buried deep within the molecular

interface and nearly 45Vo of its surface is concealed in the interface. Asparagine 803 forms

two side chain hydrogen bonds with aspartic acid-799 in the CAD and aspartic acid-346 in

the CHl domain of p300 which help to stabilise the complex. An unusual feature of the

asparagine containing interaction face is that it docks with the CHl domain via its more

polar face (Dames et al., 2OO2; Freedman et al., 2002). To further understand the structural

mechanism of repression that hydroxylation of the critical asparagine has on CHl binding

and CAD activity additional work will need to focus on elucidating the position of the

hydroxyl group on the asparagine residue. The previously identified asparaginyl

31

hydroxylase enzymes can hydroxylate both asparagine and aspartic acid residues in

epidermal growth factor (EGF) like domains found in vitamin K-dependent plasma factors

VII, IX and X, as well as a number of other proteins such as complement proteins Clr and

Cls (Stenflo, 1991). Analysis of the asparagine and aspartic acid hydroxylation products in

the EGF like domains revealed that the hydroxyl group is attached to the p carbon and

orientated with erythro stereochemistry (Przysiecki et al., 1987). If the asparagine in the

HIFo CAD were also hydroxylated in a similar manner to produce an erythro-þ-

hydroxyasparagine then this would place the hydroxyl group in an unfavourable

hydrophobic environment with no hydrogen-bonding partner. Since the non-hydroxylated

asparagine forms a network of side chain hydrogen bonding interactions with critical

amino acid residues in both the CAD and CH1 domain erythro-þ hydroxylation would be

predicted to destabilise the complex .

Apart from hydroxylating the asparagine on the erythro-B carbon position FIH-1 could

possibly also hydroxylate the asparagine on the threo-þ carbon position, or even on the

side chain amide nitrogen to form a hydroxyamic acid. As with the erythro position p

carbon, hydroxylation at the threo position would place the hydroxy group in an

unfavourable hydrophobic environment with no hydrogen bonding partners, causing the

complex to become unstable. A hydroxyl group on the side chain amide would create the

greatest disruption to the complex by disrupting both asparagine side chain hydrogen

bonds (Dames et al., 2002). Unlike other asparaginyl hydroxylases enzymes which can P-

hydroxylate both asparagine and aspartic acid residues the FIH-1 enzyme was shown to

have a clear preference for asparagine in the CAD of HIF-1cr (Hewitson et al., 2002).If the

asparagine in the CAD was substituted with an aspartic acid residue FIH-I hydroxylase

activity for the aspartic acid residue was only 77o of that obtained with asparagine

(Hewitson et al., 2002). This preference tends to suggest that FIH-1 hydroxylation of the

asparagine in the CAD may not occur on the p-carbon but instead may occur on the side

chain amide group. To fully understand the structural implications of asparagine

hydroxylation further work will need to be done to establish the position of the hydroxyl

group. The in vitro hydroxylation assay outlined in Paper IV using recombinant FIH-I and

CAD proteins could be used to generate large amounts of asparagine hydroxylated CAD.

To then determine the hydroxyl position the amino acids from the CAD could be

hydrolysed and examined using an amino acid analyser. Using asparagine standards for the

32

various hydroxy positions it may be possible to then determine the site of hydroxylation.

Elucidating the position of the hydroxyl group should help in rational drug design

strategies, which could then be used to make small molecule therapies to block CAD

activity.

Oxygen sensing

By conducting reactions in a controlled oxygen environment it has been demonstrated that

the activity of the FIPHs are sensitive to oxygen levels (Epstein et a1., 2001), moreover, the

rate of hydroxylation seems to mirror the progressive cellular increase in HIFc¿ protein

levels observed when cells are subject to similar oxygen gradients (Jiang et al., 1996b).

Therefore, it has been suggested that the HPHs may represent oxygen sensors. The finding

that the critical asparagine residue in the CAD can be hydroxylated by FIH-1, a member of

the 2-oxoglutarate dependent family of hydroxylases, raises the possibility that like the

HPHs FIH-1 may also represent an oxygen sensor. This notion is further supported from

the finding that members of this class of enzyme use molecular oxygen to modify their

target amino acid residues in polypeptides (Schofield andZbang,1999).

If FIH-1 is an oxygen sensor its negative activity on the CAD region should be suppressed

under hypoxic conditions. However, in Paper IV we found that over expression of FIH-I

was still able to block CAD activity of a GaIHIF-lcr CAD fusion protein under hypoxic

(0.5Vo Or) conditions. As well as interacting with HIF-lcr,, FIH-1 has been shown to

interact with histone deacetylases (Mahon et al., 2001), which are known to function as

transcriptional repressors. Therefore, a reason for repression of CAD transactivation

domain function under hypoxic conditions, could be that over expression of FIH-1 results

in the recruitment of histone deacetylases to the CAD to repress its activity. To gain a

better understanding of the requirement of oxygen in FIH-I activity further experiments

are required. For example, activities of FIH-1 in situations where varied oxygen levels can

be directly related to the catalytic activity of the enzyme for the asparagine residue in the

CAD would be informative. An assay that monitors the decarboxylation of 2-oxoglutarate

by p-asparagine hydroxylase enzymes has been described (Gronke et al., 1989), and could

be adapted to evaluate the activity of FIH-I towards the CAD at various oxygen levels.

This approach could then establish if the asparagine hydroxylase activity of FIH-I is

33

sensitive to graded oxygen availability. The affinity binding of oxygen to FIH-I at graded

oxygen levels will need to be established to fully characterise FIH-1 as an oxygen sensor.

Substrate specificity

It has been demonstrated in-vitro that FIH-1 can physically interact with the CAD of HIF-

1ct in a region spanning amino acid residues 157-784 (Mahon et al., 2001). Intriguingly,

this portion of HIF-1a does not contain the asparagine residue that we demonstrated is

hydroxylated by FIH-1 in paper IV. The hydroxylated asparagine residue is actually at

position 803 approximately 20-25 residues C terminal to the putative FIH-1 binding

region. This suggests that for FIH-1 to efficiently hydroxylate the asparagine residue in

HIF-Iø it may need to bind to a region in HIF-lcr adjacent to the asparagine residue. To

support this notion it has been demonstrated that the binding of p300 is not enhanced with

the asparagine hydroxylase inhibitors DMOG or iron antagonists when the putative FIH-1

binding region is removed (Sang et al., 2002). A region spanning residues 115-186 in HIF-

1cr contains an RLL motif that has been shown to be critical for the normal silencing of the

HIF-1o CAD at normoxia (O'Rourke et al., 1999). An analogous RLL motif containing

region also operates in a similar silencing fashion in HIF-2a (O'Rourke et al., 1999),

suggesting that this region of both HIF-1a and HIF-2cr, may contain important elements for

targeting FIH-I. Therefore, further work needs to be done to investigate the importance of

the RLL motif and surrounding residues in FIH-1 binding. Furthermore, the demonstration

that FIH-I must bind to HIF-lcr, in a region away from the critical asparagine residue for

efficient hydroxylation may help explain the long known phenomenon that uncoupling the

HIF-1c¿ CAD containing residues 786-826 results in a highly active CAD under normoxic

conditions (Jiang et al., 1997; Pugh et al., 1997) that binds strongly to CBP/p300

irrespective of hypoxia treatment (Kung et al.,2000).

As well as interacting with the CAD region FIH-I has also been shown to interact with

VHL via its p domain (Mahon et al., 2001). Initially this interaction was thought to be

important for the repressive activity of FIH-1 on CAD function (Mahon et al., 2001),

however, a moÍe recent analysis in VHL null cells has shown that VHL is not critical for

FIH-1 repressive activity (Sang et a1.,2002).It is possible that FIH-1 and VHL complexes

may operate in additional oxygen regulated processes that affect the transcriptional

34

response of other pathways. In support of this notion both FIH-I and VHL have been

shown to interact with chromatin modifying histone deacetylase enzymes, which are

known to play an important role in gene repression (Mahon et al., 2001). As well as

mediating the degradation of HIFo subunits, VHL has also been linked to the control of

number of other cellular processes including transcriptional elongation, mRNA stability

and extracellular matrix formation (Ivan and Kaelin, 2001). \Mhile the role of VHL in these

processes is not well characterised it is interesting to note that one of the mRNA targets

suggested to be controlled by VHL is the RNA message for VEGF (Gnarra et al., 1996).

Since VEGF message is known to be stabilised under hypoxic conditions, it will be

interesting to investigate if this oxygen-dependent process is reliant on the interaction of

VHL with FIH-I and moreover if the hydroxylase activity of FIH-1 is also required. Taken

together these observations raise the exciting possibility that FIH-I may have multiple

cellular roles other than just regulating the activity of the CAD of HIF-lcr and HIF-2c¿.

Other mechanisms of activation

The induction of HIF transcriptional activity by well established agents such as hypoxia,

cobaltous ions (cobalt chloride) and iron chelators (Dfrx and DP) can be easily explained

by our finding that FIH-I is a member of the 2-oxoglutarate-dependent family of

hydroxylase enzymes that utilise iron and oxygen to modify their target amino acid

residues. However, as mentioned earlier the activity of the CAD has also been reported to

be influenced by gas molecules (ie. NO, CO), redox (ie, thioredoxin, Ref-l) and

phosphorylation events (ie. p38 MAPK) though the understanding of how these processes

may influence FIH-I function and HIF transcriptional activity is less clear. NO is a known

analogue of dioxygen and analysis of the non heme iron (Fe2*) dependent isopenecillin N

synthase eîzyme, a closely related oxygenase to the 2-oxoglutarate family, has

demonstrated that NO can bind to the iron centre of this enzyme (Roach et al., l99l).

Since NO has only one available oxygen for use in catalysis and 2-oxoglutarate-dependent

dioxygenases normally expend two oxygen molecules for completing the hydroxylation of

their substrates, the binding of NO to the catalytic core of FIH-I may greatly impede

enzymatic activity. This may then help explain the reported positive effect NO has on

CAD activity (Huang et al., 1999). Hydroxylation of substrates by the prolyl-4-

hydroxylases have been shown to be inhibited by the artificial generation of radicals at the

35

eîzyme active site (Huang et al., 1999). Therefore, the effects on HIF transactivation

observed with redox factors such as that observed with Ref-1 in Paper I may be due to the

fact that these factors may alter the normal redox balance of the cell, which then may affect

FIH-I catalytic activity. Finally, it is possible that these other reported agents may also

target components of the coactivator complex. For example, the acetyl transferase activity

of CBP can be stimulated during various stages of the cell cycle by cycle dependent

kinases (Ait-Si-Ali et al., 1998), while phosphorylation of the nuclear acetylase GCN5 by

the DNA-dependent protein kinase can inhibit acetyltransferase activity in response to

DNA repair signals (Barlev et al., 1998).

Other possible CAD modifications

As outlined earlier we were able to identify five of the eleven tryptic peptide fragments of

the HIF2oCAD protein using MALDI-TOF MS and this represented approximately 787o

of the CAD sequence. Of the five peptide sequences we found and analysed we did not

find evidence of any other posttranslational modification of the HIF-2aCAD region. While

we believe that hydroxylation of the critical asparagine residue in the CAD represents the

crucial oxygen-dependent switch that regulates CAD activity, we do not rule out the

possibility that there may exist other CAD modifications. For example, a number of other

studies have suggested that the CADs are phosphorylated (Gradin et al., 2002; Minet et al.,

2001). However, in Paper III when we performed MALDI-TOF MS on the HIF-2c¿774-814

CAD protein we did find any evidence of phosphorylation of any of the peptides we

identified. Efficient detection of peptides by MALDI-TOF MS is known to be affected by

charge (Fenselau 1997), since phosphorylation of amino acid residues will alter the charge

of peptides it is possible that phosphorylation may have affected the ability of peptides

containing phosphorylated residues to ionise efficiently. Thus, this may be one of the

reasons why we were unable to detect phosphorylation of the CAD.

As well it is quite possible that phosphorylation and other posttranslational modifications

may be present on tryptic peptide sequences that we were unable to detect using MALDI-

TOF MS or sequence using tandem MS/MS. Since in Paper III we were analysing samples

that contained a number of mixed peptides our inability to find all HIF-2aCAD peptide

sequences is not totally unexpected. It is well known that individual peptide sequences

36

ionise differently when in mixed peptide populations (Fenselau l99l). Therefore to isolate

and examine the remaining tryptic peptide fragments may require additional strategies

such as coupling MS with liquid chromatography. In this case peptides mixes are separated

using specialised micro-bore capillary methods to isolate highly pure and homogeneous

peptide samples, which are then subjected to MS analysis (Peng & Gygi 2001). It is

possible that this type of approach may then enable the isolation and analysis of the

remaining HIF-2c¿CAD peptide fragments to determine if there exists any additional

posttranslational modifications.

Therapeutic benefits

The finding that HIF transcriptional activity is regulated by asparagine hydroxylation also

raises therapeutic possibilities especially in circumstances of aberrantly high levels of HIF

activity such as that proposed to occur in cancer. Many cancers examined to date have

been shown to have elevated protein levels of HIFcr, which is thought to promote tumor

growth through enhanced angiogenesis (Ratcliffe et al., 2000). In particular the

examination of human gliomas has found a direct link between increased HIF-lcr

expression, enhanced tumor vascularisation and poor prognos\s (Zagzag et a1., 2000).

Consequently, it is thought that therapies aimed at inhibiting the HIF transcriptional

response, in particular the transactivation of VEGF, may be clinically beneficial. Indeed

the disruption of HIF-lcr transactivation has been shown to retard tumor growth in a mouse

model (Kung et al., 2000). In this study a polypeptide derived from the CAD of HIF-lcr,

which spans HIF-lcr residues 186-826 and lacking the FIH-1 binding site, was shown to

constitutively interact with p300 via its CHl domain. The authors of this study then went

on to demonstrate that this polypeptide could block HIF mediated transactivation of

VEGF, by sequestering p300 away from mature HIF complexes, In a tumor mouse model

the expression of this same polypeptide was able to significantly retard tumor growth,

whereas a mutant polypeptide that could not bind p300 did not inhibit tumor growth (Kung

et al., 2000). 'While the anti-tumor effects of this polypeptide were shown to be HIF-lcr,

dependent the authors did concede that their strategy may also affect other p300 activities,

which in a clinical situation may lead to detrimental side effects. Thus, for this strategy to

be generally applicable, more specific pharmacological inhibitors will need to be

developed.

37

The proof of principle provided by Klung et al., (2000) suggests that further work aimed at

developing small molecule inhibitors that target the critical asparagine and surrounding

residues to mimic the hydroxylation state may lead to more specific inhibitors of HIF

coactivator complex formation. This type of strategy may therefore provide a more

selective means to inhibiting the induction of HIF target genes in cancer cells.

Interestingly, while we have demonstrated that the biological activity of FIH-1 requires 2-

oxoglutarate (Paper IV), an unusual feature of FIH-I is that it lacks arginine or lysine

residues located on the 8'h B strand of the catalytic core (Figure 1 Paper IV). These

conserved residues have previously been demonstrated to be involved in binding 5-

carboxylate of 2-oxoglutarate in many other 2-oxoglutarate dependent enzymes (Schofield

and Zhang, 1999). Since these 2-oxoglutarate binding residues are well conserved in the

HPHs (Bruick and McKnight, 2001; Epstein et al., 200I), it suggests FIH-1 may represent

a new structural sub-member of the 2-oxoglutarate dependent enzyme family raising the

possibility that selective agonists and antagonists for FIH-I can be developed. This may

therefore provide an alternative route to the development of therapies to regulate the

binding of coactivators to the CAD region.

Concluding remarks

To date one asparagine (FIH-l) and three proline (HPH-1,2,3) hydroxylase enzymes have

been discovered and found to target different portions of HIFcr, subunits affecting different

steps in the induction of the HIF complex. As a result the number of hydroxylase enzymes

and their various targets may have evolved to help manipulate the magnitude of the HIF

transcriptional response by providing a fine tuning mechanism to gradually alter the

activity of the HIFc¿ subunits in response to subtle changes in oxygen levels. Other studies

have suggested that the nuclear translocation of HIFcc subunits may also be oxygen

regulated (Kallio et al., 1998), and it will now be interesting to establish if this or other

components of HIF regulation are also influenced by either the asparagine or proline

hydroxylase enzymes or other hydroxylation mediated events.

38

RnTERENCES

Ait-Si-Ali, S., Ramirez, S., Bare, F. X., Dkhissi, F., Magnaghi-Jaulin, L., Girault, J. 4., Robin, P.,

Knibiehler, M., hitchard,L.L., Ducommun,8., et aI. (1998). Histone acetyltransferase activity of CBP is

controlled by cycle-dependent kinases and oncoprotein ElA, Nature 396, 184-6.

Alagones, J., Jones, D. R., Martin, S., San Juan, M. 4., Alfranca, 4., Vidal, F., Vara, 4., Merida, I., and

Landazuri, M. O. (2001). Evidence for the involvement of diacylglycerol kinase in the activation of hypoxia-

inducible transcription factor I by low oxygen tension, J Biol Chem 276,10548-55.

Arany, 2., Huarg, L. E., Eckner, R., Bhattacharya, S., Jiang, C., Goldberg, M. 4., Bunn, H. F., and

Livingston, D. M. (1996). An essential role for p300/CBP in the cellular response to hypoxia, Proc Natl Acad

SciUS A93,12969-73.

Barlev, N. 4., Poltoratsky, V., Owen-Hughes, T., Ying, C., Liu, L., Workman, J. L., and Belger, S. L. (1998).

Replession of GCN5 histone acetyltransferase activity via bromodomain-mediated binding and

phosphorylation by the Ku-DNA-dependent protein kinase complex, Mol Cell Biol 18,1349-58.

Beck, L, Ramirez, S., Weinmann, R., and Caro, J. (1991). Enhancer element at the 3'-flanking region controls

transcriptional response to hypoxia in the human erythropoietin gene, J Biol Chem 266,15563-6.

Beck, I., Weinmann, R., and Caro, J. (1993). Chalacterization of hypoxia-responsive enhancer in the human

erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in

erythlopoietin-producing and nonproducing cells, Blood 82, 704-|l.

Bruick, R. K., and McKnight, S. L. (2001). A conserved family of prolyl-4-hydroxylases that modify HIF,

Science 294,1331-40.

Bunn, H. F., and Poyton, R. O. (1996). Oxygen sensing and molecular adaptation to hypoxia, Physiol Rev 76,

839-85.

Carrero, P., Okamoto, K., Coumailleau, P., O'Brien, S., Tanaka, H., and Poellinger, L. (2000). Redox-

regulated recruitment of the transcriptional coactivators CREB-binding protein and SRC-l to hypoxia-

inducible factol lalpha, Mol Cell Biol20,402-15.

39

Chandel, N. S., McClintock, D. S., Feliciano, C.E., Wood, T. M., Melendez, J. 4., Rodriguez, A. M., and

Schumacker, P. T. (2000). Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-

inducible factor-lalpha during hypoxia: a mechanism of 02 sensing, J Biol Chem 275,25130-8.

Choi HJ, e. a. (1998). Crystal structure of a novel human peroxidase enzyme at2.0 A resolution, Nat Struct

Biol5,400-6.

Cockman, M. E., Masson, N., Mole, D. R., Jaakkola, P., Chang, G. V/., Cliffold, S. C., Maher, E. R., Pugh,

C. V/., Ratcliffe, P. J., and Maxwell, P. H. (2000). Hypoxia inducible factor-alpha binding and ubiquitylation

by the von Hippel-Lindau tumor suppressor protein, J Biol Chem 275,25133-41.

Compernolle, V., Brusselmans, K., Acker, T., Hoet, P., Tljwa, M., Beck, H., Plaisance, S., Dor, Y., Keshet,

E., Lupu, F., et al. (2002). Loss of HlF-2alpha and inhibition of VEGF impair fetal lung maturation, whereas

treatment with VEGF prevents fatal respiratory distress in premature mice, Nat Med 8, 102-lO.

Conrad, P. V/., Rust, R. T., Han, J., Millholn, D. E,., and Beitner-Johnson, D. (1999). Selective activation of

p38alpha and p3Sgamma by hypoxia. Role in regulation of cyclin Dl by hypoxia inPCl2 cells, J Biol Chem

274,23510-6.

Crews, S. T. (1998). Control of cell lineage-specific development and transcription by bHLH-PAS proteins,

Genes Dev 12,607-20.

Crews, S. T., Thomas, J. 8., and Goodman, C. S. (1988). The Drosophila single-minded gene encodes a

nuclear protein with sequence similarity to the per gene product, Cell 52, 143-51.

Czyzyk-Krzeska, M. F., Bayliss, D.4., Lawson, E. E., and Millhorn, D.E. (1992). Regulation of tyrosine

hydroxylase gene expression in the rat carotid body by hypoxia, J Neurochem 58, 1538-46.

Dames, S. 4., Martinez-Yamout, M., De Guzman, R. N., Dyson, H. J., and Wright, P. E. (2002). Structural

basis fol Hif-l alpha /CBP recognition in the cellular hypoxic response, Proc Natl Acad Sci U S A 99,5211-

6.

David, M., Daveran, M. L., Batut, J., Dedieu,4., Domergue, O., Ghai, J., Hertig, C., Boistard, P., and Kahn,

D. (l9SS). Cascade regulation of nif gene expression in Rhizobium meliloti, Cell 54,671-83.

Drutel, G., Kathmann, M., Helon, 4., Gros, C., Mace, S., Schwartz, J. C., and Arrang, J. M. (2000). Two

splice variants of the hypoxia-inducible factol HIF-lalpha as potential dimerization paltners of ARNT2 in

neurons, Eul J Neurosci l2,3l0l-8.

40

Ema, M., Hirota, K., Mimura, J., Abe, H., Yodoi, J., Sogawa, K., Poellinger, L., and Fujii-Kuriyama, Y

(1999). Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia

their stabilization and redox signal-induced interaction with CBP/p300, Embo J j,8, 1905-14.

Ema, M., Taya, S., Yokotani, N., Sogawa, K., Matsuda, Y., and Fujii-Kuriyama, Y. (1997). A novel bHLH-

PAS factor with close sequence similarity to hypoxia-inducible factor lalpha regulates the VEGF expression

and is potentially involved in lung and vascular development, Proc Natl Acad Sci U S A 94, 4273-8.

Epstein, A. C., Gleadle, J. M., McNeill, L. 4., Hewitson, K. S., O'Rourke, J., Mole, D. R., Muklerji, M.,

Metzen, E., Wilson, M. L, Dhanda, A., et al. (2OOl). C. elegans EGL-9 and mammalian homologs define a

family of dioxygenases that regulate HIF by prolyl hydroxylation, Cell 107,43-54.

Fandrey, J., Frede, S., Ehleben, 'W., Porwol, T., Acker, H., and Jelkmann, W. (1997). Cobalt chloride and

desfenioxamine antagonize the inhibition of erythropoietin production by reactive oxygen species, Kidney Int

51,492-6.

Feldser, D., Agani, F., Iyer, N. V., Pak, 8., Ferreira, G., and Semenza, G. L. (1999). Reciprocal positive

regulation of hypoxia-inducible factor lalpha and insulin-like growth factot 2, Cancet'Res 59, 3915-8.

Fenselau, C. (1991). MALDI MS and strategies for protein analysis. Analytical Chemistry 69,6614-6654.

Ferrara, N. (1999). Molecular and biological properties of vascular endothelial growth factor, J l[l4ollll4ed 77,

521-43.

Flamme, I., Frohlich, T., von Reutern, M., Kappel, 4., Damert, 4., and Risau, \ü. (1991). HRF, a putative

basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-l alpha

and developmentally expressed in blood vessels, Mech Dev 63,5l-60.

Freedman, S. J., Sun, Z.Y.,Poy, F., Kung, A. L., Livingston, D. M., Wagner, G., andEck, M. J. (2002).

Structural basis for recluitment of CBP/p300 by hypoxia-inducible factor- I alpha, Proc Natl Acad Sci U S A

99,5361-72.

Gibbs, FW. (1965) Joseph Priestley. Thomas Nelson Ltd, London.

Gnarra, J. R., Zhou, S., Mellill, M. J., Wagner, J. R., Krumm, 4., Papavassiliou, E., Oldfield, E. H.,

Klausnet, R. D., and Linehan, W. M. (1996). Post-transcriptional regulation of vascular endothelial growth

factor mRNA by the product of the VHL tumor suppressor gene, Proc Natl Acad Sci U S A 93, 10589-94.

4t

Goldberg, M. 4., Dunning, S. P., and Bunn, H. F. (1988). Regulation of the elythropoietin gene: evidence

that the oxygen sensor is a heme protein, Science 242, l4l2-5.

Goldberg, M. 4., Gaut, C. C., and Bunn, H. F. (1991). Erythropoietin mRNA levels are governed by both the

rate of gene transcription and posttranscriptional events, Blood 77, 27 l-7 .

Goldberg, M. 4., and Schneider, T. J. (1994). Similarities between the oxygen-sensing mechanisms

regulating the expression of vascular endothelial growth factor and erythlopoietin, J Biol Chem 269,4355-9.

Gong, rüy'., Hao, B., Mansy, S. S., Gonzalez,G., Gilles-Gonzalez, M.4., and Chan, M. K. (1998). Structure

of a biological oxygen sensor: a new mechanism fol heme-driven signal transduction, Proc Natl Acad Sci U S

A 95, 15111-82.

Gothie, E., Richard, D. E., Berra, E., Pages, G., and Pouyssegur, J. (2000). Identification of alternative

spliced variants of human hypoxia-inducible factor-lalpha, J Biol Chem 275,6922-1 .

Gradin, K., Takasaki, C., Fujii-Kuriyama, Y., and Sogawa, K. (2002). The transcriptional activation function

of the HIF-like factor lequires phosphorylation at a conserved threonine, J Biol Chem 277, 23508-14.

Greenlee, R. T., Murray, T., Bolden, S., and Wingo, P. A. (2000). Cancer statistics, 2000, CA Cancer J Clin

50,1-33.

Glonke, R. S., VanDusen, Vy'. J., Garsky, V. M., Jacobs, J. W., Sardana, M. K., Stern, A. M., and Friedman,

P. A. (1989). Aspartyl beta-hydroxylase: in vitro hydroxylation of a synthetic peptide based on the structure

of the first growth factor'-like domain of human factor IX, Proc Natl Acad Sci U S A 8ó, 3609-13

Gu, J., Milligan, J., and Huang, L. E. (2001). Molecular mechanism of hypoxia-inducible factor lalpha -p300

interaction. A leucine-rich interface regulated by a single cysteine, J Biol Chem 276,3550-4.

Gu, Y. 2., Moran, S. M., Hogenesch, J. 8., Waltman, L., and Bradfield, C. A. (1998). Molecular

characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit,

HlF3alpha, Gene Expr 7,205-13.

Guillemin, K., and Krasnow, M. A. (1997). The hypoxic response: huffing and HIFing, Cell89,9-12.

Hanahan, D., and Folkman, J. (1996). Patterns and emerging mechanisms of the angiogenic switch during

tumorigenesis, Cell 8ó, 353-64.

42

Hewitson, K. S., McNeill, L. A,., Rioldan, M. V., Tian, Y. M., Bullock, A. N., Welford, R. W., Elkins, J. M.,

Oldham, N. J., Bhattacharya, S., Gleadle, J.M., et al. (2OO2). Hypoxia-inducibleFactor (HIF) Asparagine

Hydroxylase Is Identical to Factor Inhibiting HIF (FIH) and Is Related to the Cupin Structural Family, J Biol

Chem 277, 26351-5 .

Hirota, K., and Semenza, G. L. (2001). Racl activity is required for the activation of hypoxia-inducible factor

l, J Biol Chem276,21166-12.

Hobbs, S., Jitrapakdee, S., and Wallace, J. C. (1998). Development of a bicistronic vector driven by the

human polypeptide chain elongation factor lalpha promoter for creation of stable mammalian cell lines that

express very high levels of recombinant proteins, Biochem Biophys Res Commun 252,368-72.

Hon, W. C., Wilson, M. I., Harlos, K., Claridge, T. D., Schofield, C. J., Pugh, C. V/., Maxwell, P. H.,

Ratcliffe, P. J., Stuart, D. I., and Jones, E. Y . (2002). Structural basis for the recognition of hydroxyploline in

HIF-I alpha by pVHL, Nature 417,975-8.

Huang, L. E., Arany,2. Livingston, D. M., and Bunn, H. F. (1996). Activation of hypoxia-inducible

transcription factor depends primatily upon redox-sensitive stabilization of its alpha subunit, J Biol Chem

271,32253-9.

Huang, L. E., Gu, J., Schau, M., and Bunn, H. F. (1998). Regulation of hypoxia-inducible factor lalpha is

mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway, Proc Natl Acad Sci

usA95,1981-92.

Huang, L. E., Willmore, W. G., Gu, J., Goldberg, M. A., and Bunn, H. F. (1999). Inhibition of hypoxia-

inducible factor I activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and

signaling, J Biol Chem 274,9038-44.

Huang, Z. J.,Edery,I., and Rosbash, M. (1993). PAS is a dimerization domain common to Drosophila period

and several transcdption factols, Nature 364,259-62.

Isaacs, J. S., Jung, Y. J., Mimnaugh, E. G., Martinez, 4., Cuttitta, F., and Neckers, L. M. (2002). Hsp90

Regulates a von Hippel Lindau-independent Hypoxia-inducible Factor-lalpha -degradative Pathway, J Biol

Chem 277,29936-44.

Ivan, M., and Kaelin, V/. G., JL. (2001). The von Hippel-Lindau tumor suppressor protein, Curr Opin Genet

Dev 11,21-34.

43

Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, 4., Asara, J. M., Lane, W. S., and

Kaelin, \V. G., Jr. (2001). HlFalpha targeted for VHl-mediated destruction by ploline hydroxylation:

implications for 02 sensing, Science 292, 464-8.

Iyer, N. V., Kotch, L. E., Agani, F., Leung, S. W., Laughner, E., Wenger, R. H., Gassmann, M., Gearhart, J.

D., Lawler, A. M., Yu, A. Y., and Semenza, G. L. (1998). Cellulal and developmental control of 02

homeostasis by hypoxia-inducible factor I alpha, Genes Dev 12,149-62.

Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., Kriegsheim,4., Hebestreit,

H. F., Mukherji, M., Schofield, C. J., et al. (2001). Targeting of HlF-alpha to the von Hippel-Lindau

ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292,468-12.

Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman, R. J., Mufson,,{., Seehra, J., Jones, S. S.,

Hewick, R., Fritsch, E. F., and et al. (1985). Isolation and characterization of genomic and cDNA clones of

human eryth'opoietin, Nature 313, 806-10.

Jelkmann, V/. ( 1992). Erythropoietin: structure, control of production, and function, Physiol Piev 72, M9-89.

Jiang, B. H., Rue, E., Wang, G. L., Roe, R., and Semenza, G.L. (1996a). Dimerization, DNA binding, and

transactivation propelties of hypoxia-inducible factor 1, J Biol Chem 271, 11171-8.

Jiang, B. H., Semenza, G. L., Bauer, C., and Marti, H. H. (1996b). Hypoxia-inducible factor I levels vary

exponentially over a physiologically relevant range of 02 tension, Am J Physiol 27I , Cll72-80.

Jiang, B. H.,Zheng,J.Z.,Leung, S. W., Roe, R., and Semenza, G.L. (1991). Tt'ansactivation and inhibitory

domains of hypoxia-inducible factor lalpha. Modulation of transcriptional activity by oxygen tension, J Biol

Chem 272, 19253-60.

Jiang, H., Guo, R., and Powell-Coffman, J. A. (2001). The Caenorhabditis elegans hif-l gene encodes a

bHLH-PAS protein that is required for adaptation to hypoxia, Proc Natl Acad Sci U S A 98, '7916-21.

Kadesh, T. (1993). Consequences of heteromedc intelactions among helix-loop-helix ploteins, Cell Growth

Differ 4,49-55.

Kaelin, V/. G., Jr., and Mahel, E. R. (1998). The VHL tumour-suppressor gene paradigm, Trends Genet 14,

423-6.

Kallio, P. J., Okamoto, K., O'Brien, S., Carrelo, P., Makino, Y., Tanaka, H., and Poellinger, L. (1998). Signal

transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by

the hypoxia-inducible factor-lalpha, Embo J 17,6513-86.

44

Kallio, P. J., Pongratz, L, Gradin, K., McGuire, J., and Poellinger, L. (1997). Activation of hypoxia-inducible

factor lalpha: posttranscriptional regulation and conformational change by recruitment of the Arnt

transcription factor, Proc Natl Acad Sci U S A 94, 5661-72.

Kamura, T., Sato, S., Iwai, K., Czyzyk-Krzeska, M., Conaway, R. C., and Conaway, J. V/. (2000). Activation

of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor supplessor complex, Proc

Natl Acad Sci U S 497,10430-5.

Kappel, 4., Ronicke, V., Damert, ,4,., Flamme, L, Risau, W., and Breiel, G. (1999). Identification of vascular

endothelial growth factor (VEGF) receptor-2 (Flk-l) plomoter/enhancer sequences sufficient for angioblast

and endothelial cell-specific transcription in transgenic mice, Blood 93, 4284-92.

Katschinski, D. M., Le, L., Heinrich, D., Vy'agner, K. F., Hofer, T., Schindler, S. G., and Wenger, R. H.

(2002). Heat induction of the unphosphorylated form of hypoxia-inducible factor- I alpha is dependent on heat

shock protein-9O activity, J Biol Chem 277,9262-7.

Kim, S. O., Merchant, K., Nudelman, R., Beyer, V/. F., Jr., Keng, T., DeAngelo, J., Hausladen, 4., and

Stamler, J. S. (2002). OxyR: a molecular code for redox-related signaling, Cell 109,383-96.

Kivirikko, K. I., and Myllyharju, J. (1998). Prolyl 4-hydroxylases and their protein disulfide isomerase

subunit, Matrix BioI I6,351-68.

Kotch, L. E., Iyer, N. V., Laughner, E., and Semenza, G. L. (1999). Defective vascularization of HIF-lalpha-

null embryos is not associated with VEGF deficiency but with mesenchymal cell death, Dev Biol 209,254-

61.

Kung, A. L., Wang, S., Klco, J. M., Kaelin, V/. G., and Livingston, D. M. (2000). Suppression of tumor

growth through disruption of hypoxia-inducible transcription, Nat Med 6, 1335-40.

Laughner, E., Taghavi, P., Chiles, K., Mahon, P. C., and Semenza, G. L. (2001). IIER2 (neu) signaling

increases the rate of hypoxia-inducible factor lalpha (HIF-lalpha) synthesis: novel mechanism for HIF-l-

mediated vascular endothelial growth factor expression, Mol Cell Biol 21,3995-4004.

Lee, S. H., Wolf, P. L., Escudero, R., Deutsch, R., Jamieson, S.'W., and Thistlethwaite, P. A. (2000). Early

expression of angiogenesis factors in acute myocardial ischemia and infarction, N Engl J llil:ed 342,626-33.

45

Lees, M.J., and Whitelaw, M.L. (1999) Multiple roles of ligand in transforming the dioxin receptor to an

active basic helix-loop-helix/PAS transcription factor complex with the nuclear protein Arnt. Mol Cell Biol.

19,581t-5822.

Lin, F. K., Suggs, S., Lin, C. H., Browne, J. K., Smalling, R., Egrie, J. C., Chen, K. K., Fox, G. M., Martin,

F., Stabinsky,Z., and et al. (1985). Cloning and expression of the human erytht'opoietin gene, Proc Natl Acad

SciUS A82,7580-4.

Lindebro, M. C., Poellinger, L., and Whitelaw, M. L. (1995). Protein-protein interaction via PAS domains:

role of the PAS domain in positive and negative regulation of the bHLH/PAS dioxin receptor-Arnt

transcription factor complex, Embo J 14,3528-39.

Lisztwan, J., Imbert, G., Wirbelauer, C., Gstaiger, M., and Krek, W. (1999). The von Hippel-Lindau tumor

suppressor protein is a component of an E3 ubiquitin-protein ligase activity, Genes Dev 13, 1822-33.

Liu, Y., Chlistou, H., Morita, T., Laughner, 8., Semenza, G. L., and Kourembanas, S. (1998). Carbon

monoxide and nitric oxide suppless the hypoxic induction of vascular endothelial growth factor gene via the

5' enhancer, J Biol Chem 273,15251-62.

Mahon, P. C., Hirota, K., and Semenza, G. L. (2001). FIH-l: a novel protein that interacts with HIF-lalpha

and VHL to mediate repression of HIF-l transcriptional activity, Genes Dev 15,2615-86.

Makino, Y., Cao, R., Svensson, K., Bertilsson, G., Asman, M., Tanaka, H., Cao, Y., Berkenstam, 4., and

Poellinger, L. (2001). Inhibitory PAS domain protein is a negative legulator of hypoxia-inducible gene

explession, Natule 4 I 4, 550-4.

Makino, Y., Kanopka, 4., Wilson, Vy'. J., Tanaka, H., and Poellinger, L. (2002).Inhibitory PAS Domain

Protein (IPAS) Is a Hypoxia-inducible Splicing Variant of the Hypoxia-inducible Factor-3alpha Locus, J Biol

Chem 277,32405-32408.

Masson, N., Willam, C., Maxwell, P. H., Pugh, C. V/., and Ratcliffe, P. J. (2001). Independent function of

two destluction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation, Embo J

20,5191-206.

Maxwell, P. H., Pugh, C. V/., and Ratcliffe, P. J. (1993). Inducible opet'ation of the erythropoietin 3' enhancer'

in multiple cell lines: evidence for a widespread oxygen-sensing mechanism, Ploc Natl Acad Sci U S A 90,

2423-1.

46

Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., V/ykoff, C.

C., Pugh, C. Vy'., Maher, E. R., and Ratcliffe, P. J. (1999). The tumour suppressor protein VHL targets

hypoxia-inducible factors for oxygen-dependent proteolysis, Nature 399,211-5.

Min, J. H., Yang, H., Ivan, M., Gertler, F., Kaelin, W. G., Jr., and Pavletich, N. P. (2002). Structure of an

HIF-lalpha -pVI{L complex: hydroxyproline recognition in signaling, Science 296,1886-9.

Minet, E., Arnould, T., Michel, G., Roland, I., Mottet, D., Raes, M., Remacle, J., and Michiels, C. (2000).

ERK activation upon hypoxia: involvement in HIF-l activation, FBBS Lett 468,53-8.

Minet, 8., Michel, G., Mottet, D., Raes, M., and Michiels, C. (2001). Transduction pathways involved in

Hypoxia-Inducible Factor- I phosphorylation and activation, Free Radic Biol Med 3 I , 841-55.

Morel, Y., and Barouki, R. (1999). Repression of gene expression by oxidative stress, BiochemJ 342,481-

96.

Mustonen, T., and Alitalo, K. (1995). Endothelial receptor tyrosine kinases involved in angiogenesis, J Cell

Biol 129,895-8.

Nambu, J. R., Chen, W., Hu, S., and Crews, S. T. (1995). The Drosophila melanogaster similar bHLH-PAS

gene encodes a protein related to human hypoxia-inducible factor I alpha and Drosophila single-minded,

Gene 172,249-254.

Nambu, J. R., Lewis, J. O., Wharton, K. 4., Jr., and Crews, S. T. (1991). The Drosophila single-minded gene

encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development, Cell 67, ll51-

67.

Ohh, M., Park, C. W., Ivan, M., Hoffman, M. 4., Kim, T. Y., Huang, L. E., Pavletich, N., Chau, V., and

Kaelin, W. G. (2000). Ubiquitination of hypoxia-inducible factor requires dilect binding to the beta-domain

of the von Hippel-Lindau protein, Nat Cell Biol 2, 423-1.

O'Rourke, J. F., Tian, Y. M., Ratcliffe, P. J., and Pugh, C. W. (1999). Oxygen-regulated and transactivating

domains in endothelial PAS protein 1: comparison with hypoxia-inducible factor-lalpha, J BloI Chem 274,

2060-'71.

Peng, J. and Gygi, S.P. (2001). Ploteomics: the move to mixtures. Journal of Mass Spectrometry 3ó, 1083-

1091.

47

Peng, J., Zhang, L., Drysdale, L., and Fong, G. H. (2000). The transcription factor EPAS-l/hypoxia-inducible

factor 2alpha plays an important role in vascular remodeling, Proc Natl Acad Sci U S A 97,8386-91.

Poellinger', P. (1995). Mechanisms of signal transduction by the basic helix-loop-helix dioxin receptor,

Baeuerle, P.A. ed. Edition, Volume I (Boston: Birkhauser).

Przysiecki, C. T., Staggers, J. E'., Ramjit, H. G., Musson, D. G., Stern, A. M., Bennett, C. D., and Friedman,

P. A. (1987). Occurrence of beta-hydloxylated asparagine residues in non-vitamin K-dependent proteins

containing epidermal growth factor-like domains, Proc Natl Acad Sci U S A 84, 7856-60.

Pugh, C. W., O'Rou'ke, J. F., Nagao, M., Gleadle, J. M., and Ratcliffe,P. J. (1991). Activation of hypoxia-

inducible factor- l; definition of regulatory domains within the alpha subunit, J Biol Chem 272, lL2O5-14.

Pugh, C. W., Tan, C. C., Jones, R. W., and Ratcliffe, P. J. (1991). Functional analysis of an oxygen-regulated

transcriptional enhancer lying 3' to the mouse erythlopoietin gene, Ploc Natl Acad Sci U S A 88, 10553-7.

Ratcliffe, P. J., Pugh, C. W., and Maxwell, P. H. (2000). Targeting tumors thlough the HIF system, Nat Med

6, t3t5-6.

Rechsteiner, M., and Rogers, S. V/. (1996). PEST sequences and regulation by proteolysis, Trends Biochem

Sci 21,26'7-'71.

Richard, D. E., Belra, E., Gothie, E., Roux, D., and Pouyssegur, J. (1999). p42lp44 mitogen-activated protein

kinases phosphorylate hypoxia-inducible factor lalpha (HIF-lalpha) and enhance the transcriptional activity

of HIF- l, J Biol Chem 274,32631-1 .

Richard, D. E., Berra, E., and Pouyssegur', J. (2000). Nonhypoxic pathway mediates the induction of hypoxia-

inducible factor lalpha in vascular smooth muscle cells, J Biol Chem 275,26765-1 l.

Roach, P. L., Clifton, I. J., Hensgens, C. M., Shibata, N., Schofield, C. J., Hajdu, J., and Baldwin, J. E.

(1997). Structute of isopenicillin N synthase complexed with substrate and the mechanism of penicillin

formation, Nature 387, 821 -30.

Rodliguez-Enriquez, S., and Moreno-Sanchez, R. (1998). Intermediary metabolism of fast-growth tumor

cells, Arch Med Res 29, l-12.

Ryan, H. E., Lo, J., and Johnson, R. S. (1998). HIF-I alpha is required for solid tumol formation and

embryonic vasculalization, Embo I I 7, 3OO5 - 1 5.

48

Salceda, S., and Caro, J. (1997). Hypoxia-inducible factor lalpha (HIF-lalpha) protein is rapidly degraded by

the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-

induced changes, J Biol Chem 272,22642-1.

Sang, N., Fang, J., Srìnivas, V., Leshchinsky, I., and Caro, J. (2002). Carboxyl-terminal transactivation

activity of hypoxia-inducible factor I alpha is governed by a von Hippel-Lindau protein-independent,

hydroxylation-regulated association with p300/CBP, Mol Cell Biol 22,2984-92.

Schipani,8., Ryan, H. 8., Didrickson, S., Kobayashi, T., Knight, M., and Johnson, R. S. (2001). Hypoxia in

cartilage: HIF-lalpha is essential for chondrocyte glowth arest and survival, Genes Dev 15,2865-76.

Schofield, C. J., and Zhang, Z. (1999). Structural and mechanistic studies on 2-oxoglutarate-dependent

oxygenases and related enzymes, Curr Opin Struct Biol 9,'722-31.

Schultz, 4., Lavie, L., Hochberg, L, Beyar, R., Stone, T., Skorecki, K., Lavie, P., Roguin, 4., and Levy, A. P.

(1999). Interindividual heterogeneity in the hypoxic regulation of VEGF: significance for the development of

the coronary artery collateral circulation, Circulation 1 00, 541 -52.

Schuster, S. J., Badiavas, E. V., Costa-Giomi, P., Weinmann, R., Erslev, A. J., and Caro, J. (1989)

Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure, Blood 73, 13-6.

Semenza, G. L. ( 1999a). Perspectives on oxygen sensing, Cell 98, 281-4.

Semenza, G. L. (1999b). Regulation of mammalian 02 homeostasis by hypoxia-inducible factor 1, Annu Rev

Cell Dev Biol 15, 551-78.

Semenza, G. L. (2000). HIF- I and human disease: one highly involved factot', Genes Dev 14, 1983-91

Semenza, G. L. (2001). HIF-I, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus, Cell

107, t-3.

Semenza, G. L., Duleza, R. C., Traystman, M. D., Gearhart, J. D., and Antonarakis, S. E. (1990). Human

erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting

regulatory elements, Mol Cell Biol 10, 930-8.

Semenza, G. L., Koury, S. T., Nejfelt, M. K., Gearhart, J. D., and Antonarakis, S. E. (1991). Cell-type-

specific and hypoxia-inducible expression of the human erythlopoietin gene in transgenic mice, Proc Natl

AcadSciUSA88,8125-9.

49

Semenza, G. L., and V/ang, G.L. (1992). A nuclear factor induced by hypoxia via de novo protein synthesis

binds to the human erythropoietin gene enhancer at a site required for transcriptional activation, Mol Cell

Biol12,5M7-54.

Seta, K.4., Spicer, Z.,Yuan, Y., Lu, G., and Millhorn, D.E. (2002) Responding to hypoxia: Lessons from a

model cell line. Science's STKE, http://www.stke.org/cgilcontenVfull/sigtrans;2002/146/rell.

Snyder, G. H., Cennerazzo,M. J., Karalis, A. J., and Field, D. (1981). Electrostatic influence of local cysteine

environments on disulfide exchange kinetics, Biochemistry 20, 6509- 19.

Sodhi, 4., Montaner, S., Patel, Y ., Zohar, M., Bais, C., Mesri, E. 4., and Gutkind, J. S. (2000). The Kaposi's

sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor

expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-

inducible factor lalpha, Cancer Res ó0,4873-80.

Srinivas, Y.,Zhang,L.P.,Zhu, X. H., and Caro, J. (1999). Characterization of an oxygen/redox-dependent

degradation domain of hypoxia-inducible factor alpha (HlF-alpha) proteins, Biochem Biophys Res Commun

260, 551 -61 .

Stamler, J. S., and Hausladen, A. (1998). Oxidative modifications in nitrosative stress, Nat Struct Biol 5,241-

9.

Stenflo, J. (1991). Stlucture-function relationships of epidelmal growth factor modules in vitamin K-

dependent clotting factors, Blood 78, 1631-51.

Storz, G., and Imlay, J. A. (1999). Oxidative stress, Cun Opin Microbiol2,188-94.

Sutter, C. H., Laughner, E., and Semenza, G. L. (2000). Hypoxia-inducible factor lalpha plotein expression

is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations, Pt'oc

Natl Acad Sci U S A 97, 4748-53.

Talks, K. L., Turley, H., Gatter, K. C., Maxwell, P. H., Pugh, C. V/., Ratcliffe, P. J., and Harris, A. L. (2000).

The expression and distribution of the hypoxia-inducible factot's HlF-lalpha and HlF-2alpha in normal

human tissues, cancers, and tumot'-associated macrophages, Am J Pathol I 57, 4ll-21.

Tanimoto, K., Makino, Y., Pereira, T., and Poellinger, L. (2000). Mechanism of regulation of the hypoxia-

inducible factor- I alpha by the von Hippel-Lindau tumor suppressor protein, Embo J I 9, 4298-309.

50

Taylor, B. L., and Zhulin, I. B. (1999). PAS domains: internal sensors of oxygen, redox potential, and light,

Microbiol Mol Biol Rev ó3, 419-506.

Tell, G., Scaloni, A.,Pellizzari, L., Formisano, S., Pucillo, C., and Damante, G. (1998). Redox potential

controls the structure and DNA binding activity of the paired domain, J Biol Chem 273,25062-12.

Thornton, R. D., Lane, P., Borghaei, R. C., Pease, E. 4., Caro, J., and Mochan, E. (2000). Interleukin I

induces hypoxia-inducible factor I in human gingival and synovial fibroblasts, Biochem J 350,307-12.

Tian, H., Hammer, R. E., Matsumoto, A. M., Russell, D. V/., and McKnight, S. L. (1998). The hypoxia-

responsive transcription factor EPASI is essential for catecholamine homeostasis and protection against heart

failure during embryonic development, Genes Dev 12,3320-4.

Tian, H., McKnight, S. L., and Russell, D. V/. (1991). Endothelial PAS domain protein I (EPASI), a

transcription factor selectively expressed in endothelial cells, Genes Dev 11,12-82.

Torchia, J., Rose, D. V/., Inostroza, J., Kamei, Y., rWestin, S., Glass, C. K., and Rosenfeld, M. G. (1997). The

transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function, Nature 387,611-84.

Treins, C., Giolgetti-Peraldi, S., Mut'daca, J., Semenza, G. L., and Van Obberghen, E. (2002). Insulin

Stimulates Hypoxia-inducible Factor I thlough a Phosphatidylinositol 3-KinaseÆarget of Rapamycin-

dependent Signaling Pathway, J Biol Chem 277,27915-81.

Wang, G. L., Jiang, B. H., Rue, E. 4., and Semenza, G. L. (1995a). Hypoxia-inducible factor I is a basic-

helix-loop-helix-PAS heterodimer regulated by cellular 02 tension, Proc Natl Acad Sci U S A 92, 5510-4.

'Wang, G. L., Jiang, B. H., and Semenza, G. L. (1995b). Effect of protein kinase and phosphatase inhibitors

on expression of hypoxia-inducible factor l, Biochem Biophys Res Commun 216,669-75.

Wang, G. L., and Semenza, G. L. (1993a). Characterization of hypoxia-inducible factor I and regulation of

DNA binding activity by hypoxia, J Biol Chem 268,21513-8.

Wang, G. L., and Semenza, G. L. (1993b). Desferlioxamine induces erytlu'opoietin gene expression and

hypoxia-inducible factor I DNA-binding activity: implications for models of hypoxia signal transduction,

Blood 82, 3610-5.

Wang, G. L., and Semenza, G. L. (1993c). General involvement of hypoxia-inducible factor I in

transcriptional response to hypoxia, Proc Natl Acad Sci U S A 90, 4304-8.

51

Webster, K. 4., and Murphy, B. J. (1988). Regulation of tissue-specific glycolytic isozyme genes: coordinate

response to oxygen availability in myogenic cells, Can J Zool 66, 1046-1058.

Wenger, R. H., Rolfs, 4., Spielmann,P., Zimmermann, D. R., and Gassmann, M. (1998). Mouse hypoxia-

inducible factor-lalpha is encoded by two different mRNA isoforms: expression from a tissue-specific and a

housekeeping-type promoter, Blood 9 1, 34'7 I -80.

Wiesener, M. S., Tulley, H., Allen, V/. E., Willam, C., Eckardt, K. U., Talks, K. L., Wood, S. M., Gatter, K.

C., Haris, A. L., Pugh, C. V/., et al. (1998). Induction of endothelial PAS domain protein-l by hypoxia:

characterization and comparison with hypoxia-inducible factor-lalpha, Blood 92,2260-8.

Wilk, R., rùy'eizman, I., and Shilo, B. Z. (1996). trachealess encodes a bHLH-PAS protein that is an inducer of

tracheal cell fates in Drosophila, Genes Dev 10,93-102.

Xanthoudakis, S., and Curran,'f .0992). Identification and characterizalion of Ref-1, a nuclear protein that

facilirates AP-l DNA-binding activity, EMBO J 11,653-665.

Yu, F., V/hite, S. B., Zhao, Q., and Lee, F. S. (2001a). Dynamic, site-specific interaction of hypoxia-

inducible factor- l alpha with the von Hippel-Lindau tumor suppressor protein, Cancer Res ó1, 4136-42.

Yu, F., V/hite, S. 8., Zhao, Q., and Lee, F. S. (2001b). HIF-lalpha binding to VHL is regulated by stimulus-

sensitive proline hydloxylation, Proc Natl Acad Sci U S A 98,9630-5.

Zagzag,D.,Zhong, H., Scalzitti, J. M., Laughner, E., Simons, J. V/., and Semenza, G. L. (2000). Expression

of hypoxia-inducible factor lalpha in brain tumors: association with angiogenesis, invasion, and progression,

Cancer 88,2606-18.

Zelzer, E., Levy, Y., Kahana, C., Shilo, B. 2., Rubinstein, M., and Cohen, B. (1998). Insulin induces

transcription of target genes through the hypoxia-inducible factor HIF-lalpha/ARNT, Embo J 17,5085-94.

Zheng, M., Aslund, F., and Storz, G. (1998). Activation of the OxyR transcription factor by reversible

disulfide bond formation, Science 279, 11 18-21.

Zhong, H., De Maruo, A. M., Laughner, E., Lim, M., Hilton, D. A.,Zagzag, D., Buechler, P.,Isaacs, W. 8.,

Semenza, G. L., and Simons, J. V/. (1999). Overexpression of hypoxia-inducible factor lalpha in common

human cancers and their metastases, Cancer Res 59, 5830-5.

Zhu,H, and Bunn, H. F. (1999). Oxygen sensing and signaling: impact on the regulation of physiologically

important genes, Respir Physiol I 15,239-47 .

52

AppBNnrx I

PNPE,R I

Lando, D., Pongratz, I., Poellinger, L., and Whitelaw, M.L., (2000) A redox

mechanism controls differential DNA binding activities of hypoxia-inducible factor

(HIF) 1a and the HIF-like factor.

Journal of Biological Chemistry, v. 275 (7), pp. 4618-4627.

NOTE:

This publication is included in the print copy of the thesis held

in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1074/jbc.275.7.4618

PTPER II

Lando, D., Peet, D.J., Pongratz, I., and Whitelaw, M.L., (2002) Mammalian two-

hybrid assay showing redox control of HIF-like factor.

Methods in Enzymology, v. 353, pp. 3-10

NOTE:

This publication is included in the print copy of the thesis held

in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1016/S0076-6879(02)53031-7

PTPER III

Lando, D., Peet, D.J., Whelan, D.A., Gorman, J.J., and Whitelaw, M.L., (2002)

Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch.

Science, v. 295 (5556), pp. 858-861.

NOTE:

This publication is included in the print copy of the thesis held

in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1126/science.1068592

PTPE,RIV

Lando, D., Peet, D.J., Gorman, J.J., Whelan, D.A., Whitelaw, M.L., and Bruick, R.K.,

(2002) FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional

activity of hypoxia-inducible factor.

Genes and Development, v. 16 (12), pp. 1466-1471.

NOTE:

This publication is included in the print copy of the thesis held

in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1101/gad.991402

AppnNurx II

Supplementary Data for Paper III

Figure. 1. The wild-type and GaI4DBD/HIF chimeras used in Fig. 3B are expressed at

similar levels during transient transfection. 'Whole-cell extracts of control or transfected cells

were separated by SDS-PAGE, and proteins were detected by immunoblotting with

polyclonal antibodies directed against the COOH-terminus of HIF-1c (A)or HIF-2 "

(B).

BA

Ecoo

GaIDBD/HlF-1cr727-826

wt N8034

ecoo

GaIDBD/HlF-2cr77^-874

wt N8514

32.5 -32.5 -

Figure. 2. Expression levels of wild-type and amino acid-substituted HlF-løproteins

during the transient transfection process of Fig. 4. Whole-cell extracts from the indicated

untreated or DP-treated transfected cells were separated by SDS-PAGE, and HIF-

la proteins were detected with a polyclonal antibody directed against the HIF-lcx, COOH-

terminus. A similar pattem was obtained for expression levels of the respective wild-type

and amino acid-substituted HIF-2a proteins (data not shown).

efbos wt P5644 N8O3A P564AN8O3A

-++ + + +

1 2 3 4 5 6 7 8 I 10

100¡rM DP

Table 1. Theoretical and observed masses of the indicated ions derived from tandem MS

sequencing of the hydroxylated (2106 column) and nonhydroxylated (2090 column) forms of

the peptide YDCEVNVPVPGS STLLQGR.

TABLE 1 Masses of fragment ions derived from MS/MS sequencing of 2090 and 2106 peptides

Fragment mlz

Theory

2090

Observed

2090 2106

Difference

2090 2106

yly2y3y4y5y6y7y8y9

vl0vllv12v13v14vl5v16vl7vl8v19

t7s.tt9232.140360.199473.283586.367687.415774.447861.479918.5001015.5531114.6221211.6741310.7431424.7861523.8541652.8971812.9271927.9s42091.018

164.071279.098439.128568.171667.239781.282880.351977.4031076.4721173.5251230.546t3t7.5781404.6101505.6581618.742t73t.826I 859.8841916.9062073.007

t75.ll8232.142360.1 99473.279586.272687.410774.447861.473918.4881015.5511114.6321211.6781310.7301424.7691523.8341652.919

279.094439.129568.176667.233781.283880.349977.4061076.467

l75.ll8232.140360.199473.278586.363687.402774.440

1015.5501114.6221211.6721310.734t440.7711539.8361668,87

279.tos439.129568.1 73667.237797.283896.344993.4061092.4581189.4971246.5261333.611

0.0010.00200.0040.0050.00500.0060.0020.0020.010.0040.0130.0170.020o.ol2

0.0010

0

0.0050.0040.0130.007

0.00300.0020.009r6.00616.00215.973

0.0070.001

0.0020.00216.00115.99316.00315.98615.97215.98

16.033

blb2b3b4b5b6b7b8b9bl0bllbl2b13bl4b15bl6b17bl8bl9

0.0040.0010.0050.0060.0010.0020.0030.006

1618.691731.8321 859.89

1634.751747.7161875.824

0.0520.0060.006

16.008

15.89

ts.94

AppBNDIX III

Comments on Methodology

The methods and materials used in this thesis are described in each paper. Here, some key

techniques are described in more detail.

Purification of HIF-2c¿ CAI)

In Paper III we decided to employ mass spectrometry (MS) to investigate the possibility that

an oxygen dependent post-translational modification may target the CAD region. To achieve

this we engineered a stable cell line, derived from human embryonic kidney HEK293T cells

to express the carboxy-terminal 100 amino acid residues of HIF-2cr (HIF-2u774-874). An

expression vector previously described was employed (Hobbs et al., 1998). This expressed

protein also contained an N terminal 6x histidine (His) and myc epitope (Myc) tag to

facilitate purihcation and detection by western blot, respectively. To screen for post-

translational modifications by MS we envisaged needed approximately 100ng (10 pmoles) of

relatively pure HIF-2a774-874 protein. To obtain enough HIF-2I774-874 protein sample for

analysis after a range of cell treatments (ie. 20% oxygen, l00pM DP, <lo/o oxygen) we

started with 20x 17 5 cm2 T flasks Q.{algene) of HIF-2cr7 7 4-87 4 expressing stable cell line at

50%o cell confluency. After treatment flasks were washed once with l0 mls of ice chilled

phosphate buffered saline solution. Cells were then immediately lysed with 20 mls of

binding buffer (100mM Na-phosphate (pH8.0), 8M urea, 0.1% NP40, 0.15M NaClz, 5 mM

imidazole, protease (Boehringer) and phosphatase (Sigma) inhibitor cocktail) per flask by

gently rocking flasks for 20 minutes at 25"C. To minimise the effects of reoxygenation low

(<l%) oxygen treated samples were washed and cells lysed in an hypoxic workstation MkIII

(Don Whitely Scientific). Pooled cell lysates were then clarified by filtering lysate through a

0.2 micron filter (Nalgene) to remove genomic DNA and lipophilic protein. Clarified protein

lysate ("50 mg) was then applied to a 0.5 ml Ni-agarose column (Scientifix) with a 0.5 ml

per minute flow rate. The column was then washed with 500 mls of wash buffer (100mM

Na-phosphate þH8.0), 8M urea, 0.5M NaCl2, 20 mM imidazole). Bound proteins were then

eluted with 4x lml of elution buffer (100mM Na-phosphate (pH8.0), 8M urea, 200 mM

imidazole). Proteins eluted from the Ni-agarose column were then loaded onto a butyl C4

high-performance liquid chromatography column (Brownlee PerkinElmer) equilibrated in

0.1% trifluoroacetic acid (TFA). Proteins were then separated with an increasing gradient of

acetonitrile (0-60%180 minutes, flow rate lml/min) in 0.Io/o TFA. The HIF-2I774-874

protein eluted at 40%o acetonitrile in a hnal volume of I-2 mls. Using this strategy we

purified on average 100-200 ng of HIF-2a774-874 protein in acetonitrileiTFA solution. To

screen for post-translational modifications the collected HIF-2a174-874 protein sample was

then digested with trypsin and the peptide fragments subjected to MS analysis as outline in

Paper III.

,,

Amendments

The following is a list of amendments that should be referred to when reading this thesis

ENtitICd ..CHARACTERISING MECHANISMS OF REGULATION OF HYPOXIA-

INDUCIBLE FACTORS" by David Lando'

Figurel, page4 word Tryrosine amend to TYrosine

Glucose Transporter -1,-2 amendto Glucose Transporter -1' -3

Page10, line4 word their amend to there

Pagel5, para2, linel I word inhibitor amend to analogue

Pagel7,hrc2l word intermidiary amend to intermediary

Page27,hne20 word prevents amend to Prevent

Figure6, page31,

legend, lineT

Page36, linel

Reference citation

von Hippel Lindaul amend to von Hippel-Lindau

reference Huang etal.,1999 amend to Wu et al',7999

on page 52 after Wilk et al., reference add additional reference'

'Wu, H., Moon, H.S', Begley, T.P', Myllyharju, J', and Kivirikko'

K.I. (1999). Mechanism -based inactivation of the human prolyl-

4-hydroxylase by 5-oxaproline-containing peptides: evidence for

a prolyl radical intermediate, J' Am. Chem' Soc' 121, 587-588'