Biochem. J. (2008) 414, 19–29 (Printed in Great Britain)
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doi:10.1042/BJ20081055
REVIEW ARTICLE
Regulation of gene expression by hypoxia
Niall Steven KENNETH and Sonia ROCHA1
College of Life Sciences, Wellcome Trust Centre for Gene Regulation and Expression, MSI/WTB/JBC Complex, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Hypoxia induces profound changes in the cellular gene expression
profile. The discovery of a major transcription factor family
activated by hypoxia, HIF (hypoxia-inducible factor), and the
factors that contribute to HIF regulation have greatly enhanced
our knowledge of the molecular aspects of the hypoxic response.
However, in addition to HIF, other transcription factors and
cellular pathways are activated by exposure to reduced oxygen.
In the present review, we summarize the current knowledge of
how additional hypoxia-responsive transcription factors integrate
with HIF and how other cellular pathways such as chromatin
remodelling, translation regulation and microRNA induction,
contribute to the co-ordinated cellular response observed following hypoxic stress.
INTRODUCTION
HIF-1β, also known as ARNT (aryl hydrocarbon receptor
nuclear translocator), also possesses several splice variants and is
constitutively expressed [2,4]. HIF-α subunits share highly similar
domain regions, characterized by the presence of bHLH (basic
helix–loop–helix)–PAS (Per/ARNT/Sim) domains (Figure 1). In
addition, they possess an ODD (oxygen-dependent degradation
domain), rendering these proteins labile in the presence of oxygen
(see below). Whereas HIF-1α and HIF-2α contain two transactivation domains, HIF-3α lacks the C-terminal transactivation
domain, and, as such, is thought to act as an inhibitor of HIF-1α
and HIF-2α [2]. HIF-1 and HIF-2 have non-redundant functions
in the cell, and, although HIF-1α is the best understood isoform,
recent studies have unveiled important functions for HIF-2α [5,6].
A number of recent reviews have focused on HIF-mediated targets
and function [2,3,7] and therefore we will not focus on this matter.
Instead, we discuss how HIF integrates with other transcription
factors and how HIF is, or could be, modulated by other hypoxiainduced cellular processes which contribute to the gene expression
pattern.
Hypoxia, or decreases in the oxygen concentration, activates a
variety of complex pathways at both the cellular and organism
level, with the ultimate aim of reinstating oxygen homoeostasis.
Although physiological responses to hypoxia have been appreciated for a long time, the molecular processes activated within
the cells are still under investigation. However, this area has
been greatly advanced by the discovery of a major transcription
factor that responds to hypoxia, HIF (hypoxia-inducible factor).
Importantly, the finding of hypoxia-responsive HIF regulators
has uncovered the complex mechanism of control over this
transcription factor family. A novel class of dioxygenases called
PHDs (prolyl hydroxylases) were identified as molecular oxygen
sensors within the cell. How these enzymes are regulated within
the cell and what other possible targets they possess is now under
intense investigation. Apart from the HIF system, other transcription factors are hypoxia-responsive. In addition, other
molecular processes are activated following hypoxia that will
ultimately determine the transcription profile of a given cell. In
the present review, we describe several of the pathways that are
activated following hypoxic stress that influence the pattern of
gene expression achieved.
THE HIF SYSTEM
Molecular research into hypoxia-induced cellular responses was
greatly enhanced by the seminal discovery of HIF-1 by the
Semenza laboratory in the early 1990s [1]. Since this discovery, a
number of laboratories have contributed to the delineation of the
HIF system as we presently know it. The HIF family of transcription factors are composed of a heterodimer of α and β
subunits [2,3]. There are three known isoforms of HIF-α: HIF1α, HIF-2α and HIF-3α. HIF-3α has several splice variants.
Key words: chromatin, hypoxia, hypoxia-inducible factor (HIF),
microRNA (miRNA), transcription, translation.
PHD AND FIH-1 (factor inhibiting HIF-1) REGULATION OF HIF
The discovery of HIF led to an intensification of the research
on the molecular mechanisms behind hypoxia cellular sensing.
The identification of the players involved in HIF regulation
was the subject of extreme interest. The identification of a novel
class of dioxygenases called PHDs made these proteins prime
candidates for the task of cellular oxygen sensors. PHDs catalyse
the hydroxylation reactions in the ODD of HIF [8,9]. They require
molecular oxygen, iron and 2-oxoglutarate as co-factors in this
reaction [9,10]. The hydroxylation of HIF acts as a signal for
recognition by the tumour suppressor VHL (von Hippel–Lindau
protein). VHL acts as an E3 ligase, promoting Lys48 -linked
ubiquitination and proteasomal-mediated degradation (Figure 2).
Abbreviations used: AMPK, AMP-activated protein kinase; AP-1, activator protein 1; ARNT, aryl hydrocarbon receptor nuclear translocator; ATF,
activating transcription factor; ATM, ataxia telangiectasia mutated; bHLH, basic helix–loop–helix; ChIP, chromatin immunoprecipitation; eIF, eukaryotic
initiation factor; 4E-BP, eIF4E-binding protein; EPO, erythropoietin; FIH-1, factor inhibiting hypoxia-inducible factor-1; HAT, histone acetyltransferase;
HDAC, histone deacetylase; HIF, hypoxia-inducible factor; IL, interleukin; IRES, internal ribosome entry site; miRNA, microRNA; mTOR, mammalian target
of rapamycin; NF-κB, nuclear factor κB; ODD, oxygen-dependent degradation domain; PHD, prolyl hydroxylase; PKR, interferon-inducible double-stranded
RNA-activated kinase; PERK, PKR-like endoplasmic reticulum kinase; TNFα, tumour necrosis factor α; UPR, unfolded protein response; VEGF, vascular
endothelial growth factor; VHL, von Hippel–Lindau protein.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
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Figure 1
N. S. Kenneth and S. Rocha
Domain structure of HIF proteins
Schematic diagram showing structural domains of HIF family members HIF-1α, HIF-2α, HIF-3α and HIF-1β. The modifications of specific residues are highlighted above each protein, and the
proteins that perform those modifications are shown. Coloured bars below each protein delineate particular interaction regions within HIF proteins. CTAD, C-terminal transactivation domain; LZIP,
leucine zipper; NLS, nuclear localization signal; NTAD, N-terminal transactivation domain; PAS, Per/ARNT/Sim domain; PAC, PAS-associated C-terminal domain.
Figure 2
Regulation of HIF-1α in normoxia and hypoxia
Schematic diagram of the key molecules involved in HIF function. Molecules are not drawn to scale. Under normoxia, PHDs and FIH enzymes use O2 to hydroxylate key residues within the HIF-1α
subunit. Hydroxylation of the ODD signals for VHL binding and ubiquitination occurs, leading to proteasomal degradation. In hypoxia, PHDs and FIH are inhibited, and HIF-1α is stabilized and able
to dimerize with HIF-1β and activate target gene transcription through recruitment of co-activators. CTAD, C-terminal transactivation domain; PAS, Per/ARNT/Sim domain; PAC, PAS-associated
C-terminal domain; ubq, ubiquitination. An animated version of this Figure can be seen at http://www.BiochemJ.org/bj/414/0019/bj4140019add.htm.
There are four PHD isoforms identified so far: PHD1–
PHD4. Only isoforms 1, 2 and 3 have been shown to hydroxylate
HIF [8]. Genetic and biochemical studies have demonstrated
differences between these enzymes [11–14]. Biochemical studies
have shown that PHD2 has higher affinity for HIF-1α, whereas
c The Authors Journal compilation c 2008 Biochemical Society
PHD1 and PHD3 have higher affinity for HIF-2α [11]. In
addition, the importance of PHDs in development was also
elucidated recently [12–14]. Although homozygous deletion
of PHD2 results in embryonic lethality between E (embryonic
day) 12.5 and E14.5, PHD1- or PHD3-knockout mice are viable
Regulation of gene expression by hypoxia
Figure 3
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Cross-talk among hypoxia-responsive transcription factors
Although HIF is at the centre of the hypoxic response, other transcription factors are also hypoxia-responsive. Co-operation and antagonism between these transcription factors will result ultimately
in a co-ordinate cellular response. The duration and intensity of hypoxia exposure determines who wins in the transcription battle. Although AP-1 and NF-κB mostly co-operate with HIF, p53 seems
to antagonize it, whereas c-Myc has HIF-α subunit preference. CTAD, C-terminal transactivation domain; PAS, Per/ARNT/Sim domain; PAC, PAS-associated C-terminal domain.
[12]. However, despite not causing embryonic lethality, a double
deletion of PHD1 and PHD3 induced moderate erythrocytosis
[13]. In a more recent study, PHD3 deletion was shown to result
in abnormal sympathoadrenal development [14]. This study
also confirmed the previous suggested increased affinity for
HIF-2α [14]. These genetic models will be extremely helpful in
determining the importance of the individual PHDs in terms of
acute compared with prolonged hypoxia.
In addition, to the prolyl hydroxylation catalysed by the PHDs,
another oxygen-dependent modification occurs in the most Cterminal transactivation domain of HIF-α [15–17]. This is an
asparaginyl hydroxylation, catalysed by another dioxygenase
called FIH-1. Asparaginyl hydroxylation prevents binding of
HIF to its co-activators p300 or CBP [CREB (cAMP-responseelement-binding protein)-binding protein], and hence prevents
full target gene activation (Figure 2). However, several studies
have shown that, in the absence of FIH-1 [18] or absence of p300
binding [19], a number of target genes are still HIF-inducible.
These studies have raised the possibility that either the N-terminal
transactivation domain is sufficient for induction of certain target
genes or, alternatively, binding of unknown co-activators can
occur even in the presence of FIH-1-mediated asparaginylmodified HIF. Further exploration of these possibilities over the
next few years should provide valuable insights into this aspect
of HIF regulation. As with the PHDs, a genetic knockout model
would reveal the importance of this HIF regulator in development
and hypoxia responses.
Since the discovery of this class of dioxygenases, a large
number of research laboratories have been investigating alternative and novel targets to HIF. Interestingly, FIH-1 has been
shown to be able to modify a number of other targets, all of which
contain ankyrin repeats, such as IκB [inhibitor of NF-κB (nuclear
factor κB)]-like molecules [20] and Notch [21]. Again, future
studies will determine the relative contribution of these novel
targets to the gene expression profile following hypoxic stress.
HYPOXIA-RESPONSIVE TRANSCRIPTION FACTORS
Apart from the HIF family, hypoxia activates a number of other
important transcription factors, such as NF-κB, AP-1 (activator
protein 1), p53 and c-Myc, among others (Figure 3). Given the
importance of HIF for the cellular response to hypoxia, cross-talk
between these different transcription factors should exist, and, in
fact, it has been documented for several of them. In this section,
we will review the recent findings in this area.
NF-κB family
NF-κB is the collective name for a family of seven proteins,
encoded by five genes: RelA, RelB, c-Rel, NF-κB1 (p105/p50)
and NF-κB2 (p100/p52) (reviewed in [22]). These proteins all
share a conserved region of homology at their N-terminus called
the Rel homology domain. The C-terminus of these proteins is
poorly conserved between family members, with three of the proteins possessing transactivation domains (RelA, RelB and c-Rel).
The remaining two (NF-κB1 and NF-κB2) require proteolytic
cleavage to achieve their active forms (p50 and p52). However,
since p50 and p52 do not possess transactivation domains, they
generally need to heterodimerize to activate transcription. NFκB is best known for its role in the immune system and in
inflammatory responses. However, recent studies have demonstrated its prominent role in other disorders such as cancer [23].
Hypoxia-mediated induction of NF-κB was reported over
a decade ago [24]. Despite this, the mechanism of hypoxiainduced NF-κB has not been clearly defined, although a number
of different laboratories have demonstrated that NF-κB can
contribute to the cellular response to hypoxia [25–28]. Although
in some tissues and cell systems, hypoxia-induced NF-κB acts
as a survival signal [25], in neuronal systems, NF-κB acts as a
pro-death factor [26,27,29]. This dual nature of NF-κB is now
well established [23]; however, what triggers the different and
c The Authors Journal compilation c 2008 Biochemical Society
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N. S. Kenneth and S. Rocha
often opposing responses in NF-κB function is a matter of intense
research in the field. Most studies have concentrated on the RelA–
p50 heterodimer of NF-κB [25,28,30]. However, to date, no study
has investigated the function of NF-κB2 (p100/p52) in hypoxia.
This research could have important implications, as NF-κB2 has
significant links to cancer and cell-cycle progression [31–33]. The
function of RelB in hypoxia has also not been investigated so far.
Importantly, RelB activation was recently shown in invasive breast
tumours [34], suggesting that this NF-κB subunit also plays a role
in cancer. Only a few studies have documented the involvement
of c-Rel in the hypoxia response. These have shown that the
c-Rel–p50 heterodimer regulates the expression of a known target
and important anti-apoptotic gene, Bcl-X [35]. A more in-depth
analysis of NF-κB complexes would reveal which of the subunits
are important for HIF co-operation.
Interestingly, we, and others, have shown that NF-κB can
directly modulate HIF-1 transcriptionally [36–38]. This occurs
not only in response to stimuli such as TNFα (tumour necrosis
factor α) [36,38] and H2 O2 [37], but also in response to hypoxia
[38]. Whether HIF-1 or HIF-2 can regulate directly any of the
NF-κB subunits has not been investigated so far. It is possible
that HIF can induce NF-κB via the activation of HIF targets
such as TNFα and IL (interleukin)-1β [39,40], two potent NFκB inducers. Studies over the next few years should determine
whether a reciprocal and more direct link exists.
AP-1
AP-1 refers to the combination of dimers formed between the
Jun, Fos and ATF (activating transcription factor) families of transcription factors. As such, its regulation and biological functions
are highly complex. AP-1 activation has been associated with
a great number of biological processes, such as proliferation,
apoptosis and even tumorigenesis. Genetic knockout and transgenic experiments have determined the importance of some AP-1
members for development [41]. Although c-Jun, JunB and Fra-1
are indispensable for embryonic development, c-Fos, FosB and
JunD are dispensable, despite harbouring other defects (for a
review, see [41]).
It is well accepted that hypoxia induces AP-1 activity (reviewed
in [42,43]). The mechanisms of AP-1 activation, however, are
generally cell-type-specific and even dependent on the experimental conditions used. Transcriptional activation for c-Jun [44]
and JunB [30] has been demonstrated in certain cell types, whereas
increased post-translational modifications owing to activation of
upstream signalling cascades has been shown to occur for c-Jun
[45].
In general terms, AP-1 exerts its effects by acting in cooperation with additional transcription factors to modulate their
activity. In fact, co-operation between AP-1 and HIF-1 has been
reported [46,47] and is thought to occur in hypoxia. Although
physical association between these transcription factors has
not been formally demonstrated, functional co-operation has
been widely documented [46,47]. In addition, AP-1 and NFκB co-operation is observed in the activation of common target
genes, such as IL-8 [40]. The impact of AP-1 on the gene
expression profile following hypoxia has not been established.
Given the number of AP-1 family members, this question is
almost impossible to answer. However, depletion of certain AP-1
components does not seem to affect the cellular response to
hypoxia [42].
p53
One additional and important transcription factor activated by
hypoxia is the tumour suppressor p53. Although the activation
c The Authors Journal compilation c 2008 Biochemical Society
of p53 following hypoxia is to be expected, given its sensitivity
to most types of stress within the cell (reviewed in [48]), p53
activation following hypoxia is very atypical. Hypoxia-induced
p53 has been suggested to be dependent on the DNA-checkpoint
kinases ATM (ataxia telangiectasia mutated)/ATR (ATM- and
Rad3-related). However, hypoxia does not induce detectable DNA
damage, the stimulus for these sensor kinases. Hypoxia mostly
induces a G1 arrest in the cell cycle, through HIF-1-dependent and
-independent mechanisms [49]. In addition, hypoxia-induced p53
has been shown to be both dependent and independent of HIF-1,
in several different studies (reviewed in [50]).
Surprisingly, experiments investigating the transcriptional
activity of p53 found that, under hypoxic conditions, p53 did not
induce the same set of genes as a typical p53-activating stimulus
such as UV light [50,51]. Most studies have found that p53
activated by low oxygen levels acts to repress certain genes,
such as α-tubulin, while failing to induce others, such as p21
[51]. However, it has been demonstrated that p53 does activate
a subset of genes different from those normally seen with DNAdamaging agents [52–54]. For example, BNIP3L (Bcl2/adenovirus E1B 19 kDa interacting protein 3-like) was identified as
a key p53 target gene activated under hypoxia that is responsible for p53-mediated apoptosis following hypoxic stress [52].
Furthermore, the Harris laboratory has shown that hypoxiainduced p53 activates a very similar subset of genes to those
seen following nitric oxide exposure [53]. Interestingly, ChIP
(chromatin immunoprecipitation) experiments have shown that
p53 is recruited to target gene promoters, despite no increase in
mRNA production being detected [55,56]. Therefore alternative
mechanisms for p53-mediated responses in hypoxia, which
do not necessitate an increase in mRNA production, warrant
consideration. These mechanisms would include regulation of
translation rates, mRNA maturation processes or even miRNA
(microRNA) induction (see below).
Although the transcriptional targets are currently not well
defined, it is clear that p53 is important for hypoxia-induced
apoptosis. However, further and more detailed analysis will be
required to identify the mechanisms by which p53 mediates
hypoxia-induced apoptosis.
The Myc family
Genes of the Myc family of transcription factors are involved
in a wide variety of cellular processes such as cell growth,
cell proliferation, inhibition of cell differentiation, promotion of
angiogenesis and genomic instability (reviewed in [57,58]), and
as such are subject to very tight control [58].
In mammalian cells, there are four members of the Myc
family: c-Myc, N-Myc, L-Myc and S-Myc [57]. No monomers or
homodimers of Myc proteins are found in vivo; instead, Myc
proteins heterodimerize with a partner protein, Max, through
contacts between their bHLHZ (bHLH zipper) domains [59,60].
Myc–Max heterodimers bind to specific DNA sequences, termed
E-boxes, within the promoters of target genes to activate or repress
transcription [61]. Myc can also bind to non-consensus sequences
through protein–protein interactions to alter expression of target
genes [62]. Max itself is also under tight regulation through
protein–protein interactions with Mad1, Mxi1 (Mad2), Mad3
or Mad4, which compete for binding to Max, and function to
antagonize Myc activity ([63–65], but see [65a]).
Mammalian cells respond to hypoxic stress by reducing cell
growth and by inducing gene products, which causes cell-cycle
arrest. Both of these activities are usually promoted by high Myc
activity [66–68]. A body of work has suggested that Myc function
Regulation of gene expression by hypoxia
is compromised under low oxygen conditions and therefore
contributes to the hypoxic response. This antagonism of Myc can
occur through a variety of mechanisms. Under hypoxic conditions,
cell proliferation ceases through the induction of cyclin-dependent
kinase inhibitors normally repressed in proliferating cells under
normoxic conditions [66,68]. Myc and HIF complexes can
compete for binding sites at the promoters of target genes to
alter their expression profile. This has been demonstrated in the
regulation of the cyclin-dependent kinase inhibitor p21. Under
normoxic conditions, p21 is repressed by c-Myc binding to its
promoter, but when cells are exposed to low oxygen, c-Myc is
replaced by HIF-1, resulting in induction of p21 expression and
hence cell-cycle arrest [69]. Indeed, HIF-1 overexpression, even
in normoxia, is sufficient to induce p21 expression [69]. Interestingly, HIF-1α transcriptional activity or its DNA binding are not
essential for the cell-cycle arrest, indicating a novel function for
HIF-1α [69].
HIF-1α can block Myc binding to its DNA-binding partners directly. HIF can bind to Myc itself, but also to its binding partners
Max and Sp1, in hypoxic cells [68–71]. This acts to disrupt
Myc–Max complex formation and DNA binding and hence reduce
expression of Myc target genes. HIF can also disrupt Myc function
indirectly by mediating the induction of the Myc antagonist, Mxi
[72]. This results in a decrease in the levels of several Myc target
genes and inhibition of transformation [73].
In contrast with HIF-1, elevated HIF-2 can promote c-Myc
activity in a variety of cell lines [71]. It can achieve this by
binding to and stabilizing the Myc–Max heterodimer, allowing
it to transactivate its target genes. HIF-2α was shown to act in
concert with Myc to promote transformation of mouse embryonic
fibroblasts [71]. As HIF-1 is expressed ubiquitously and HIF-2 is
only expressed in a subset of cell types, the different effects of
these factors on Myc activity in hypoxia may reflect the different
requirements of different tissues.
Although there are many genes and processes in which Myc
and HIF have opposite effects, both are elevated in tumour cell
types and promote processes such as angiogenesis. Furthermore,
it has been shown that HIF-1α is essential for c-Myc-mediated
tumorigenesis [74]. Detailed analysis of cells expressing elevated
Myc showed that HIF and c-Myc can co-operate to induce shared
target genes VEGF (vascular endothelial growth factor), HK2
(hexokinase 2) and PDK1 (pyruvate dehydrogenase kinase 1)
[75]. These observations reveal a complex interplay between the
Myc and HIF transcription factors, which will ultimately be celltype- and cell-context-dependent.
The co-operative or antagonistic nature of the connection
between HIF and the additional transcription factors is bound
to determine the gene expression profile of each cell (Figure 3).
Whether a cell progresses or stops its cell cycle, survives or dies
following hypoxia exposure is tightly linked to the cross-talk
between HIF and transcription factors such as p53, NF-κB and
c-Myc, among others.
CHROMATIN CHANGES
We have discussed how hypoxia activates HIF, as well as other
transcription factors, which act in concert to alter the gene
expression profile of the cell. Although the identities of some of
these DNA-binding factors are known, it is still unclear by which
precise mechanisms they activate or repress transcription of their
target genes. For transcription factors to activate gene expression,
they must be recruited to the promoters or enhancers of their target
genes through direct binding to specific DNA sequences. As a
mammalian cell packages approx. 2 m of DNA into a compact
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chromatin structure that is contained in a nucleus of less than
10 μm in diameter, these sequences may not always be accessible.
Therefore different mechanisms exist to allow the transcription
machinery to access these areas.
The fundamental unit of the chromatin is the nucleosome,
which consists of 146 bp of DNA wrapped twice around an
octomer formed from the globular domains of two molecules
of each of histones H2A, H2B, H3 and H4 [76]. The globular
domains of the histones form the core of the nucleosome, and the
basic flexible tail regions of the histones protrude from the octomer. These tail regions are thought to be associated with the
negatively charged DNA wrapped around the outside [77]. Each
nucleosome is connected with 10–80 bp of linker DNA associated
with a linker histone, such as histone H1 or H5. These complexes
are progressively folded into higher-order and more condensed
structures [77]. The higher-order packed chromatin is generally
repressive for transcription, because of the inaccessibility of the
DNA [78,79]. Appropriate gene expression therefore requires
interplay with complexes that when recruited by transcriptional
activators, or repressors, can adjust the chromatin structure to alter
its accessibility.
Chromatin structure can be altered by three main mechanisms:
through mobilization of the nucleosomes utilizing energy from
ATP [80]; post-translational modification of histone proteins
(predominantly within the flexible tail regions) [81]; or replacement of histone proteins in the octomer with histone variants [82].
Nucleosome remodelling is performed by various complexes,
which utilize the energy from ATP to alter the contacts between
the histones and the DNA, thus facilitating disruption of the
nucleosome structure [80]. These can be subdivided into three
groups on the basis of biochemical properties and the conservation
of their catalytic subunits: SWI/SNF, ISWI and MI-2/CHD groups
(for a review, see [83]). In general terms, the SWI/SNF complexes
are involved in gene activation, whereas ISWI and MI-2/CHD are
involved in transcriptional repression.
Another way to change the accessibility of DNA within the
chromatin is through post-translational modification. Residues
on histones have been found to be phosphorylated, acetylated,
methylated, ubiquitinated, SUMOylated and poly(ADP-ribosyl)ated. These covalent modifications of histones correlate with profound effects on transcription, either changing the charge and
structure of the chromatin or providing binding sites for co-activator or co-repressor proteins to be targeted to the DNA. Each
one of these modifications can be correlated with an effect on
transcription and it has been proposed that the combination of
modifications on the histones surrounding a gene can fine-tune its
expression [81].
It is therefore surprising, considering the profound effects that
chromatin has on transcription, that almost no work has been done
to elucidate the effects of chromatin on gene expression following
hypoxia.
The HAT (histone acetyltransferase) p300 has been shown to
interact with HIF and is required for full activation of target genes
[84]. Several studies have demonstrated an increase in localized histone acetylation surrounding activated genes following
hypoxia (Figure 4) [85–87], but the observation that disrupting the
HIF–p300 interaction only alters expression of 35–50 % of HIFresponsive genes suggests that p300-induced acetylation of promoter histones is not required for all HIF target genes [19].
Several studies have also investigated the effects of HDACs
(histone deacetylases) on HIF-1 function [88,89]. One of these
studies identified HDAC7 as an interacting partner of HIF-1,
and revealed its importance for HIF-1 activation. This apparent
paradox between HDACs and HATs both being co-activators
for HIF activity may reflect differing co-factor requirements for
c The Authors Journal compilation c 2008 Biochemical Society
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Figure 4
N. S. Kenneth and S. Rocha
Chromatin changes in response to hypoxia
Schematic diagram illustrating the different chromatin-remodelling complexes that can be recruited to HIF responsive genes in hypoxia. (A) The SWI/SNF complex co-operates with HIF to activate
target genes. (B) Hypoxia results in a reduction of nucleosome density around its target gene promoters, and also in a reduction of repressive histone modifications such as histone H3 Lys27
trimethylation (H3K27 Me). In addition, hypoxia induces an increase in histone H3 Lys4 trimethylation (H3K4 Me) and an enrichment in histone acetylation, modification marks associated with
actively transcribing genes. (C) The HDAC inhibitor reduces HIF activity, and HDAC7 interacts with the HIF–p300 complex, which contributes to the transcription of HIF target genes. Ac, acetylated
histones; Pol II, RNA polymerase II.
different target genes. The situation is complicated further by
the demonstration, by co-immunoprecipitation analysis, that HIF,
HDAC7 and p300 can exist in the same complex at the same time
(Figure 4) [88,90]. This may reflect another level of control of
the HIF response with different combinations of co-factors being
available to activate different target genes. In vivo ChIP assays
identifying co-factor recruitment to HIF target genes would help
to elucidate how co-repressors and co-activators are targeted.
A recent study has also demonstrated changes in histone
methylation on HIF target genes when cells are challenged with
low oxygen [87]. In this study, the promoters of the hypoxiainduced genes VEGF and EGR1 (early growth response 1) were
investigated [87]. When cells were exposed to hypoxia, there
was an observed increase in the proportion of promoter histones
displaying H3K4 (histone H3 Lys4 ) trimethylation, a mark of
actively transcribing genes [81], and a decrease in H3K27 (histone
H3 Lys27 ) trimethylation, a modification generally associated with
genes in the inactive state (Figure 4) [81]. The identity of the
methylases and demethylases involved in this switch is unknown,
but may indicate how important epigenetic changes can be in the
regulation of hypoxia-induced genes.
The contribution of ATP-dependent chromatin-remodelling
enzymes to the gene expression changes induced by hypoxia has
also been severely underinvestigated. The one report to date that
has investigated how this class of enzymes can alter transcription
in hypoxia has focused on the EPO (erythropoietin) gene [85].
This study demonstrated that the SWI/SNF complex is required
for full induction of the EPO mRNA following hypoxia. It is also
interesting to note that there was an increase in localized histone
acetylation at the promoter of the EPO gene following hypoxia
[85], perhaps indicating a series of chromatin changes induced by
hypoxic stress. Although not looking directly at ATP-dependent
remodellers, a study examining genes both repressed and activated by exposure to low oxygen has demonstrated an inverse
relationship between nucleosome occupancy and transcriptional
activation of these genes (Figure 4) [87]. These findings may
c The Authors Journal compilation c 2008 Biochemical Society
indicate a hypoxia-responsive remodelling of chromatin, with a
link to the activation of downstream target genes.
TRANSLATION
Another important aspect of gene expression is translation.
Following hypoxia, global protein synthesis is slowed down or
inhibited (Figure 5). This is part of an energy-saving mechanism,
by which cells adapt and attempt to survive stressful conditions
such as lack of oxygen or nutrients.
Mechanistic studies on translation and overall protein synthesis
have been mostly performed following severe lack of oxygen,
ranging from 0.5 to 0 % O2 [91,92]. However, moderate hypoxia
(1–5 % O2 ) can produce the same effects, although with delayed
kinetics [93,94].
Translation is a complex process with multiple levels of
regulation. Molecularly, the process of mRNA translation can
be divided into three steps: initiation, elongation and termination.
Initiation is the most complex step and is very tightly controlled
by a number of molecules termed eIFs (eukaryotic initiation factors). In eukaryotes, most translation is cap-dependent, where the
assembly of an active eIF4 at the m7 GpppN mRNA structure
(5 -end of the RNA) is required. This complex consists of a capbinding protein eIF4E, a scaffold protein eIF4G and an ATPdependent helicase eIF4A. This complex facilitates the recruitment of the 43S pre-initiation complex, composed of the small 40S
ribosomal subunit, the initiation factor eIF3, and the ternary complex [eIF2–GTP–Met-tRNAi (initiator-methionyl tRNA)] [95].
The function of the 43S complex is to scan through the 5 -UTR
(untranslated region) of the mRNA until it encounters the AUG
initiation codon. eIF5 then promotes the hydrolysis of GTP-bound
eIF2 into GDP, releasing the initiation factors and promoting the
recruitment of the large 60S ribosomal subunit. This defines
the beginning of the elongation step.
The eIF4 and eIF2 complexes constitute the two major control
points in initiation of translation. eIF4E protein levels are limiting,
Regulation of gene expression by hypoxia
Figure 5
25
Hypoxia-induced pathways of translational control
Hypoxia can induce a variety of pathways leading to translational control. The severity and duration of hypoxia usually determines which pathways are activated first. These pathways will eventually
lead to preferential translation of key proteins required for adaptation and survival to hypoxic stress. S6K, S6 kinase; S6 RP, S6 ribosomal protein; 5 TOP, 5 -terminal oligopyrimidine; TSC1/2,
tuberous sclerosis complex 1/2.
and the formation of the eIF4 complex can be prevented by a
family of 4E-BPs (eIF4E-binding proteins), which compete with
eIF4G for binding. 4E-BPs are regulated by phosphorylation,
mediated mainly by mTOR (mammalian target of rapamycin), a
global integrator of several signalling pathways [96].
mTOR is a serine/threonine protein kinase that responds to
changes in energy status, nutrient availability, insulin and growth
factors, and its activity is associated with cell-cycle progression
and cell growth. mTOR-mediated phosphorylation of 4E-BPs
induces a reduction in their affinity for eIF4E, thereby allowing it
to bind eIF4G. In addition, mTOR also phosphorylates S6 kinase,
which stimulates translation via the ribosomal protein S6 and the
ribosomal recruitment protein eIF4B [96].
In hypoxia, mTOR activity is inhibited, leading to hypophosphorylation of S6K, S6 ribosomal protein and 4E-BP1 [93]. The
mechanism of mTOR inhibition is complex. However, the data
published so far suggest that hypoxia can induce a decrease in
cellular energy levels, leading to activation of the energy-responsive kinase AMPK (AMP-activated protein kinase) [93]. AMPK
phosphorylates the TSC1/2 (tuberous sclerosis complex 1/2),
which down-regulates mTOR activity. AMPK activation is mainly
regulated by the tumour suppressor protein kinase LKB1 [97].
Additional kinases are also involved, although their identities are
currently unknown [93].
Hypoxia also induces hyperphosphorylation of Ser51 of eIF2α
[91,93]. This results in translation inhibition, since phosphorylation of eIF2α at this site prevents the exchange of GDP for GTP
and sequesters eIF2B in an inactive complex. Phosphorylation
of eIF2α can be achieved by several different kinases: PKR
(interferon-inducible double-stranded RNA-activated kinase),
HRI (haem-regulated inhibitor of translation), GCN2 (kinase
activated by nutrient starvation) and PERK (PKR-like endoplasmic reticulum kinase), which is actively involved in the UPR
(unfolded protein response). All of these kinases can respond to
different types of stress. PERK is the main kinase responsible
for eIF2α phosphorylation following hypoxia [91,98,99]. Several
studies have identified the UPR as a component of the cellular
response to hypoxia, leading to more mechanistic insights as to
how cells survive such harsh conditions. Genetic studies with
loss-of-function models for PERK and ATF4 (a transcription
factor that is up-regulated following hypoxia in a HIF-independent
manner and is also involved in the UPR) demonstrated that these
proteins contribute to overall survival. In addition, cells deficient
for these factors are more sensitive to hypoxia-induced cell death,
and tumour xenograft models have demonstrated that PERK
contributes to tumour growth in vivo [91]. These studies have
identified the UPR as a good candidate for therapeutic targeting
in solid tumours.
Interestingly, a recent study demonstrated that 4E-BP1 and
eIF4G are highly overexpressed in human breast cancers [100].
This study suggested that this facilitates cap-independent translation in hypoxic tumours, and promotes translation of IRES
(internal ribosome entry site)-containing mRNAs allowing
survival and angiogenesis to occur [100]. However, IRES-containing mRNAs cannot account for all the preferential translation that
takes place in hypoxia. In fact, a more recent study investigated
the activity of previously documented IRES elements in certain
mRNAs that are preferentially translated in hypoxia [101]. This
study demonstrated that hypoxia-selective mRNA translation was
independent of IRESs, even for IRES-containing transcripts such
as VEGF and HIF-1α [101]. One additional mechanism for controlling gene expression, which is dependent on eIF2α phosphorylation, was described recently [102]. In this study, nonsensemediated RNA decay was found to be inhibited by hypoxia,
giving rise to increased stability of a number of mRNAs of stressinduced genes such as ATF4, ATF3 and CHOP [C/EBP (CCAAT/
enhancer-binding protein)-homologous protein 10] [102]. This
suggests yet another pathway modulated by hypoxia (Figure 5).
c The Authors Journal compilation c 2008 Biochemical Society
26
Figure 6
N. S. Kenneth and S. Rocha
Schematic diagram of miRNA biogenesis
miRNAs are encoded in the genome and possess a critical hairpin structure. After being transcribed, this primary miRNA (Pri-miRNA) is processed by the enzyme Drosha, to a precursor miRNA
(Pre-miRNA). This is then transported to the cytoplasm, where it is processed further by another enzyme Dicer, leading to the mature miRNA and incorporation into the RISC (RNA-induced silencing
complex). This will then target direct sequences in mRNA leading to mRNA cleavage (RNA interference) or target transcripts that are being translated, leading to inhibition of translation or changes
in mRNA stability.
miRNAs
The fact that translation still occurs in hypoxia by mechanisms
that are cap-independent and IRES-independent gives rise to the
possibility that other pathways contribute to the gene expression
in these conditions. One such pathway is miRNA modulation.
miRNA is a non-coding RNA composed of 21–24 nt, which is derived from an endogenous hairpin RNA precursor (usually approx.
70 nt long). The process of miRNA maturation involves several
classes of proteins with enzymatic capability such as the RNaseIII-type enzyme called Dicer and multifunction proteins such as
Argonautes (for reviews on the mechanisms of miRNAs, see
[103,104]). The miRNA mode of action seems to be more of
a fine-tuning of expression rather than degradation of mRNA, a
mechanism by which RNA interference works [104]. miRNAs can
prevent translation by several mechanisms, either by preventing
initiation, or by preventing elongation by inducing ribosome drop
from the polypeptide (Figure 6).
Despite the relatively recent discovery of this form of gene
regulation, hypoxia induction of miRNAs has been documented
[105–109]. Some studies have even identified a miRNA signature
associated with hypoxia in certain cell types [108,110]. Furthermore, some miRNA targets have been identified, with VEGF
being one of the most prominent [105]. A study investigating
mechanistic manipulations of miRNAs in hypoxia has been conducted recently [109]. However, this study only focused on one
particular miRNA, mir-210. The next few years should determine
how much of the hypoxia-induced gene expression is dependent
on miRNA function. In addition, the next big question shall
be the determination of which miRNAs are important for cell
transformation and tumour survival in hypoxic situations. One
additional point to consider is the fact that several of the
transcription factors activated by hypoxia can induce miRNAs;
these include p53 and NF-κB, as well as c-Myc [111–114].
Induction of transcription-factor-mediated miRNA has not been
investigated in the context of hypoxia stimulation. Despite this
fact, miRNA induction is a very important aspect of the biology
c The Authors Journal compilation c 2008 Biochemical Society
Figure 7
Hypoxia is at the centre of a multitude of cellular pathways
Hypoxia induces changes in transcription factor activity, transcription, translation, miRNAs and
even global chromatin changes. All of the pathways are integrated, and a co-ordinated cellular
response is achieved.
of these transcription factors that needs to be taken into account
while testing for hypoxia responses.
CONCLUSION
Traditionally, gene expression analysis following hypoxia has
focused on HIF-mediated transcriptional responses; however,
many pathways are activated by hypoxic stress within the cell
(Figure 7). In order to achieve a co-ordinated response under such
stressful conditions, all of these pathways must be integrated.
Chromatin reorganization, translational processes and miRNA
induction are all new aspects that have to be considered in the
Regulation of gene expression by hypoxia
analysis of the hypoxia response (Figure 7). In addition, crosstalk with other transcription factors also modulates HIF activity.
Future research in these areas will offer much needed insight into
how the gene expression profile of a cell under hypoxia is
determined, with the hope of applying this knowledge to
pathological situations.
We thank Professor Neil Perkins, Dr Cari Culver, Mrs Sharon Mudie and Mr Patrick
van Uden for valuable comments on this manuscript. We thank all of the members
of the Wellcome Trust Centre for Gene Regulation and Expression at the College of
Life Sciences, University of Dundee, for their support and encouragement. Work in our
laboratory is supported by the International Association for Cancer Research (AICR), the
Medical Research Council (MRC), Research Councils UK (RCUK) and the University of
Dundee.
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Received 29 May 2008/6 June 2008; accepted 9 June 2008
Published on the Internet 29 July 2008, doi:10.1042/BJ20081055
c The Authors Journal compilation c 2008 Biochemical Society