The interplay between hypoxia-inducible factor and E2F
Transcriptional crosstalk under hypoxic conditions
Almar Neiteler
The interplay between hypoxia-inducible factor and E2F
Transcriptional crosstalk under hypoxic conditions
Author:
Almar Neiteler
Study:
Molecular and Cellular Life Sciences
Graduate School of Life Sciences
Faculty of Science
Utrecht University
Written at:
Department of Pathobiology
Faculty of Veterinary Medicine
Utrecht University
Head of department:
Prof. A. de Bruin
Supervisor/examiner: Dr. W. J. Bakker
Date:
7 June 2013
Figure on the front page is described in supplementary figure S1.
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Contents
Chapter
Abstract
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1. Introduction .
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1.1 Hypoxia
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1.2 HIF .
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1.3 E2F .
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1.4 Aims of this study
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2. Coordinated cell cycle control by HIF and E2F .
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2.1 pRB promotes cell cycle arrest via HIF-1
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2.2 HIF-2 promotes tumor growth by inducing cyclin D1 expression
2.3 HIF-1 promotes cell cycle arrest by inhibiting cyclin E .
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2.4 Feedback loops between HIF, E2F and the PI3K-pathway
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2.5 HIF regulates E2F indirectly through c-Myc .
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2.6 HIF regulates E2F activity indirectly through TGF-β .
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2.7 HIF regulates E2F-regulated genes indirectly through Sp1
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3. Regulation of cell metabolism by HIF and E2F .
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3.1 Regulation of the glycolysis and the TCA cycle under hypoxia .
3.2 Feedback loops between HIF, E2F and PPAR .
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3.3 HIF and E2F regulate COX activity
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4. Crosstalk between DNA repair, HIF and E2F pathways
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4.1 HIF-1α and E2F colocalize with BRCA1
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4.2 ATM stabilizes HIF and E2F .
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4.3 PARP-1 induces HIF and E2F-responsive gene expression
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4.4 HIF and E2F regulate mismatch repair
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4.5 HIF and E2F regulate hTERT .
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5. Regulation of cell death by HIF and E2F
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5.1 HIF and E2F regulate p53
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5.2 HIF regulates E2F indirectly through DP4
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5.3 Feedback loops between HIF, E2F and FOXO .
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5.4 HIF and E2F regulate BH3-only Bcl-2 family proteins .
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5.5 Feedback loops between HIF, E2F and NF-κB .
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5.6 Feedback loops between HIF, E2F and ASK1 .
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6. Discussion .
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References
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Abbreviations .
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Supplementary information
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Abstract
Hypoxia is an important condition in both developmental and pathological settings. It is crucial for
cell survival to adapt to hypoxic stress. A key response to hypoxia is regulation of gene expression.
The transcription factor HIF is involved in the regulation of most hypoxia-sensitive genes. HIF is
known to regulate genes involved in the cell cycle, angiogenesis, metabolism, DNA repair and cell
death. The E2F family of transcription factors also regulate genes in these cellular processes.
Intriguingly, recently it was shown that E2F7/8 regulate angiogenesis through a transcriptional
cooperation with HIF (Weijts and Bakker, 2012). It is therefore likely that these important
transcription factors are involved in many of the same pathways under hypoxic conditions. However,
not much is known about the interplay between HIF and E2F. This literature study investigates and
reveals a broad relationship between HIF and E2F in the cell cycle, metabolism, DNA repair and cell
death. Elucidation of the relationship between HIF and E2F may provide a better understanding of
the role of hypoxia in development and disease.
1. Introduction
1.1 Hypoxia
Oxygen is a key requirement for cell survival. Oxygen serves as primary electron acceptor in many
intracellular biochemical reactions and is utilized by the cell to generate ATP through aerobic
metabolism in mitochondria. For this reason oxygen homeostasis needs to be tightly controlled and
is therefore regulated by erythropoiesis, vascular development and metabolic adaptation. Low
oxygen tension (hypoxia) is defined as a decreased level of oxygen compared to normal oxygen levels
(normoxia). Physiological normoxia ranges from 2-9% oxygen in most mammalian tissues1.
Homeostasis of oxygen needs to be tightly controlled in the cell. Cells respond therefore to hypoxia
through coordinated changes in gene expression. The ability to adapt to hypoxia is important in both
developmental and pathological settings.
Hypoxia plays a central role in normal development. In the developing and adult embryo hypoxic
microenvironments create specific niches that regulate stem cell maintenance and differentiation1.
Stem cells are known to reside in specialized microenvironments, or niches, and are regulated by
factors inherent to their microenvironments. Hypoxic microenvironments occur naturally preceding
the formation of the circulatory system. Hypoxia is important in organogenesis as it plays a crucial
role in the regulation of energy metabolism, heart development, angiogenesis, trachea development,
chondrogenesis, bone formation and cell death1, 2. Also in adult tissues localized areas of hypoxia can
be found resulting from a low level of vascularization or from highly metabolic active cells that run
out of their oxygen supply. Oxygen availability in these areas are known to affect cell proliferation,
survival, and differentiation of stem and progenitor cells3. Oxygen level regulation is therefore not
only important for cellular metabolism, but is also essential for cell fate regulation.
Hypoxia is also an important aspect of a variety of pathological conditions. Low oxygen levels or
the absence of oxygen (anoxia) can be found in a wide range of pathological conditions including
stroke, tissue ischemia, inflammation and solid tumors. Hypoxic stress is thought to be a common
phenomenon in human tumorigenesis4. Oxygen levels are known to vary substantially in tumors and
can have diverse effects on the pathology5. Uncontrolled proliferation causes tumors to outgrow
their blood supply4. Also aberrant blood vessels commonly found in tumors can limit the supply of
blood4. These combined events typically create microenvironments in tumors of severe hypoxia or
anoxia. Extended exposure to anoxia leads to necrosis, but necrotic zones in tumors are often
surrounded by a hypoxic zone6. Cancer cells undergo genetic changes to adapt to the hypoxic
conditions that allow them to survive and even proliferate in a hypoxic environment4. The
malignancy and aggressiveness of the tumor is often increased by this adaptation4. It is therefore
4
important to learn more about the cellular response to hypoxia in order to understand more about
the progression of these pathological conditions.
Cells induce a variety of biological changes in response to hypoxic conditions. An important way
for cells to respond to hypoxia is by adapting their gene expression profile. The transcription factor
hypoxia-inducible factor (HIF) plays a central role in the regulation of hypoxia-sensitive genes7. Genes
that are upregulated during hypoxia are typically involved in cell proliferation, angiogenesis,
metabolism, DNA repair, cell death, and migration4, 8.
A transcription factor family that is involved in the regulation of many of the same cellular
processes as HIF is the E2 factor (E2F) family. Particularly, both transcription factors have overlapping
regulatory functions in the cell cycle, angiogenesis, metabolism, DNA repair and cell death. It is
therefore likely that both transcription factors have a pronounced relationship in these cellular
processes during hypoxia. It is possible that there is crosstalk between HIF and E2F pathways and
that there are direct or indirect interactions between the two transcription factors. Moreover, recent
studies already show that HIF can directly associate with specific E2Fs9. This interaction was shown to
form a transcriptional complex important for angiogenesis during the embryonic development of
zebrafish and mice. E2F has also recently been shown to regulate the expression of HIF under certain
conditions10. Surprisingly, there is however still not much known about the involvement of HIF and
E2F transcription factors in the same regulatory pathways. It is thought that interactions between HIF
and E2F are also involved in the regulation of other cellular processes than angiogenesis. The
regulation of HIF and E2F are discussed below.
1.2 HIF
HIF was identified as a transcription factor
binding to the promoter of the human
erythropoietin gene after hypoxic exposure11,
12
. HIF is a heterodimeric transcription factor
consisting of an oxygen-labile HIF-α subunit
and a constitutively expressed HIF-β subunit13,
14
. There are three isoforms of the HIF-α
subunit (HIF-1α, HIF-2α, and HIF-3α)
expressed in mammals. HIF-1α is expressed in
nearly all cell types. In contrast, HIF-2α and
HIF-3α expression is more restricted to certain
tissues6. HIF-1α and HIF-2α have overlapping
target genes as well as unique target genes15.
In contrast to HIF-1α and HIF-2α, not much is
yet known about HIF-3α. HIF-α subunits
heterodimerize with a HIF-β subunit, also
known as aryl hydrocarbon nuclear
translocator (ARNT). There are also three
isoforms of HIF-β (HIF-1β/ARNT, ARNT2, and
Figure 1. Regulation and function of HIF. HIF-α is
ARNT3). HIF-α subunits mainly dimerize with
hydroxylated by PHD under normoxic conditions, which allows
the binding of VHL (A). VHL promotes the ubiquitination and
HIF-1β, and also to a lesser degree with
subsequent proteasomal degradation of HIF-1α. Under
ARNT26, while for ARNT3 there is not much
hypoxia, HIF-α is not hydroxylated by PHD and can therefore
evidence for cooperative gene regulation. HIFs
bind to HIF-β (B). This heterodimer can translocate to the
are helix–loop–helix (HLH) proteins and
nucleus, associate with p300/CBP, and can regulate gene
belong to the PER–ARNT–SIM (PAS) protein
expression by binding to the HRE in promoter regions.
family. The HLH and PAS domains are involved
in the dimerization. The HIF heterodimers recognize and bind promoter regions bearing hypoxia
responsive elements (HREs) which contain the core sequence G/ACGTG16. HIF heterodimers bind to
HREs in association with coactivators and thereby regulate the expression of hypoxia-sensitive genes.
5
The HIF-α subunits are hydroxylated at conserved proline residues under normoxia (Fig. 1A). HIFspecific prolyl-hydroxylases (PHDs) mediate this reaction in the presence of oxygen16. The oxygen
dependent PHD proteins belong to the Fe(II) and 2-oxoglutarate-dependent dioxygenase
superfamily. The E3 ubiquitin ligase Von Hippel-Lindau (VHL) protein complex recognizes and marks
the hydroxylated HIF-α for proteasomal destruction16, 17. Under hypoxic conditions the rate of HIF
hydroxylation is suppressed by limited PHD activity. Accordingly, HIF-α subunits can accumulate and
dimerize with HIF-β subunits (Fig. 1B). This HIF heterodimer subsequently translocates to the nucleus
where it regulates a broad range of genes involved in various cellular processes.
In addition to promote HIF-α ubiquitination and degradation, VHL also forms a complex with HIFα and factor inhibiting HIF-1 (FIH-1). VHL together with FIH-1 mediate the repression of HIF-α
transcriptional activity18. Oxygen-dependent hydroxylation of HIF-α by FIH-1 blocks the interaction of
HIF-α with the coactivators p300 and CBP under normoxic conditions19. FIH1 is, like the PHD family
members, a Fe(II)- and 2-oxoglutarate-dependent dioxygenase. FIH1 is therefore also dependent on
oxygen availability for its activity. Together, PHDs and FIH-1 are often considered as the oxygen
sensors that regulate the stability of HIF-α.
Stabilization of the HIF-α subunit is but one step in the regulation of the HIF heterodimer. HIF can
have multiple posttranslational modifications which can affect its stability as well as its activity.
Several protein kinase pathways can modulate the activity of HIF20, 21. HIF is known to be
phosphorylated, but phosphorylation is not necessary for HIF stabilization under hypoxia11, 21, 22. HIFα can also be acetylated and sumoylated under hypoxia which influences HIF-α stability23-25. After the
stabilization the HIF heterodimer translocates to the nucleus where it associates with coactivators
including p300/CBP26, 27 and the SCR-1 family28. This complex subsequently binds to the HRE in the
promoter region of hypoxia-sensitive genes (Fig. 1B). Coactivators are important regulators of the
transcription-inducing function of HIF.
Hypoxia regulates the expression of many different genes via HIF, which function in diverse
important cellular processes, including cell proliferation, angiogenesis, metabolism, DNA repair, cell
death, and migration4, 8. Not only the upregulation of genes during hypoxia is important. Also
hypoxia-downregulated genes merit consideration29. Induction and repression of expression by HIF
can be dependent on the cellular context and is also dependent on the severity and the length of
hypoxia. The broad range of genes that are targeted by HIF reflects the complex and extensive
changes that cells undergo in response to hypoxia.
1.3 E2F
The E2Fs are a large family of transcription factors that regulate the expression of genes involved
in a wide range of cellular processes, including cell cycle progression, DNA replication, DNA repair,
differentiation, and apoptosis. All E2F family members possess a DNA-binding domain that binds to
the E2F consensus binding sequence. Different E2F family members therefore regulate many of the
same target genes. However, differences in E2F sites provide preferential affinity for specific
members of the E2F family30. E2F family members are divided in two subgroups, based on their
potential function. E2F1, E2F2 and E2F3a were originally classified as activating E2Fs, because they
are shown to be essential for transactivation of genes involved in the G1/S transition and thus the
initiation of DNA replication31. By contrast, E2F3b, E2F4, E2F5, E2F6, E2F7 and E2F8 were considered
to have repressive activity. However, it is now known that E2F family members can function as either
activators or repressors of transcription, depending on the cellular context32, 33. Although E2Fs that
are classified as activators function in most cases as activators, while E2Fs classified as repressors
mainly have a repressive function.
6
Most E2F family members bind DNA as
heterodimers with one of three related dimerization
partner proteins (DP1, DP2/3, and DP4). The E2F/DP
heterodimer function is thought to be primarily
dictated by the E2F subunit32. The function of each
of the different DP subunits in regulating E2F activity
is not yet fully understood, but it appears that
dimerization is required for high-affinity and
sequence-specific binding of E2F to the promoters of
target genes34.
E2F1, E2F2, E2F3, E2F4 and E2F5 can interact with
the retinoblastoma (RB) family of proteins, also
called pocket proteins. The pocket proteins to which
E2Fs bind are pRB, p107 and p130. E2F1, E2F2 and
E2F3 primarily bind to pRB and not to the other
pocket proteins35. E2F4 and E2F5 can associate with
each of the three pocket proteins36, 37. When an
Figure 2. Regulation of E2F-responsive genes.
E2F/DP heterodimer associates with a pocket
Hypophosphorylated pocket proteins associate with
protein, the transcriptional activation domain is
E2F/DP heterodimers which can bind to the promoter of
masked and therefore the transcriptional activity is
E2F-responsive genes (A). This complex also recruits
blocked32 (Fig. 2A). Association with a pocket protein
cofactors such as histone acetyl transferases (HDACs) to
repress the expression of the target genes. Pocket
prevents E2F/DP from recruiting the basal
proteins can be hyperphosphorylated by cyclin/CDK
transcription factor TFIID, as well as transcriptional
complexes and this causes the release of E2F/DP (B).
cofactors such as the histone acetyl transferases
E2F/DP bound to the promoter of target genes can
(HATs) p300/CBP38, 39. Association with pocket
recruit cofactors such as histone acetyl transferases
(HATs) to induce expression.
proteins can also convert E2F/DP heterodimer
activators into repressors by recruiting various
cofactors to the promoter of E2F responsive genes, such as histone deacetylases (HDACs)32, 40, 41. E2Fs
are released when pocket proteins are phosphorylated by cyclin-dependent kinases (CDKs)31 (Fig.
2B). These phosphorylation events thus modulate the expression of E2F target genes. E2F6, E2F7 and
E2F8 lack a pocket protein binding domain and therefore are regulated in a pocket protein
independent manner42, 43.
E2F7 and E2F8 are considered atypical E2Fs as they do not associate with DPs and have two DNAbinding domains instead of one44, 45. Atypical E2Fs can bind an E2F consensus binding sequence in a
DP-independent fashion because of the presence of two DNA-binding domains42. Moreover, atypical
E2Fs are shown to dimerize with each other and can form homo- or heterodimers46. One of the two
DNA-binding domains in atypical E2Fs appears to be mutated in such a way that both DNA-binding
domains together can mimic the DNA binding interface of the E2F/DP heterodimer42. Atypical E2Fs
are known to play important roles during development, the control of cell size, endocycle,
proliferation, angiogenesis and DNA repair9, 42.
E2Fs are not only regulated by pocket proteins. E2Fs are also subjected to posttranslational
modifications which regulate their activity, such as acetylation and phosphorylation events47, 48.
These posttranslational modifications can have positive as well as negative effects on E2F activity.
Regulation of E2Fs is important because an E2F can have diverse key functions that can lead to
contrasting effects on cell fate. E2F1 for example can act both as a tumor suppressor and as an
oncogene49, 50. Moreover, E2F1 has the ability to stimulate cell proliferation as well as the ability to
induce apoptosis49, 50. Regulation of this E2F, either by pocket proteins or posttranslational
modifications, is therefore important and the actual function is dependent on the cellular context.
E2Fs are especially known for their role in the cell cycle (Box 1). The best studied and understood
function of E2Fs is their ability to regulate the G1/S transition and S-phase entry during the cell cycle.
Many E2F family members are inhibited in function in the G0 and early G1-phase, as pocket
protein/E2F complexes are prevalent in these phases of the cell cycle51. However, pocket proteins are
7
phosphorylated in late G1-phase by cyclin/CDK
complexes31. In turn, E2Fs regulate the expression of
cyclins52. E2Fs control the expression of numerous Sphase genes, including various genes encoding for DNA
replication proteins51. In addition, E2Fs also regulate
several genes that are important for mitosis53. Towards
the end of M-phase pocket proteins are
dephosphorylated by the phosphatase PP-1 and
therefore can repress E2Fs again54.
E2Fs also have functions in other important cellular
processes. In addition to regulating the cell cycle, E2Fs
also regulate many other genes from pathways involved
in angiogenesis, metabolism, DNA repair and
apoptosis55, 56. The involvement of E2Fs in these cellular
processes reflects the broad range of cellular changes
that occur during the cell cycle.
Box 1. The cell cycle and its control
system.
The cell cycle is divided in four sequential
phases: G1, S, G2, and M. Chromosomes are
duplicated in the S phase and mitosis
occurs in the M phase. The extra gap
phases, G1 and G2, allow for more cell
growth. In addition, the gap phases provide
time for the cell to monitor the internal and
external environment to ensure that the
conditions are suitable for the cell to
commit to enter the S or M phase. An
important process such as the cell cycle
needs a control system. There are three
checkpoints in the cell cycle control system:
Start (in late G1; also known as restriction
point), G2/M checkpoint, and metaphaseto-anaphase transition. The control system
blocks progression through each of these
checkpoints if it detects problems inside or
outside the cell until conditions become
favorable. Cyclin-dependent kinases (CDKs)
are central components of the cell cycle
control system. The activity of these
kinases changes throughout the cell cycle,
leading to cyclical changes in the
phosphorylation of important proteins that
initiate or regulate the cell cycle. CDKs are
regulated by cyclins which protein levels
changes during the cell cycle.
1.4 Aims of this study
HIFs and E2Fs are important transcription factors that
play central roles in many vital cellular processes. Recent
studies show that HIFs and E2Fs operate in many of the
same pathways and especially under hypoxic conditions.
HIFs and E2Fs can cooperate in biological complex by
directly or indirectly forming a transcriptional complex.
In addition, they may also interact by regulating similar
biological processes and even target genes without the
formation of a common complex. Diverse cellular
processes that are regulated by both HIFs and E2Fs are
cell cycle control, angiogenesis, cellular metabolism,
DNA repair and cell death. The cooperation between
HIFs and E2Fs in angiogenesis has already been
reviewed recently57. This literature study therefore investigates the interplay of HIFs and E2Fs in cell
cycle control, cellular metabolism, DNA repair and cell death (Fig. 3) under hypoxia and highlights the
importance of the interactions between these transcription factors.
Figure 3. HIF and E2F regulated pathways. HIF and E2F regulation of genes
overlaps in pathways that are involved in the cell cycle, DNA repair, metabolism
and cell death.
8
2. Coordinated cell cycle control by HIF and E2F
Hypoxia is known to alter the cell cycle. Increasing evidence shows that HIF is responsible for this.
The involvement of HIF in many different pathways that influence the cell cycle suggests that
regulation of the cell cycle is an important function of HIF. The major mechanisms through which HIF
is linked to the cell cycle also include the involvement of E2Fs. These major mechanisms are
discussed below.
2.1 pRB promotes cell cycle arrest via HIF-1
The pocket protein pRB is a potent inhibitor of E2F-dependent transcription32. This transcriptional
repression leads to cell cycle arrest. Conversely, pRB can also function as a transcriptional activator.
pRB increases the DNA-binding and the transactivational function of various transcription factors58.
The mechanism by which pRB can promote transcriptional activation is however not yet fully
understood. It is possible that pRB, in addition to its repressive function, also has a transcriptional
activator function that is involved in cell cycle regulation.
Hypoxia can halt cellular proliferation in two distinct manners by triggering cell cycle arrest or by
inducing programmed cell death59. Cell cycle arrest in the event of hypoxia is mainly caused by the
decrease of cyclin/CDK complex activity which leads to hypophosphorylation of pRB. HIF-1 plays a
role in this process. CDKs can be inhibited in activity by CDK inhibitors (CKIs) and HIF-1 is involved in
inducing the expression of certain CKIs59, 60. Furthermore, HIF-1α was shown to be able to bind to
hypophosphorylated pRB in vitro58. This interaction is thought to induce the transactivational ability
of HIF-1α. An interaction between HIF-1α and pRB would therefore promote cell cycle arrest or cell
death through the induced expression HIF-regulated genes.
The role of pocket proteins in the cell cycle is well studied. In G0 and early G1,
hypophosphorylated pRB is associated with E2F1, E2F2, and E2F3 and thereby repress the activation
of E2F-responsive genes (Fig. 2A). In addition, transcriptional repressor complexes are formed in G0
and early G1 by the pocket proteins p107 and p130 in conjunction with E2F4 or E2F561. These
repressor complexes bind and repress most, if not all, E2F-responsive promoters. Pocket proteins are
generally phosphorylated and dissociate from E2Fs in late G1-phase. This causes the relocation of
E2F4 and E2F5 to the cytoplasm, and the promoters of E2F-responsive genes are subsequently
generally bound and activated by E2F1, E2F2 and E2F3. E2F1, E2F2 and E2F3 activate genes by
recruiting HATs to the promoter and thereby increase histone acetylation, which allows for
transcriptional activation62 (Fig. 2B).
Hypophosphorylation of pRB, which is promoted by hypoxia, maintains the inactive state of
activator E2Fs. Moreover, hypophosphorylated pRB is thought to also bind to HIF-1α which would
lead to more hypophosphorylation of pRB. Thus, this establishes a positive feedback loop that
promotes cell cycle arrest. It is however unknown if a complex between HIF-1α and
hypophosphorylated pRB also includes an E2F, since activator E2Fs are generally bound to
hypophosphorylated pRB. Activator E2Fs in this complex could possibly recruit HATs to promoter
sites, which could explain the increased transactivational ability of HIF-1α.
2.2 HIF-2 promotes tumor growth by inducing cyclin D1 expression
Cyclin D1 activates CDK4 and CDK6 and plays a key role in the cell cycle regulation during the G1/S
transition63. One important way by which cyclin D1/CDK4/6 promotes G1 progression is by
phosphorylating pocket proteins. After cells progress through the late G1 restriction point, cyclin D1
becomes unnecessary to complete the cell cycle. This implies that the most important function of
cyclin D1 is in G1 to sense if the cell is ready to commit to enter the S-phase.
The gene CCND1, which encodes cyclin D1, is a HIF-regulated gene in renal carcinoma cells64, 65.
HIF-2 was found to bind to HREs in an active enhancer of CCND1 and thereby promote the expression
of cyclin D164 (Fig. 4). Moreover, CCND1 was found to be one of the most HIF-regulated genes on
chromosome 11. Although the HIF-dependent enhancer of CCND1 can bind both HIF-1 and HIF-2,
9
only HIF-2 is transcriptionally active66. However, induction of cyclin D1 expression by HIF-2 appears to
be cell type specific64. It was also found that CCND1 has three HREs in its promoter region that can
bind to HIF-2 in vitro67. Also VHL regulates the expression of cyclin D1 under hypoxia67-69 and can do
this also in a HIF-independent manner70. The precise mechanism by which VHL regulates cyclin D1
expression independently of HIF is not yet known. Regulation by VHL appears also to be cell type
specific.
E2F1 and E2F4 can bind the promoter of
CCND1 and regulate its expression71, 72. In
turn, cyclin D1/CDK4/6 can regulate E2F
activity by phosphorylating pocket proteins
to release E2Fs in early G1 phase73, 74 (Fig. 4).
E2F1 can induce or repress cyclin D1
expression, depending on the cellular
context72, 75. In addition to the ability to
induce cyclin D1 expression, E2F1 can also
induce the expression of other cyclins in G1.
The induced expression of cyclin D1 and the
subsequent release of E2F1 from pRB
Figure 4. Regulation of cyclin D1 expression by HIF-2 and E2F.
provides a strong positive feedback loop75,
HIF-2 can bind to HREs in an active enhancer of CCND1 in renal
76
. In contrast, repression of cyclin D1
carcinoma cells. E2F1 and E2F4 can bind to the promoter region
expression by E2F1 would form a negative
and regulate the expression of cyclin D1 as well. Cyclin D1 can
promote E2F activity by hyperphosphorylating pRB in
feedback loop72.
conjunction with CDK4/6.
In G0 and early G1, E2F4/DP1/p130
complexes are bound to promoters of E2F-regulated genes and thereby inhibit transcription62, 77.
When activated in mid G1, cyclin D1/CDK4 can phosphorylate p130 and therefore causes the
dissociation of E2F4/DP1 from p130. The DNA binding capability of E2F4/DP1 could therefore be
potentially disrupted78. This subsequently causes derepression of E2F-regulated genes and that could
clear the E2F-binding sites for other E2Fs to bind in the late G1-phase.
In conclusion, both HIFs and E2Fs can regulate the expression of cyclin D1. For HIF-2 this was at
least shown in renal cell carcinoma. In the event of hypoxia, it is possible that HIF-2 induces extra
expression of cyclin D1. The extra expression of cyclin D1 could amplify the already existing feedback
loops with E2F1. Depending on the cellular context, more E2Fs could become functionally active and
promote cell cycle progression under hypoxia.
2.3 HIF-1 promotes cell cycle arrest by inhibiting cyclin E
E-type cyclins are also involved in the HIF and E2F pathways. Cyclin E is only expressed in the late
G1-phase and is considered to promote S-phase entry61, 79. Cyclin E in conjunction with CDK2 can
phosphorylate substrates involved in transcriptional control, DNA replication, centrosome
duplication and cell survival regulation79, 80. It is therefore important that cyclin E expression and
activity are tightly regulated. Cyclin E is a substrate of the ubiquitin ligase containing F-box and WD40
domain protein 7 (Fbw7). Ubiquitination by Fbw7 marks cyclin E for proteasomal degradation. The
activity of cyclin E can be regulated by CKIs, which inhibit the function of cyclin/CDK complexes.
When Fbw7-dependent degradation of cyclin E is impaired in human mammary epithelial cells, it
was found that HIF-1α expression was highly induced10. This suggests that cyclin E is involved in the
regulation of HIF-1α expression. The same study also shows that the HIF1A gene, encoding for HIF1α, has potential E2F-binding sites in its promoter. Moreover, E2F1 was found to occupy the E2Fbinding sites and induce expression in the setting of dysregulated cyclin E10 . Thus, HIF-1α expression
seems to be regulated by cyclin E and E2Fs. HIF-1α was also less degraded via the VHL pathway in
mammary epithelial cells with dysregulated cyclin E10. Therefore, when cyclin E is not degraded by
Fbw7, HIF-1α is more expressed by E2F1 and is also less degraded.
HIF-1 was shown to downregulate the expression of cyclin E81. HIF-1 can increase the expression
of p21Cip1 and p27Kip1 which are CKIs59, 60 (Fig. 5). The p21Cip1 and p27Kip1 proteins inhibit the catalytic
10
activity of cyclin/CDK2 complexes. Both CKIs can bind to CDK2 and control its kinase activity,
depending on the cell cycle phase. HIF-1 can therefore function as one of the primary regulators at
the G1/S interface as p21Cip1 and p27Kip1 can promote cell cycle arrest under hypoxia (0.5% - 3%
oxygen)59, 60. Furthermore, cyclin E/CDK2 can phosphorylate p27Kip1 on a specific site and thereby
cause its degradation82. This suggests that cyclin E/CDK2 can partly regulate its own expression.
Figure 5. Inhibition of cyclin E/CDK2 function by HIF-1. HIF-1 can induce the expression of CKIs that control
cyclin E/CDK2 activity. This prevents the hyperphosphorylation of pocket proteins by cyclin E/CDK2. Pocket
proteins in complex with E2Fs repress the expression of E2F-responsive genes, such as CCNE1, which
encodes for cyclin E. HIF1A is also thought to be an E2F-responsive gene, at least when cyclin E is
dysregulated.
Cyclin E is regulated by two E2F-binding sites in its promoter region. One of these sites is occupied
in G0 and early G1 by E2F4 or E2F5 in complex with pocket proteins and therefore the expression of
cyclin E is repressed83, 84. The other E2F-binding site is constitutively occupied by E2F1-3 in complex
with pRB85 (Fig. 5). In late G1, pRB is phosphorylated and E2F1, E2F2 and E2F3 are thought to remain
bound to the cyclin E promoter site and activate the expression. Cyclin E/CDK2 is also known to
further phosphorylate pRB in preparation for progression into the S-phase74, 86, 87. Additionally, cyclin
E/CDK2 can also directly phosphorylate E2Fs. Phosphorylation of E2F5 by cyclin E/CDK2 has been
shown to facilitate the recruitment of p300/CBP88. Recruitment of p300/CBP to a promoter region is
known to induce expression.
Pocket proteins are also involved in the regulation of cyclin/CDK2 complexes. The pocket proteins
p107 and p130 can inhibit CDK2 in a similar fashion as the p21Cip1 and p27Kip1 CKIs61. Additionally, pRB
was found to increase the expression of p27Kip1 by stabilizing the protein89. Pocket proteins can
therefore also be seen as important regulators of the G1/S transition.
In conclusion, it can be said that the interaction between cyclin E, HIF and E2F is quite complex.
Regulation between cyclin E, HIF and E2Fs is controlled by several feedback loops. Under hypoxic
conditions, HIF-1 induces the expression of p21Cip1 and p27Kip1, and therefore indirectly controls E2F
function and cell cycle progression. Because p21Cip1 and p27Kip1 can inhibit the phosphorylation of
pRB by cyclin E/CDK2, these CKIs therefore maintain the interaction between pRB and E2F and inhibit
cell cycle progression. E2Fs can induce the expression of cyclin E and, at least under certain
conditions, HIF-1. This induced expression by E2F is therefore also inhibited by CKIs under hypoxia.
Thus, HIF-1 and E2F can regulate the expression of each other with cyclin E playing a role in this.
2.4 Feedback loops between HIF, E2F and the PI3K-pathway
An important signaling pathway for cell survival is initiated by phosphatidylinositide 3′-OH kinase
(PI3K)90 (Fig. 6). PI3K can be activated by several types of transmembrane receptors. PI3K can also be
induced by direct interaction with Ras90, 91. Akt, also known as protein kinase B (PKB), is an important
target of PI3K and is a crucial mediator for cell survival. Akt can phosphorylate and inactivate
glycogen synthase kinase-3 (GSK-3). The PI3K/Akt/GSK-3 pathway causes stabilization and nuclear
accumulation of cyclin D191. Akt can also induce the activity of mammalian target of rapamycin
11
(mTOR)92. mTOR is a serine/threonine kinase that binds to raptor and mLST8. This complex (mTORC1)
regulates cap-dependent translation, transcription, cell cycle progression, and survival93.
It was shown that PI3K indirectly induces HIF1α expression and phosphorylation under hypoxic
conditions (0.1% oxygen)8, 21, 94 (Fig. 6). The PI3Kpathway can regulate HIF-1α without affecting
VHL, suggesting that there are other pathways
involved in HIF-1α stabilization and expression94.
HIF-1-dependent gene expression was also shown
to be increased by Akt and FRAP, a mTOR
protein95. Moreover, this mTOR was shown to
increase the translation of HIF-1α96. The induction
of HIF-1α expression is thought to be mTORdependent, at least in certain cell types97.
Moreover, another downstream target of Akt,
GSK-3, was shown to stabilize HIF-1α98. GSK-3 can
directly phosphorylate the HIF-1α oxygendependent degradation domain which causes
stabilization. HIF-1-dependent gene expression
was inhibited by the PI3K-pathway inhibitor
PTEN95, 99, showing the importance of this
pathway for HIF-1 function. The PI3K-pathway
Figure 6. The PI3K-pathway in HIF-1 and E2F1 regulation.
The PI3K-pathway can phosphorylate and stabilize HIF-1α.
does not affect HIF-2α expression94. Thus, the
This pathway can also activate E2F1. Akt can also inhibit
PI3K-pathway can increase HIF-1 expression via
E2F1. E2F1 can induce this pathway by stimulating the
two different downstream mediators, mTOR and
upstream adapter protein Gab2.
GSK-3, which achieve this increased expression
via different mechanisms.
PI3K/Akt/GSK-3 pathway can upregulate the expression of E2Fs by promoting cyclin D1 activity91
(Fig. 6). In addition to promoting cell cycle progression, Akt is also known for promoting cell survival
by inhibiting E2F1-induced apoptosis100. E2Fs are able to induce the transcription of an adapter
protein, Gab2, that relays the signal from activated transmembrane receptors to PI3K and Akt101 (Fig.
6). Thus, activated transmembrane receptors likely can transmit more signals due to E2Fs. E2F
activity was also found to be required for Akt phosphorylation, resulting in Akt activation101.
Therefore, a positive feedback loop is established between Akt and E2Fs. Furthermore, activation
of mTOR was shown to be dependent on E2F activity, at least in vitro102. Thus, E2Fs regulate multiple
steps in the PI3K-pathway.
The PI3K-pathway can also regulate the activity of E2Fs. The CKIs p21Cip1 and p27Kip1, are also
phosphorylated by Akt82 (Fig. 6). Phosphorylated p21Cip1 and p27Kip1 translocate out of the nucleus.
The translocated CKIs are subsequently unable to inhibit cyclin/CDK2 complexes, which leads to cell
cycle progression (see also paragraph 2.3). In addition, p27Kip1 is also transcriptionally regulated by
the PI3K pathway82. Moreover, the PI3K pathway also plays a role in regulating the stability of the
p27Kip1 protein82. Thus, the PI3K-pathway also regulates E2F activity through CKI regulation.
In conclusion, the PI3K-pathways positively regulate both HIF-1α and E2F expression under
hypoxia. In addition, Akt can induce the expression of certain CKIs that regulate cyclin/CDK2
complexes. This generates more complexes such as cyclin E/CDK2, which amplifies the feedback
loops between this complex and both HIF-1 and E2F under hypoxic conditions (see also paragraph
2.3). Moreover, there is also a positive feedback loop between the PI3K/Akt/GSK-3 pathway and
E2Fs. The PI3K/Akt/mTOR pathway can also induce the expression of HIF-1 and mTOR activity is
thought to be E2F-dependent. Thus, under hypoxic conditions multiple feedback loops could be
established between the PI3K-pathways, cyclin E/CDK2, HIF-1, and E2Fs. In addition, the PI3Kpathways upregulate the expression of FOXO and NF-κB transcription factors to promote cell
12
survival90, 103. Both FOXO and NF-κB transcription factors are also regulated by HIF-1 and E2F (Fig. 21
and 23).
2.5 HIF regulates E2F indirectly through c-Myc
c-Myc plays a central role in the cellular processes growth, differentiation, metabolism and
apoptosis104. It is overexpressed in many human tumors. c-Myc can activate transcription of its target
genes when it associates with Max (Fig. 7). This complex binds to an E-box consensus sequence,
CACGTG. The c-Myc/Max complex can also recruit Miz1 and thereby repress transcription (Fig. 7).
The c-Myc/Max/Miz1 complex can bind to promoter sequences that lack the E-box sequence.
Figure 7. The role of HIFs and E2Fs in the c-Myc-pathway. HIF-1 and HIF-2 both have opposite effect on c-Myc
function. c-Myc can active and inhibit CKI expression and thereby indirectly control E2F activity. c-Myc can also
directly regulate E2F expression by binding to the promoter of E2F genes and by inducing the miRNAs that inhibit E2F
expression. E2Fs can transcriptionally regulate the expression of c-Myc.
HIF-1 is known to transcriptionally upregulate the expression of the CKIs p21Cip1 and p27Kip1 (Fig.
5). In contrast, the c-Myc/Max/Miz1 complex is able to repress p21Cip1 and p27Kip1 expression by
binding to the promoter sequence of the genes CDKN1A and CDKN1B respectively (Fig. 7). Moreover,
HIF-1 is able to induce the expression of CDKN1A and CDKN1B by displacing the c-Myc/Max/Miz1
complex104, 105. HIF-1 is also able to promote a shift of inductive c-Myc/Max complexes to repressive
Mad1/Max and Mxi1/Max complexes104. In contrast, HIF-2α is not able to displace c-Myc from
CDKN1A or CDKN1B. Moreover, HIF-2α can promote the interaction of c-Myc with its binding
partners and thereby increase the c-Myc/Max/Miz1 complex activity106 (Fig. 7). HIF-2α can therefore
increase the repressive function of the c-Myc/Max/Miz1 complex on the expression of p21Cip1 and
p27Kip1.
E2F1, E2F2 and E2F3a are c-Myc-activated genes and the induction is thought to be direct as they
have E-box consensus sequences in their promoter regions107, 108 (Fig. 7). c-Myc can also induce the
expression of a subset of micro RNAs (miRNAs) from the miR-17-92 cluster that can downregulate
E2F expression. The miRNAs miR-17-5p and miR-20a that are expressed by c-Myc can target E2F
mRNA and downregulate its translation109, 110. Also E2Fs regulate the miR-17-92 cluster and thereby
creating a negative feedback loop110 (Fig. 7). c-Myc can also promote the release of E2Fs from pocket
proteins111. Furthermore, c-Myc appears to be essential for the binding of E2F1 to the promoter
region of E2F regulated genes108. Indeed, the known function of c-Myc in inducing the S-phase and
13
apoptosis is mainly mediated via E2Fs112. E2Fs can also regulate the expression and function of cMyc. E2Fs can bind to the promoter of c-Myc and induce its expression113, 114.
HIF-1 and HIF-2 both have opposite effects on the activity of c-Myc. HIF-1 represses c-Myc
activity, while HIF-2 activates it. E2F1, E2F2 and E2F3a expression is regulated by c-Myc and
therefore these E2Fs are indirectly regulated by HIFs under hypoxia. Furthermore, HIF-1 and c-Myc
also regulate E2F function indirectly by regulating CKI activity (see also paragraph 2.3). In addition,
the E-box sequence in E2F promoter regions is very similar to the HRE sequence. It is therefore
thought that HIF-1 can also bind directly to the promoter of E2Fs containing an E-Box sequence. The
functional activity of E2F1 is also regulated by c-Myc and under hypoxia indirectly by HIFs. c-Myc can
also inhibit E2F translation with miRNAs. Thus, not only c-Myc expression needs to be regulated, also
c-Myc activity needs to be controlled in order to regulate the function of c-Myc. Under hypoxia both
HIF-1 and HIF-2 directly interact with c-Myc and regulate its function. It is possible that hypoxia could
favor E2F expression or repression by tightly regulating c-Myc activity.
2.6 HIF regulates E2F activity indirectly through TGF-β
Transforming growth factor-β (TGF-β) is a superfamily of homodimeric proteins that are involved
in diverse cellular processes, including proliferation, differentiation, migration, angiogenesis, and
apoptosis115. The first member and prototype of this superfamily, TGF-β, has various types expressed
from different genes in various tissues. The TGF-β signal is transduced from plasma membrane to the
nucleus via a heterodimeric serine/threonine kinase receptor. This receptor mediates the
phosphorylation of Smad2 and Smad3, which subsequently form a complex116. This complex
translocates to the nucleus were it associates with Smad4. The Smad2/Smad3/Smad4 complex can
transcriptionally regulate various target genes.
HIF-1 can induce the expression of TGF-β1 in mesenchymal stem cells117. It was shown that HIF-1
can bind to the HRE in the TGF-β1 promoter (Fig. 8). HIF-1 was also shown to induce the expression
of TGF-β2 in HUVECs and TGF-β3 in specific human choriocarcinoma cell lines by binding to their
respective promoters118, 119. TGF-β1 can induce both HIF-1α and HIF-2α expression, at least in certain
cell lines120. This is likely achieved by the inhibition of PHD2 expression by TGF-β1121 (Fig. 8). TGF-β1
was also shown to induce the expression of the HIF-regulated gene VEGF. Moreover, TGF-β1 was
shown to promote VEGF expression via a cooperation of Smad3 and HIF-1α under hypoxic
conditions122 (Fig. 8). However, under normoxic conditions, VEGF expression was induced via a
cooperation of Smad3 and HIF-2α120. This synergistic function of Smad3 and HIF-2α was found to be
inhibited by HIF-1α. It is thought that HIF-1α and HIF-2α compete for the binding to Smad3 on the
VEGF promoter during normoxia. It is however not clear why HIF-1α has an opposite role during
normoxic conditions.
Figure 8. HIF-1 and E2F
are regulated by the
TGF-β-pathway. Smad3,
which is downstream in
the TGF-β-pathway, can
regulate the expression
and activity of HIF-1.
Smad3 can also regulate
E2F-activity together
with Smad4. HIF-1 can
transactivate the
expression of TGF-β
under hypoxia.
14
TGF-β can regulate complex formation between E2Fs and pocket proteins and thereby inhibit the
expression of several target genes123-126. This enables TGF-β to inhibit the cell cycle in several ways.
TGF-β can induce the association of E2F4 or E2F5 with p107, Smad3, Smad4, and a HDAC123 (Fig. 8).
This complex can inhibit the expression of c-Myc, an important regulator of the CKIs p21Cip1 and
p27Kip1 (Fig. 7). Moreover, TGF-β can also induce p15Ink4b and p21Cip1 expression127. By regulating the
expression of p15Ink4b, TGF-β can also influence the function of p27Kip1. Another way TGF-β inhibits
the cell cycle is by inducing the formation of a complex between E2F4, p130, and a HDAC which can
repress the expression of cdc25A, an activator of G1 CDKs124. Inhibition of CDKs by TGF-β leads to
hypophosphorylation of pocket proteins and this subsequently promotes E2F/pocket protein
complex formation. TGF-β can also inhibit telomerase activity by inducing complex formation
between E2F1 and pRB that causes the repression of hTERT125 (see also paragraph 4.6).
Under hypoxic conditions, HIFs can upregulate the expression of TGF-β. In turn, TGF-β can induce
the expression of HIFs and the activity of E2Fs. TGF-β can use both HIFs and E2Fs to regulate target
genes that are involved in important cellular processes. HIFs are known to regulate p21Cip1 and p27Kip1
expression via c-Myc (see also paragraph 2.3). HIFs can also regulate the expression of these CKIs via
transactivation of TGF-β, as TGF-β can directly influence p21Cip1 expression and indirectly p27Kip1
function. Moreover, TGF-β is also able to inhibit c-Myc expression. Repression by TGF-β requires
E2Fs, suggesting an E2F function downstream of HIF. Both HIFs and TGF-β promote the association of
E2Fs with pocket proteins and thereby induce cell cycle arrest (see also paragraph 2.3).
HIFs and E2Fs were shown to cooperate with Smad3, a downstream effector of TGF-β. HIF-1α can
associate with Smad3 and this complex can induce the expression of VEGF. Interestingly, VEGF was
recently also shown to be induced by a complex of HIF-1 and E2F7 or E2F89, 57. It is however unknown
if these atypical E2Fs also associate with a complex of HIF-1α and Smad3. Similarly, E2F4 and E2F5
can associate with Smad3 but it is unknown if these E2Fs can form a complex with HIF-1α. The TGF-βpathway could thus play an important role in the interplay between the HIF and E2F pathways.
2.7 HIF regulates E2F-regulated genes indirectly through Sp1
The specificity protein (Sp) family of transcription factors are known to be involved in regulating
the cell cycle, hormonal activation, apoptosis and angiogenesis129. Sp1 plays a role in regulating genes
involved in cell cycle progression and cell growth. Sp1 is specifically highly expressed in G1 when
compared to the other cell cycle phases and was shown to be involved in the regulation of the
expression of cyclin D1 and E2F1. Sp1 can also associate with c-Myc and thereby regulate specific
promoters104.
HIF-1α and HIF-2α regulate the activity of c-Myc under hypoxia (Fig. 7). Therefore, HIF-1α and HIF2α indirectly also regulate the activity of Sp1. HIF-1α competes with c-Myc for binding to the
transcription factor Sp1104, 130 (Fig. 9). In contrast, HIF-2α can increase the binding affinity of c-Myc to
Max and Sp1 and thereby may favor the binding of Sp1 to c-Myc/Max rather than to HIF-1α104, 106.
Therefore, HIFs seem important for the regulation of Sp1 activity.
15
Figure 9. Regulation of Sp1 by HIF and E2F. HIF-1α competes with c-Myc for the binding to Sp1,
whereas HIF-2α promotes the binding of c-Myc to Sp1. Sp1 can also bind to specific E2Fs and thereby
can regulate specific genes. E2Fs also regulate the expression of Sp1. Sp1 can also bind p107, which
modulates the activity of Sp1.
Sp1 can bind to the N-terminal domain of E2F1, E2F2 and E2F3, and for the activation of some
promoters this interaction seems necessary131-133 (Fig. 9). E2F4 and E2F5 do not have a Sp1-binding
domain in their N-terminus and therefore do not bind Sp1. An important gene for the cell cycle that
is regulated by Sp1 is CCND1. Both E2F1 and Sp1 need to bind to the promoter region of this gene for
repression of cyclin D1 expression72. The transcription of Sp1 was found to be regulated by pRB134.
This regulation is probably mediated by E2Fs, because E2Fs are shown to bind in the promoter region
of the Sp1 gene135 (Fig. 9). E2Fs thereby positively regulate the transcription of Sp1. Also a pocket
protein, p107, was found to directly regulate Sp1 by associating with it (Fig. 9).
In conclusion, during hypoxia, HIF-1α and HIF-2α can indirectly regulate the activity of Sp1. Sp1 is
important for the activation of certain E2F-regulated genes, such as CCND1 that encodes for cyclin
D1. Thus, under hypoxia these genes could be indirectly regulated by HIF-1α and HIF-2α. E2Fs can
also bind and induce the Sp1 promoter, which is also regulated by Sp1 itself135. This feedback loop
could therefore also be regulated under hypoxia.
16
3. Regulation of cell metabolism by HIF and E2F
Hypoxia is known to change cellular metabolism. Cells switch to oxygen-independent metabolism
under hypoxic conditions136. HIF-1 regulates the transcription of various genes involved in glucose
and energy metabolism. Also proliferating cells have increased glycolysis. It is therefore not
surprising that important cell cycle transcription factors such as E2Fs also have a role in regulating
metabolism. E2F1 can inhibit oxidative metabolic gene expression and promote glycolytic gene
expression under stressful conditions137. Thus, HIF and E2F can have similar effects on metabolism
changes and therefore possibly cooperate with each other. These possible collaborations between
the transcription factors are discussed below.
3.1 Regulation of the glycolysis and the TCA cycle under hypoxia
Glycolytic metabolism is used as a primary mechanism for ATP production. Glycolytic enzymes
metabolize glucose into pyruvate (Fig. 10). After glycolysis, pyruvate can be converted into either
acetyl coenzyme A (AcCoA) or lactate. Pyruvate is converted into AcCoA by pyruvate dehydrogenase
(PDH). PDH activity is regulated by pyruvate dehydrogenase kinases (PDKs). PDKs can phosphorylate
and therefore inactivate the catalytic domain of PDH. AcCoA enters the tricarboxylic acid (TCA) cycle
to produce ATP and NADH. The TCA cycle is succeeded by oxidative phosphorylation.
Figure 10. The involvement of
HIF and E2F in regulating
glycolysis. HIF-1 can increase the
amount of glucose that enters
the glycolysis by inducing the
expression of GLUT. HIF-1 also
induces the expression of
glycolytic enzymes and therefore
promotes the glycolysis. HIF-1
and E2F1 also regulate the
expression of glycolytic genes via
c-Myc. Products of the glycolysis
stabilize HIF-1. HIF-1 and E2F1
inhibit the production of AcCoA
and therefore the TCA cycle. HIF1 can also promote the
conversion of pyruvate to lactate
via LDHA.
Hypoxia stimulates anaerobic metabolism by increasing glycolysis via HIF-1. HIF-1 can induce the
expression of glucose transporter 1 (GLUT1) and GLUT3, and therefore increase the glucose flux into
the glycolysis15, 138 (Fig. 10). In addition, HIF-1 can also induce the expression of HK1 and HK2 which
encodes hexokinase, the first enzyme of the glycolysis138. The expression of many glycolytic enzymes
is induced by HIF-1136, 139. In addition, c-Myc is known to promote the expression of glycolytic genes
and HIFs regulate c-Myc function140 (Fig. 7). It is therefore not unlikely that HIFs can also exert their
17
regulatory effect on the expression of glycolytic genes via c-Myc. Moreover, HIF-1 and c-Myc can
cooperate together to induce the expression of certain glycolytic enzymes141. HIF-1 also regulates the
conversion of pyruvate. The PDK1, PDK3 and PDK4 genes have been shown to be induced by HIF-1142144
. It is hitherto unknown if PDK2 is also regulated by HIF-1. Thus, HIF-1 inhibits the production of
AcCoA via PDK upregulation (Fig. 10). AcCoA is important for the TCA cycle and therefore this process
is subsequently also inhibited by HIF-1. Furthermore, HIF-1 promotes the conversion of pyruvate into
lactate by inducing the expression of the enzyme lactate dehydrogenase A (LDHA)15, 138 (Fig. 10).
Pyruvate and lactate can stabilize and therefore promote HIF-1α expression145, 146. Since HIF-1
expression leads to elevated pyruvate and lactate levels, a positive feedback loop is established (Fig.
10). Thus, hypoxia promotes anaerobic metabolism and inhibits aerobic metabolism.
E2F1 is thought to indirectly regulate the expression of genes involved in the glycolysis such as
GLUT1, HK2, PKM2, and LDHA during cell cycle progression147. E2F1 is known to induce the
expression of c-Myc113, 114 (Fig. 7). c-Myc can subsequently induce the expression of these glycolytic
genes147 (Fig. 10). E2F1 is also involved in the conversion of pyruvate. E2F1 regulates the expression
of PDK4148. Moreover, E2F1 was shown to bind directly to the promoter of PDK4. It is still unknown if
other PDKs are also regulated by E2Fs. Regulation of PDK4 expression by E2F1 suggests that this E2F
is indirectly involved in the inhibition of pyruvate conversion into AcCoA and is thereby also involved
in the regulation of the TCA cycle (Fig. 10). Thus, E2Fs also promote anaerobic metabolism and inhibit
aerobic metabolism.
The glycolysis is directly and indirectly via c-Myc regulated by HIFs under hypoxia. Also E2F1 can
indirectly regulate glycolysis via c-Myc. Both HIF-1 and E2F1 can inhibit the TCA cycle by promoting
the expression of PDK4. The glycolysis provides two ATP molecules. In contrast, the TCA cycle and the
subsequent oxidative phosphorylation together provide 36 ATP molecules. Hypoxic cancer cells favor
glycolysis over the TCA cycle136, 149, 150. Hypoxic cells compensate for the inefficiency of the glycolysis
by increasing the flux through the pathway by taking up more glucose151. However, increased
glycolysis is also thought to be a normal response to proliferation and also migrating cells use this
pathway as main energy source152. It is therefore also not surprising that E2Fs are involved in the
regulation of the glycolysis as well. Furthermore, the intermediary metabolites of the glycolytic
pathway provide the precursors for synthesis of glycine, serine, purines, pyrimidines, and
phospholipids4, 153. All of these molecules are essential for cell growth and maintenance of cells under
stress.
3.2 Feedback loops between HIF, E2F and PPAR
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that, upon binding to a
ligand, function as transcription factors154, 155. Three types of PPAR can be found in man and mouse:
PPARα, PPARδ and PPARγ. There are two isoforms of PPARγ which are derived from the same gene
by alternative promoter usage and splicing. PPARs regulate the metabolism of lipids and lipoproteins.
PPARs also regulate the expression of PDK4156. Furthermore, PPARs regulate glucose homeostasis
and influence cellular proliferation, differentiation and apoptosis.
PPARα was shown to suppress HIF-1α activity in cancer cells157 (Fig. 11). In turn, HIF-1α can
suppress the expression of PPARα-activated genes158. This is likely caused by HIF-1-mediated
transcriptional downregulation of PPARα159 (Fig. 11). PPARα regulates genes that are involved in fatty
acid β-oxidation, a process that generates AcCoA from fatty acids. This process is therefore also
inhibited by HIFs. PPARα, but not PPARγ, was found to form a complex with HIF-2α in cancer cells160.
The PPARα/HIF-2α complex was found to bind to HREs and regulate gene expression (Fig. 11).
18
Figure 11. HIF and E2F are involved in PPAR regulation. HIF-2α can bind to PPARα, and this complex
can regulate hypoxia-responsive genes by binding to the HRE in promoter regions. HIF-1 can
downregulate PPARα and PPARα can inhibit HIF-1 activity. PPARγ is thought to be required for HIF-1regulated gene expression in adipocytes. PPARγ can also negatively regulate HIF-1 and E2Fs. PPARγ is a
HIF-1 and E2F-regulated gene and certain functions of PPARγ require HIF-1. PPARγ can form a complex
with pRB and HDAC3 to repress PPARγ-regulated genes.
PPARγ plays a role in adipogenesis, the differentiation of preadipocytes into adipocytes.
Adipocytes are fat cells that are important for storing energy. In adipocytes, PPARγ is thought to be
required for the activation of multiple HIF-1α target genes161. However, PPARγ can also have a
negative influence on HIF-1α expression and function162, 163 (Fig. 11). In turn, HIF-1α is known to
cause increased PPARγ expression164. PPARγ was found to be a direct transcriptional target of HIF-1
(Fig. 11). Genes involved in lipid anabolism are regulated by PPARγ and this regulation was shown to
be HIF-1-dependent164. In contrast, HIF-1 is also found to be required for the inhibition of PPARγ2
expression165. Thus, HIF-1 could have a differential role in the regulation of PPARγ, depending on the
type of PPARγ.
PPARγ is able to indirectly inhibit E2F expression166 (Fig. 11). The expression of PPARγ is in turn
regulated by E2Fs167. E2Fs were found to bind to an E2F-binding site in the promoter of PPARγ1. The
expression of PPARγ was found to be dependent on E2F activity (Fig. 11). E2F1 and E2F3 are able to
induce the expression of PPARγ in early adipogenesis, while E2F4 can inhibit the expression at the
terminal stage. This reflects the importance of E2Fs in regulating adipogenesis through the activation
of PPARγ. Moreover, hypophosphorylated pRB was shown to bind to PPARγ and inhibit expression by
recruiting a HDAC168 (Fig. 11). The PPARγ/pRB/HDAC3 complex was able to inhibit adipogenesis.
PPARγ can regulate the expression of both HIF-1 and E2Fs, and these transcription factors can in
turn regulate the expression of PPARγ. Under hypoxic conditions, HIF-1 can upregulate the
expression of PPARγ. PPARγ can subsequently repress the expression of HIF-1α and E2Fs. This
establishes two negative feedback loops because E2Fs can also induce the expression of PPARγ. Thus,
HIF-1 and E2Fs can indirectly regulate their expression via PPARγ. The expression of PPARγ is
therefore tightly regulated under hypoxia. This is not surprising since PPARs are involved in
important cellular metabolic processes such as lipid and lipoprotein metabolism and glucose
homeostasis. By repressing PPARα-activity, HIF-1 can inhibit the synthesis of AcCoA as well and
therefore inhibit oxidative metabolism. HIF-1 and E2F can also do this via PDK (Fig. 10). In addition,
19
PPARs play a role in proliferation, differentiation and apoptosis. Important transcription factors such
as PPARs therefore need to be controlled under stress conditions.
3.3 HIF and E2F regulate COX activity
During oxidative phosphorylation, electrons are transferred through different cytochromes. This
establishes a proton gradient in the mitochondrial membrane that can be used to generate ATP. The
final electron acceptor of this process is oxygen and this subsequently results in the production of
water. This last step is catalyzed by cytochrome c oxidase (COX). COX is a dimer in which each
monomer consists of 13 subunits169. In contrast to the other subunits, subunits I, II and III are
encoded by the mitochondrial genome and form the catalytic core of the enzyme. Subunit IV is
thought to have an important regulatory function of COX activity170.
HIFs can regulate the expression of COX subunit IV (COX4).
The expression of two paralogue genes that encode alternative
isoforms of COX4, COX4/1 and COX4/2, are regulated by HIFs171
(Fig. 12). The gene LON encodes for a mitochondrial protease
that degrades isoform COX4-1. HIF-1 was shown to induce the
expression of this protease and thereby negatively regulates
COX4-1 expression171 (Fig. 12). Two potential HREs were found in
the COX4/2 gene171. Expression of COX4-2, but not COX4-1, was
shown to be upregulated during hypoxia171. This suggests that
COX4-2 is directly transcriptionally regulated by HIF-1.
The promoter of COX4/1 was shown to have several potential
E2F-binding sites172. At least one of these E2F-binding sites was
Figure 12. Regulation of COX by
HIF and E2F. E2Fs can
suggested to be important for COX4-1 expression. This suggests
transcriptionally regulate COX4-1
that E2Fs can regulate COX enzyme activity by regulating COX4-1
expression. HIF-1 can regulate the
expression (Fig. 12). E2F was also shown to bind to the promoter
expression of COX4-1 via Lon. HIFof the gene that encodes for COX subunit VIIc173.
1 can also transcriptionally
Both HIF-1 and E2Fs are shown to be important for the
regulate the expression of COX4-2.
expression of COX4 isoforms. This subunit is thought to be an
important regulator of COX enzymatic activity170. Therefore, both HIF-1 and E2Fs can regulate the
important last step of the oxidative phosphorylation by regulating COX activity via different isoforms
of COX4.
20
4. Crosstalk between DNA repair, HIF and E2F pathways
Hypoxia can cause increased mutagenesis and can impair DNA repair. This is mainly caused by the
inhibition of various important proteins from different DNA repair systems by HIFs. Hypoxia
influences the two pathways that are mainly involved in the repair of DNA double strand breaks
(DSBs): homologous recombination and non-homologous end joining (NHEJ). In addition, hypoxia is
also involved in base excision repair (BER) and mismatch repair (MMR) A number of E2F family
members are known to be responsive to DNA damage. Especially the involvement of E2F1 in DNA
repair has been well characterized. E2F1 plays a role in the same DNA repair pathways that are
regulated by HIF. The relation between HIF and E2Fs in these pathways are discussed below.
4.1 HIF-1α and E2F colocalize with BRCA1
An important regulator in genome integrity is BRCA1. BRCA1 is involved in DNA DSB repair via HR
and is essential in cell cycle checkpoint control174. BRCA1 harbors tandem BRCA1 carboxyl-terminal
(BRCT) domains that binds phosphorylated proteins. BRCT domains are present in many proteins that
are involved in DNA damage response. BRCA1 localizes at DNA breaks and can form several different
complexes with distinct and overlapping functions in DNA repair and cell cycle checkpoint regulation.
BRCA1 can form a complex with HIF-1α under hypoxic and normoxic conditions175 (Fig. 13).
Moreover, under hypoxic conditions (0.1% oxygen) BRCA1 is necessary for HIF-1α stability175.
However, in BRCA1 germline mutation related breast cancer, functional HIF-1α overexpression is
seen under hypoxia176. This suggests that the stabilizing function of BRCA1 is more complex. HIF-1α is
also able to indirectly inhibit BRCA1 by functionally antagonizing c-Myc105 (Fig. 13). The stabilization
of HIF-1α by BRCA1 and the inhibitory function of HIF-1α effectively creates a negative feedback
loop. One reported function of the association of HIF-1α with BRCA1 is the induction of the VEGF
promoter175.
21
Figure 13. Regulation of BRCA1 function and expression by HIF and E2F. HIF-1α can directly
associate with BRCA1 and regulate the expression of VEGF. This complex could possibly also
regulate other hypoxia-responsive genes. E2F1 can bind to BRCA1 through TopBP1. BRCA1 can
therefore recruit E2F1 and possibly also HIF-1α to DNA DSBs. HIF-1α and E2F1 can regulate BRCA1
expression via c-Myc. E2F1 can also directly regulate BRCA1 expression in cooperation with
MCPH1. E2F4 can inhibit BRCA1 expression.
E2Fs are involved in the regulation of BRCA1 expression177. BRCA1 is a c-Myc regulated gene, and
c-Myc activity is regulated by E2Fs (Fig. 7). It is therefore possible that E2Fs indirectly regulate BRCA1
via c-Myc. E2Fs can also bind to two conserved binding sites in the BRCA1 proximal promoter
region178, 179 (Fig. 13). Hypoxic stress can favor a higher occupancy of the promoter by certain
members of the E2F family, suggesting different regulatory roles for the different E2Fs178. Moreover,
BRCA1 expression is repressed under hypoxia caused by E2Fs binding to the BRCA1 promoter
region178. E2F4 and p107/p130 complexes predominantly bind the BRCA1 promoter and inhibit its
activity179. The E2F4/p130 complex is known to bind a conserved E2F site in the proximal promoter of
BRCA1 (Fig. 13). This conserved binding site is also present in the promoter of the homologous repair
gene RAD51 which is also shown to be regulated by the E2F4/p130 complex180.
In addition to regulating the BRCA1 promoter, E2F1 also colocalizes with BRCA1 in which the
protein TopBP1 plays an important role (Fig. 13). TopBP1 can bind to E2F1 and thereby inhibit several
activities of this transcription factor181, 182. TopBP1 also contains several BRCT motifs and therefore
can associate with BRCA1 at stalled replication forks. Thus, TopBP1 likely inhibits several E2F1
functions by recruiting E2F1 to a BRCA1-containing repair complex (Fig. 13). This also suggests that
E2F1 plays a role in DNA repair in association with BRCA1. Indeed, E2F1 can promote the recruitment
of DNA repair factors to DNA DSBs183. Another protein with BRCT motifs is MCPH1. This protein binds
together with E2F1 to the BRCA1 promoter184 (Fig. 13). However, it is not yet known if MCPH1 is also
part of the BRCA1-containing repair complex.
In conclusion, both HIF-1α and E2F1 colocalize with BRCA1 in a complex. However, it is hitherto
unknown if HIF-1α and E2F1 associate to the same complex with BRCA1. It is also possible that HIF1α and E2F1 could compete in binding to BRCA1. Nonetheless, this implies that both HIF-1α and E2F1
influence the function of BRCA1. E2Fs regulate the expression of BRCA1 and BRCA1 can regulate the
stabilization of HIF-1α, therefore E2Fs could be indirectly involved in the stabilization of HIF-1α. E2Fs
were shown to repress BRCA1 expression under hypoxia. However, downregulation of the expression
of BRCA1 by E2F in response to hypoxia is not dependent on HIF-1178, 180. BRCA1 expression is also
regulated by c-Myc, and c-Myc activity is regulated by HIFs and E2Fs (Fig. 7). Thus, HIF and E2F are
likely also indirectly involved in the regulation of BRCA1 expression.
4.2 ATM stabilizes HIF and E2F
The primary response to repair DNA damage is carried out by kinases of the phosphatidylinositol3-OH kinase-related family, which includes ataxia teleangiectasia mutated kinase (ATM), ATM- and
Rad3-related kinase (ATR), and DNA-dependent protein kinase (DNA-PK)185. ATM and DNA-PK in
general respond to DSBs in DNA, while ATR mediates the repair of single-stranded DNA185. ATM and
ATR phosphorylate and activate two checkpoint kinases, CHK1 and CHK2, which are also critical in
transferring the DNA damage signal to effector molecules186, 187 (Fig. 14).
22
Figure 14. ATM regulates HIF and E2F function. ATM can phosphorylate both HIF-1α and E2F1 on specific sites
and thereby stabilize these transcription factors. HIF-1α can repress mTORC1 when it is phosphorylated on this
specific site. HIF-1α can also cause the phosphorylation of ATM. ATR and CHK2 can also phosphorylate E2F1.
When phosphorylated on a specific site, E2F1 can associate with BRCA1 and induce DSB repair. E2F1 can also bind
to 14-3-3τ, which leads to apoptosis.
ATM expression is essential for HIF-1α activity188. ATM is shown to phosphorylate HIF-1α on a
serine-696 site which stabilizes this transcription factor188 (Fig. 14). Under hypoxic conditions,
phosphorylated HIF-1α downregulates mTORC1 which is important for cell survival188 (Fig. 14). This
possibly leads to hypoxia-mediated cell death. HIF-1α is also able to influence the ATM pathway as is
evident by the reduced phosphorylation of ATM in the absence of HIF-1α189 (Fig. 14). However, the
precise mechanism behind this phenomenon is hitherto unknown. Thus, by activating ATM, HIF-1α is
also involved in promoting DSB repair.
ATM and ATR have been shown to phosphorylate E2F1 on a site not conserved in other E2F family
members48 (Fig. 14). E2F1 associates with the 14-3-3τ protein specifically when it is phosphorylated
on this unique serine-31 site. The binding of the 14-3-3τ protein prevents ubiquitination and thus
proteasomal degradation of E2F1190. By contrast, E2F1 is instable when 14-3-3τ is absent and
apoptosis is therefore inhibited in response to DNA damage. Phosphorylation of E2F1 at the serine31 site also creates a TopBP1 binding site, which recruits E2F1 to BRCA1181 (Fig. 14) (see also
paragraph 4.1). Also a downstream mediator of ATM, CHK2, is able to phosphorylate and thereby
stabilize E2F1191 (Fig. 14). Phosphorylation of E2F1 by ATM and ATR can thus promote both DSB
repair via BRCA1 and apoptosis.
ATM is important for the stabilization of both HIF-1α and E2F1. This response towards DNA
damage is likely initiated when the damage cannot be repaired by the DNA repair mechanisms.
Stable HIF-1α downregulates mTORC1 which is needed for cell cycle progression and cell survival. In
addition, stable E2F1 can promote apoptosis192, 193. ATM can however also repair DNA DSB and can
also stimulate E2F1 to do so via BRCA1. In addition, HIF-1α can also promote the phosphorylation of
ATM and is therefore also indirectly involved in activating the DSB repair pathway.
23
4.3 PARP-1 induces HIF and E2F-responsive gene expression
Poly(ADP-ribose) polymerase 1 (PARP-1) functions as a sensor and signal transducer of DNA
damage194. PARP-1 plays a central role in BER, as it recruits repair effectors to DNA lesions. PARP-1
activation leads to modification of chromatin and subsequent loosening of its structure. In response
to BER, PARP-1 also recruits important factors that function in BER such as XRCC1, DNA ligase III and
DNA polymerase β. PARP-1 also associates with DNA polymerase β and thus is also directly involved
in BER.
PARP-1 was shown to form a complex with HIF-1α by directly binding to it195 (Fig. 16). This
association did not influence the binding of HIF-1 to HREs. Furthermore, PARP-1 was shown to
function as a coactivator in HIF-1-dependent transcriptional regulation (Fig. 16). PARP-1 also
regulates the stabilization of HIF-1α by controlling oxidative stress and nitric oxide production196.
Therefore, PARP-1 can be considered as a regulator of HIF-1 function.
Figure 16. PARP-1 regulates HIF and E2F-dependent expression. PARP-1 can associate with both
HIF-1 and E2F1 and functions as a coactivator. PARP-1 can also stabilize HIF-1α. Repression of
certain genes by PARP-1 requires E2F4/p130.
PARP-1 plays a role in the regulation of E2F expression. It was shown that PARP-1 was not binding
to the promoter of E2F1, but directly to E2F1197 (Fig. 16). This coincided with increased E2F1
promoter activity and upregulation of E2F1-regulated genes. E2F1 is known to be able to bind to its
own promoter and PARP-1 was shown to act as a cofactor to promote this interaction, which also
leads to induced expression of E2F1-regulated genes (Fig. 16). Inhibition of PARP-1 also suppresses
the expression of BRCA1 and RAD51. This suppression mediated by PARP-1-inhibition was found to
be caused by E2F4/p130198 (Fig. 16). The binding of PARP-1 to E2F1 was found to increase upon
PARP-1-inhibition. Moreover, inhibition of PARP-1 decreases the interaction of E2F1 to the promoter
of E2F1-regulated genes198. These promoters were instead found to be occupied by E2F4/p130. Thus,
PARP-1 can regulate the function of E2Fs and thereby can also regulate the expression of E2F target
genes.
In conclusion, under hypoxia and in the presence of DNA damage, PARP-1 functions as a cofactor
of both HIF-1 and E2F1-responsive genes. PARP-1 thereby directly binds to either HIF-1 or E2F1. It is
not known if PARP-1 can bind to HIF-1 and E2F1 simultaneously. PARP-1 seems also to be involved in
DNA DSB repair by regulating BRCA1 and RAD51. This is regulated by E2Fs, thus E2Fs can regulate
BRCA1 either directly or indirectly through PARP-1 (see also paragraph 4.1). PARP-1 is however not
only involved in regulation of gene expression. PARP-1 also localizes at damaged DNA sites and
recruits factors that are involved in BER. In addition, PARP-1 is also directly involved in BER by
24
associating with DNA polymerase β. The direct binding of PARP-1 to HIF-1 or E2F1 suggests that these
transcription factors could also play a role in regulating PARP-1 activity in BER.
4.4 HIF and E2F regulate mismatch repair
The DNA MMR system is essential for the safeguarding of genomic integrity. The mammalian
proteins of the MMR system include MLH1, PMS2, MSH2, MSH3, and MSH6199. These MMR proteins
form the functional heterodimers MSH2/MSH6 (MutSα), MSH2/MSH3 (MutSβ), and MLH1/PMS2
(MutLα). The MutSα heterodimer recognizes both base-base mismatches and insertion and deletion
mismatches, whereas MutSβ recognizes only insertion and deletion mismatches. The heterodimers
MutSα and MutLα can form a complex that is thought to play a role in the initiation of the MMR
system.
HIF-1α functions as a transcriptional repressor of MSH2 and MSH6 under hypoxic conditions130.
However, HIF-1α likely does not bind directly to the promoter regions of these genes, as these
promoters lack HREs. The transcription factor Sp1 was found to associate with c-Myc (Fig. 9) and this
complex binds to the promoter of MSH2 (Fig. 17). c-Myc is a positive regulator of MSH2 expression
under normoxic conditions. HIF-1α was found to displace c-Myc from Sp1 under hypoxic conditions130
(Fig. 9). This suggests that HIF-1α, in complex with Sp1, can function as a negative regulator of MSH2
expression by binding to its promoter under hypoxic conditions (Fig. 17). The tumor suppressor
protein p53 was found to be essential for this repression130. Furthermore, HIF-1 can also indirectly
downregulate the expression of MLH1200.
25
Figure 17. Regulation of the mismatch repair pathway by HIF and E2F. HIF-1α and E2Fs can regulate the
expression of MSH2 and MSH6 through Sp1 and c-Myc. E2Fs can also directly regulate the expression of
MSH2, MSH6, and to a lesser extent, PMS2. HIF-1 can indirectly regulate the expression of MLH1. E2F1
and E2F4 were found to bind to the promoter of MSH2 and MLH1, suggesting that these E2Fs are
involved in the regulation.
E2F1 and E2F3 were shown to be able to regulate the expression of MSH2, MSH6 and PMS2201
(Fig. 17). Especially MSH2 and MSH6 were shown to be strongly induced by these E2Fs. Two E2Fbinding sites were found in the MSH2 promoter and the promoter was shown to be regulated by at
least E2F1. Furthermore, E2F1 and E2F4 were also shown to bind to the promoter of MLH1 and
MSH2173 (Fig. 17). These findings show that all the functional heterodimers in the DNA MMR system
are subjected to regulation by different E2Fs.
Both HIF-1α and E2Fs can regulate the expression of different MMR proteins that form the
functional heterodimers MutSα, MutSβ and MutLα. Although thus far E2Fs were found to be able to
induce the expression of many of these proteins, E2Fs in combination with pocket proteins are also
implicated to function in the repression of these proteins. In contrast, HIF-1α mainly represses the
transcription of the different subunits of the heterodimers under hypoxia. This implies that under
hypoxic conditions the MMR system is mainly repressed.
4.5 HIF and E2F regulate hTERT
Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, an
enzyme complex that synthesizes telomere repeats202. The telomerase ribonucleoprotein enzyme
complex also contains human telomerase RNA component (hTR) in its catalytic core.
HIF-1 can enhance the expression of hTERT by transactivation of the hTERT promoter203, 204 (Fig.
18). The hTERT promoter was found to possess two HREs to which HIF-1 can bind. Also hTR is
regulated in this manner205 (Fig. 18). HIF-1α can however also inhibit hTERT expression by
antagonizing c-Myc105. In addition, HIF-1α was also shown to regulate hTERT expression posttranscriptional by regulating alternative splicing (Fig. 18). HIF-1α was shown to change the ratio of
hTERT spliced transcripts in favor of the active variant205. However, the mechanism and function of
hTERT alternative splicing and the role of HIF-1 herein is currently unknown. Thus, HIF-1 can regulate
the expression of the telomerase complex in various ways.
Figure 18. Regulation of
telomerase by HIF and E2F. HIF-1
can transactivate the expression of
hTERT and hTR. E2Fs can induce or
repress the expression of hTERT.
HIF-1α and E2Fs can also regulate
hTERT expression through c-Myc.
E2F1 can inhibit hTERT expression
with Sp1. HIF-1 can affect hTERT
alternative splicing in favor for
active hTERT.
E2F1 was also shown to regulate the expression of hTERT206. Two putative E2F-binding sites were
found in the promoter region of hTERT. E2F1 was shown to negatively regulate the expression of
26
In conclusion, both HIF-1 and E2Fs can regulate the expression of hTERT and thereby also regulate
the function of telomerase. Although it seems that E2Fs can both stimulate and repress hTERT
expression, under hypoxic conditions HIF-1 mainly enhances the expression of hTERT. It therefore
seems that active telomerase is important during hypoxia. Moreover, hTERT is a c-Myc and Sp1regulated gene. c-Myc and Sp1 activity is regulated by both HIFs and E2Fs (see also paragraph 2.5 and
2.7). Furthermore, p21Cip1 has been shown to regulate hTERT209, and thus is regulated by the HIF/cMyc/p21Cip1/E2F-pathway (Fig. 7). This suggests that the regulation of hTERT is more complex under
hypoxic conditions.
27
5. Regulation of cell death by HIF and E2F
Hypoxia is known to induce apoptosis via HIF-1α. However, HIF-1α also has an important role as
suppressor of apoptosis. It is therefore not surprising that HIF-1α regulates a subset of both proapoptotic factors as well as anti-apoptotic factors. Hypoxia also influences autophagy pathways via
HIF-1α. This can lead to another form of programmed cell death, autophagic cell death. E2Fs also
have important roles in the regulation of apoptosis. Especially E2F1 is known for its function in
different pathways that lead to apoptosis. HIF and E2F are therefore involved in many of the same
pathways that regulate programmed cell death. These pathways are discussed below.
5.1 HIF and E2F regulate p53
A key regulator in the DNA damage response is p53210-213. The protein levels of p53 are generally
kept low owing to its ubiquitination by the E3-like ubiquitin ligase MDM2, leading to subsequent
proteasomal degradation. Various stresses can inhibit p53 degradation or can modify p53 in order to
stabilize and activate the protein. Stabilization and activation of p53 leads to cell cycle arrest or
apoptosis. Depending on the cellular context, p53 can also have anti-apoptotic functions214.
Hypoxia is known to regulate the p53 pathway, either in a HIF-dependent or independent
manner215. HIF-1 is thought to stabilize p53 under severe hypoxic conditions (<0.2% oxygen) via the
factor Nucleophosmin (NPM)216, 217. Increased protein level of p53 is often associated with the
induction of apoptosis217. However, HIF-1-mediated upregulation of NPM decreases hypoxia-induced
apoptosis. Induction of p53 by hypoxia is not always observed, and seems to be dependent on the
severity and duration of hypoxia and the cellular context215. HIF-1α can also functionally antagonize
p53, depending on the cellular context215, 218, 219 (Fig. 19). Hypoxia is thought to increase p53
degradation by upregulating MDM2 expression220 (Fig. 19). In addition, HIF-1 is thought to compete
with p53 for the binding to the coactivators p300/CBP, which affects the expression of p53-regulated
genes221. HIF-2α is also suggested to play a role in p53 regulation. HIF-2α likely also promotes cell
survival by repressing p53 function222 (Fig. 19). Thus, HIFs generally inhibit p53 function, especially
with regard to apoptosis.
Figure 19. Regulation of p53mediated apoptosis by hypoxia
and E2Fs. Hypoxia can regulate
p53 either through HIF or via
HIF-independent pathways. The
mode in which hypoxia regulates
p53 is dependent on the severity
and the length of this condition.
E2F1 generally promotes p53
expression, but can also
indirectly inhibit p53. E2F7 and
E2F8 can inhibit p53-mediated
apoptosis. p53 generally inhibits
HIF and E2F1 activity.
p53 can also regulate the expression of HIF-1, by promoting ubiquitination and proteasomal
degradation of HIF-1α223, 224 (Fig. 19). In addition, p53 can also modulate the expression of HIF-1β225,
226
. p53 was shown to upregulate the expression of the miRNA miR-107 that suppresses HIF-1β
expression225. Also HIF-1 activity can be repressed by p53. The competition of HIF-1 with p53 for the
coactivators p300/CBP also negatively influences the expression of HIF-1-regulated genes221, 227. HIF28
1α can also form a ternary complex with p53 and MDM2223. This complex is thought to promote the
degradation of HIF-1α. p53 therefore generally inhibits HIF-1 activity (Fig. 19). Moreover, p53 can
also inhibit the proapoptotic factor BNIP3 and thereby inhibit hypoxia-induced cell death228 (see also
paragraph 5.4).
E2F1 can also regulate the expression of p53. E2F1 can promote the phosphorylation of p53229.
E2F1 is thought to induce p53 phosphorylation through ATM230. Moreover, E2F1 induces p53mediated apoptosis via CHK2, a downstream mediator of ATM. In fact, E2F1 can also upregulate the
expression of the Chk2 gene231. E2F1 activation in response to DNA damage also leads to the
increased expression of ARF232. ARF can modulate the activity of MDM2, and therefore inhibit the
proteasomal degradation of p53. Thus, E2F1 is thought to inhibit MDM2-mediated p53 degradation
by phosphorylation via ARF233 (Fig. 19). However, the induction of ARF was found not to be necessary
for E2F1-mediated apoptosis via p53229. In the presence of p53, ARF can also associate with E2F1 to
inhibit its transcriptional activity and promote its proteasomal degradation234, 235 (Fig. 19). This
provides a functional feedback loop. E2F1 can also transactivate the miRNA miR-449, which can
activate p53236. E2F1, E2F2 and E2F3 can also directly bind to p53 and influence its activity237.
Phosphorylation of p53 at a specific site allows for the binding of E2F1 and retains p53 in the
nucleus238. This promotes p53 DNA binding and transactivation of p53-regulated genes. Several E2Fregulated genes are also involved in the regulation of p53 activity, such as PIN1, SIRT1, and SKP2237.
PIN1 can induce the pro-apoptotic function of p53. In contrast, SIRT1 and SKP2 are thought to inhibit
p53 activity. Thus, E2F1 generally promotes p53 activity, which can lead to p53-mediated apoptosis
(Fig. 19).
E2F1-activated p53 leads specifically to apoptosis rather than cell cycle arrest237. This bias in p53
function was attributed to specific E2F1-regulated genes. In addition to stabilizing and activating p53,
E2F1 can also directly regulate various genes that promote the pro-apoptotic function of p53 (Fig.
19). The p53 cofactors ASPP1 and ASPP2 specifically induce p53-mediated apoptosis and are
upregulated by E2F1239. Although E2F1 is generally thought to promote apoptosis via p53, E2F1 can
also cooperate with p53 to induce apoptosis237. E2F1 and p53 regulate an overlapping set of proapoptotic genes and both also regulate distinct genes that are involved in apoptosis. E2F7 and E2F8
were shown to be involved in the repression of E2F1 and p53-mediated apoptosis46 (Fig. 19).
p53 also regulates the expression of E2Fs in more ways than via ARF. p53 can induce the
expression of miR-34. This miRNA and miR-449 can inhibit the E2F pathway in a negative feedback
loop236. Expression of the CKI p21Cip1 is also induced by p53209, 240. This CKI indirectly prevents the
release of E2Fs from pRB (Fig. 5). p53 can therefore also regulate hTERT209 (see also paragraph 4.6).
After DNA damage, p53 can also induce the expression of BTG3. This p53-regulated gene can block
E2F1 DNA-binding activity241. p53 therefore generally inhibits E2F activity, providing a feedback loop
(Fig. 19).
In conclusion, both HIF and E2F can regulate the activity of p53 and thereby p53-mediated
apoptosis. Hypoxia generally represses p53 activity, especially with regard to apoptosis. HIF-1 can
stabilize p53 but inhibit its apoptotic function. Moreover, stabilized p53 generally inhibits E2F
activity. It is therefore likely that HIF-1 can inhibit E2F1-mediated cell death via p53. E2F1 generally
promotes p53 activity, which specifically leads to apoptosis. However, p53 specifically inhibits
hypoxia-mediated cell death. p53 generally inhibits HIF-1 and E2F activity and thus provides a
negative feedback loops. HIF-1 can therefore indirectly regulate E2F1 through p53 and vice versa.
However, during hypoxia it is likely that excess of HIF-1 can control the p53-pathway and thereby
inhibit apoptosis.
5.2 HIF regulates E2F indirectly through DP4
DP4, also known as transcription factor dimerization partner 3 (TFDP3), is functionally distinct
from DP1 and DP2/3242, 243. In contrast to the other DP family members, DP4 inhibits E2F-mediated
transcriptional activity and therefore cell cycle progression. The main reason for this is that E2F/DP4
heterodimers are inhibited in binding to DNA (Fig. 20). The exact mechanism of the inhibition of
29
E2F/DP4 DNA binding is unknown. It is thought that some amino acid differences in the DNA binding
domain of DP4 are the reason for this242, 243.
HIF-2α was shown to transcriptionally repress
the expression of DP4244 (Fig. 20). This repression
was shown to induce apoptosis in hepatocellular
carcinoma. In contrast, HIF-1α did not seem to be
able to regulate the expression of DP4. This
suggests that, at least in hepatocellular
carcinoma, hypoxia can induce E2F1-mediated
apoptosis via HIF-2α.
Association of DP4 with E2F1 can inhibit the
function of E2F1 in promoting p53-mediated
Figure 20. HIF regulates E2F activity through DP4.
apoptosis245 (Fig. 19). This is caused by the ability
HIF-2α can inhibit the expression of DP4. DP4
of DP4 to inhibit the transcriptional activity of
dimerizes with E2F1 and can thereby inhibit the DNAbinding capability of this E2F.
E2F1 (Fig. 20). Reduced transcriptional activity of
E2F1 leads subsequently to the diminished
accumulation of p53 and therefore reduces p53-mediated apoptosis.
HIF-2α was shown to promote the transcriptional activity of E2F1 by repressing the expression of
DP4 in hepatocellular carcinoma244. In this cellular context, hypoxia seems to be important for the
regulation of E2F1. Moreover, this regulation can lead to hypoxia-mediated apoptosis.
5.3 Feedback loops between HIF, E2F and FOXO
Forkhead box O (FOXO) transcription factors are involved in the regulation of cell-cycle
progression, stress resistance, metabolism, and apoptosis246. FOXO transcription factors have an
approximate 100-amino acid Forkhead domain, which is a characteristic DNA-binding motif. The
FOXO family of transcription factors contains the four members FOXO1, FOXO3a, FOXO4, and FOXO6.
Under hypoxic stress FOXO3a expression is
upregulated247. The FOXO3a promoter region was found
to possess nine HREs, which suggests that the gene is
regulated by HIFs (Fig. 21). Furthermore, FOXO3a was
shown to induce the expression of CITED2, a protein that
competes with HIF-1 for the binding to p300/CBP247, 248
(Fig. 21). Thus, by inducing CITED2 expression, FOXO3a
was shown to indirectly inhibit HIF-1-induced apoptosis247.
Furthermore, FOXO3a can inhibit reactive oxygen species
(ROS) production caused by hypoxia249. ROS production by
hypoxia is required for the stabilization of HIF-1α. Thus,
FOXO3a indirectly inhibits HIF-1α expression. FOXO3a was
also shown to directly form a complex with HIF-1α and
p300, and directly inhibit HIF-1 transcriptional activity250.
E2F1 was found to regulate the expression of FOXO1
and FOXO3a251 (Fig. 21). Both FOXO1 and FOXO3a contain
conserved E2F-binding domains in their promoters and
Figure 21. FOXO regulates HIF and E2F and vice
E2F1 was shown to bind to the promoter of FOXO genes.
versa. HIF-1 and E2F1 induce the expression of
In addition, E2F1 was found to induce the expression of
FOXO3a. E2F1 can also induce the expression of
these FOXO genes. FOXO genes can transcriptionally
FOXO1. FOXO3a can inhibit the expression of
activate p27Kip1 (Fig. 21), a CKI that indirectly represses
HIF-1 and E2F1. Also FOXO1 can inhibit the
expression of E2F1.
E2F activity252, 253 (Fig. 5). These FOXO transcription factors
Kip1
can therefore regulate the cell cycle via p27 . E2F1 can
also use the HATS p300/CBP, and therefore likely also competes with CITED2 for the binding. This
suggests that E2F1 and FOXO transcription factors are involved in a negative feedback loop.
30
Hypoxia is shown to induce FOXO3a expression. In turn, FOXO3a negatively regulates HIF-1
function and expression and this was at least demonstrated for HIF-1-induced apoptosis. Also E2F1 is
involved in a negative feedback loop with FOXO transcription factors. Thus, this suggests that E2F1 is
indirectly involved in the regulation of HIF-1 function and vice versa. When FOXO3a expression is
induced under hypoxia, both HIF-1 and E2F function are repressed. FOXO3a expression is at least
upregulated by E2F1 and possibly also by HIF. This suggests that FOXO3a ultimately represses its own
expression by repressing HIF-1 and E2F activity. Induction of FOXO3a can also lead to the expression
of BNIP3 and BNIP3L254. This could lead to cell survival by autophagy or autophagic cell death (see
also paragraph 5.4).
5.4 HIF and E2F regulate BH3-only Bcl-2 family proteins
Bcl-2 homology 3 (BH3)-only proteins are pro-apoptotic factors255. BH3-only proteins are a
subfamily of the Bcl-2 family of proteins and induce apoptosis in response to diverse stimuli. The
BH3-only members are BNIP3, BNIP3L, Beclin 1, NOXA, PUMA, SPIKE, BMF, HRK, BIM, BIK, BID and
BAD. Some of these proteins mediate apoptosis via the p53 pathway, while others mediate apoptosis
in a p53-independent fashion.
HIF regulates the expression of the BH3-only members BNIP3, BNIP3L, NOXA, PUMA, BMF, HRK,
BIM, and BID256-262 (Fig. 22). It is not yet known if SPIKE is also regulated by HIF. Beclin 1 and BIK
expression is upregulated during hypoxia263, 264, but it is not yet known if HIF is directly involved in the
regulation. Although BAD is downregulated under hypoxic conditions, HIFs do not seem to be
required for its downregulation262 (Fig. 22). The promoters of BNIP3 and BNIP3L contain HREs and
were shown to be upregulated by HIF-1α256, 257. Also NOXA has a HRE in its promoter region and is
upregulated by HIF-1, which leads to hypoxic cell death258. The expression of PUMA was also shown
to be upregulated by HIF-1α259. It is thought that if HIF-1α is more expressed relative to HIF-1β, HIF1α can bind to p53. This association with HIF-1α stabilizes p53 (see also paragraph 5.1), leading to
BH3-only members upregulation that are dependent on p53, such as NOXA and PUMA259. Hypoxia
also downregulates some BH3-only members. BMF and BIM expression was shown to be inhibited by
HIF-1260. HIF-2 downregulates HRK expression261, however if HIF-1 also regulates HRK remains to be
determined. BID is also downregulated by HIF-1262. Thus, about half of the BH3-only members are
upregulated by HIF and half are downregulated. These separate studies are however not done under
the exact same conditions, for example in the same cell lines. It is therefore possible that induction
or repression by HIF is dependent on the cellular context. Nonetheless, these studies show the
importance of HIF in the regulation of the BH3-only proteins.
Hypoxia can also promote cell survival by inducing autophagy or mitophagy263, 265, 266. This is
thought mainly to occur by the induction of BNIP3 and BNIP3L via HIF-1267. Moreover, BNIP3 and
BNIP3L induce autophagy via Beclin 1268. Instead, hypoxia can also promote cell death via these BH3only members, leading to autophagic cell death269, 270 (Fig. 22). BNIP3 can also function as a mediator
for necrotic cell death271.
31
Figure 22. Regulation of BH3-only proteins by hypoxia and E2F. Hypoxia can regulate the expression of many of the
BH3-only proteins via HIF. E2F1 can induce the expression of many of these proteins. Induction of these proteins lead
typically to apoptosis or another form of cell death.
E2F1 was shown to regulate the expression of the BH3-only members BNIP3, Beclin 1, NOXA,
PUMA, HRK, BIM, BIK, BID and BAD269, 272-276 (Fig. 22). These BH3-only members were also found to
have E2F-binding sites in their promoter regions. BNIP3L expression was found to be unaffected by
E2F1, at least in ventricular myocytes277. If SPIKE and BMF are also regulated by E2F1 remains to be
determined. Upregulation of these proteins can result in apoptosis, albeit mediated in response to
different stimuli. E2F1 displaces E2F4 under hypoxic conditions on the BNIP3 promoter, and also pRB
is recruited to the promoter269. The E2F1/pRB complex subsequently represses BNIP3, which
promotes cell survival. However, depending on cellular context, E2F1 can also activate BNIP3
expression and thereby induce apoptosis277. E2Fs were shown to bind to the promoter region of
Beclin 1 and to upregulate its expression272, 278. E2F1 was also shown to upregulate NOXA, PUMA,
HRK, and BIM expression274. Inhibition of NOXA or PUMA diminished E2F1-mediated apoptosis,
suggesting that these BH3-only proteins play a major role in E2F1-mediated apoptosis274. BIK was
shown to be transcriptionally activated by E2F1275. BID and BAD are also upregulated by E2F1276.
Involvement of other E2Fs and pocket proteins in the regulation of BH3-only members remains to be
investigated.
Hypoxia regulates cell fate via the BH3-only subfamily. It is however not yet clear if hypoxia
generally promotes cell survival or cell death via these proteins. Hypoxia upregulates the expression
of some of the BH3-only members, while others are downregulated. Furthermore, upregulation of
for example BNIP3 by hypoxia could also either promote cell survival or promote cell death. It is
possible that hypoxia could decide the cell fate depending on the cellular context, such as the
severity of hypoxia or the amount of cellular damage. E2F1 generally upregulates the expression of
the BH3-only members and thereby promotes cell death. However, the E2F/pRB complex can also
prevent cell death. E2F/pRB was shown to prevent hypoxia induced autophagic cell death by
inhibiting BNIP3 and BNIP3L expression269. However, E2Fs can also directly induce the expression of
Beclin 1 and therefore promote autophagy. It seems therefore that E2F1 could act as an important
regulator of cell fate during hypoxia.
32
5.5 Feedback loops between HIF, E2F and NF-κB
Nuclear factor-κB (NF-κB) is a key mediator in immune and inflammatory responses. In addition,
NF-κB is also involved in cell survival279. NF-κB is activated by a wide variety of different stimuli,
including tumor necrosis factor-α (TNF-α) and various kinds of stresses, such as infections,
inflammation and hypoxia280, 281. NF-κB is a heterodimer comprising p50 and p65, also known as RelA
(Fig. 23). Inhibitor of NF-κB (IκB) can sequester NF-κB in the cytoplasm. Stress can activate IκB kinases
(IKKs), comprising the catalytic subunits IKKα and IKKβ, which can phosphorylate IκB. Phosphorylated
IκB is subsequently polyubiquitinylated and degraded by the proteasome. Release of NF-κB from IκB
permits the translocation of NF-κB to the nucleus where it activates a large number of genes. NF-κB
is especially in the immune system important for its anti-apoptotic function279.
Figure 23. Regulation of
NF-κB by hypoxia and E2F.
Hypoxia can regulate the
expression of NF-κB
through HIF-1 and also
independent of HIF-1. E2F1
represses the expression of
NF-κB via IκB and by
inhibiting p65. E2F1 can
also upregulate p50. IKKs
can induce E2F repressor
complexes. NF-κB can
induce HIF-1 expression
and can repress E2F1.
Hypoxic conditions influence NF-κB activity280 (Fig. 23). Hypoxia increases NF-κB protein
expression and activity in neutrophils. Moreover, hypoxic upregulation of NF-κB was shown to be
dependent on HIF-1α282. HIF-1α upregulates the expression of IKKα, which is important for NF-κB
expression282 (Fig. 23). Thus, in neutrophils, hypoxia is thought to promote cell survival via NF-κB. In
contrast, in cardiomyocytes, p65 protein levels decrease under hypoxia and also NF-κB-dependent
gene transcription is reduced283 (Fig. 23). Cell fate regulated by hypoxia seems therefore dependent
on the cellular context.
NF-κB activated by TNF-α was shown to cause accumulation of the ubiquitinated form of HIF-1α
under normoxia284. However, no noticeable increase in HIF-1α mRNA transcripts were observed,
suggesting that NF-κB regulates HIF-1α expression posttranscriptionally. In contrast, constitutively
active NF-κB was found to be able to bind to the NF-κB-binding site in the HIF1A promoter and
activate expression285. NF-κB was also found to upregulate HIF-1α mRNA transcripts when activated
via the PI3K/AKT pathway286. Moreover, NF-κB was found to be an important transcriptional activator
of HIF-1α and essential for HIF-1α accumulation under hypoxia287, 288 (Fig. 23). This suggests that NFκB regulates HIF-1α both transcriptionally and posttranscriptionally.
E2F1 can act as a repressor of NF-κB activity and thereby promote apoptosis. The physical
interaction of E2F1 with a subunit of NF-κB, p65, is thought to modulate the activity of NF-κB156 (Fig.
23). Moreover, E2F1 can stabilize IκB and can thereby also inhibit NF-κB function289 (Fig. 23). It is also
shown that E2F1 could interact with the other subunit of NF-κB, p50, when cells are stimulated by
33
lipopolysaccharides (LPS)290. This interaction, however, is thought to promote the activation of NFκB-regulated genes (Fig. 23). It is hitherto unknown if other E2Fs also play a role in regulating NF-κB
activity.
NF-κB is also involved in the regulation of E2F activity. IKKα and IKKβ can activate NF-κB, but also
the repressor complex E2F4/p130291 (Fig. 23). This causes displacement of E2F1 by this repressive
complex at the E2F-binding domain of E2F-responsive genes. In addition, NF-κB activated by IKKs can
disrupt the interaction between E2Fs and TRRAP, a component of various HAT complexes291 (Fig. 23).
E2F1 is known to promote cell cycle progression and apoptosis. A possible mechanism for the cell to
switch between these opposite effects of E2F1 is regulated by NF-κB. NF-κB is also thought to
promote cell survival by antagonizing E2F1 at the promoter of BNIP3292. BNIP3 is an important
proapoptotic factor (see also paragraph 5.4). Thus, the IKK/NF-κB signaling pathway is also involved
in the regulation of E2F function.
Depending on the cellular context, NF-κB activity can be induced or repressed under hypoxia. HIF1α is indirectly involved in the induction of NF-κB. E2F1 can directly and indirectly regulate NF-κB
activity. NF-κB can also regulate the activity of HIF-1 and E2Fs, thus establishing feedback loops.
Upregulation of NF-κB during hypoxia causes enhanced expression of HIF-1α, which leads to more
expression of NF-κB. Enhanced expression of NF-κB could therefore cause the repression of E2Fregulated genes and could inhibit E2F functions such as apoptosis. This also inhibits the NF-κB
repressive activity of E2F1. Upregulated NF-κB could inhibit apoptosis. In contrast, downregulation of
NF-κB during hypoxia could lead to less expression of HIF-1α. In addition, E2F-responsive genes and
E2F activity would be less repressed by reduced NF-κB expression. E2F1 could therefore also repress
NF-κB activity under hypoxic conditions and promote apoptosis. Thus, hypoxia could promote or
inhibit apoptosis by regulating NF-κB activity via HIF-1α and E2Fs, which is cell-type specific.
5.6 Feedback loops between HIF, E2F and ASK1
The sequential activation of protein
kinases, such as mitogen-activated
protein kinases (MAPKs), is a common
mechanism of signal transduction. These
protein kinase cascades are used in many
different cellular processes and are
typically activated by extracellular agents.
Apoptosis signal-regulating kinase 1
(ASK1) is a MAPK kinase kinase (MAPKKK)
that is activated in response to various
stresses, such as oxidative stress, LPS and
endoplasmic reticulum (ER) stress293, 294.
ASK1 can also be activated by TNF-α. ASK1
can activate different signaling pathways
via MAPKK 4 (MKK4)/MKK7/c-Jun aminoterminal kinase (JNK) and
MKK3/MKK6/p38294, 295 (Fig. 24). These
pathways have important roles in cellular
processes such as apoptosis, cell survival,
differentiation, and inflammation293, 294.
ASK1 can indirectly activate the MAPK
Figure 24. Regulation of ASK1 by hypoxia and E2F. Hypoxia
p38, which in turn can phosphorylate and
regulates the ASK1-pathway through HIF-1 and independent from
HIF-1. HIF-1 can both induce and inhibit ASK1, depending on the
thereby stabilize HIF-1α296 (Fig. 24). In
length of hypoxia. E2Fs can induce ASK1 expression and ASK1 can
turn, HIF-1 seems important for the
activate E2Fs. p38 from the ASK1-pathway can also induce HIF-1
297-299
regulation of ASK1 as well
. Hypoxia
and E2F activity.
induces transient activation of the
ASK1/MKK4/JNK pathway297 (Fig. 24). Moreover, hypoxia also enhances the interaction between
34
ASK1 and serine/threonine protein phosphatase type 5 (PP5), a protein that can inactivate ASK1297.
Prolonged hypoxic periods (30 min to 1 h) lead to the expression of PP5 induced by HIF-1 which can
bind to the HRE in the promoter of PP5297. Thus, HIF-1 can indirectly inactivate ASK1 after prolonged
hypoxia. ASK1 can therefore no longer stabilize HIF-1α, hence creating a negative feedback loop (Fig.
24).
ASK1 is known to be a E2F-regulated gene, at least under normoxic conditions55, 56, 300. At least
E2F1, E2F2, E2F3 and E2F4 have been shown to bind to the promoter of ASK1300 (Fig. 24). ASK1 is
thought to play no major role in E2F1-mediated apoptosis, as knockdown of ASK1 yielded little effect
on this process. It is however possible that E2F1 regulates ASK1 for the other processes that it is
involved in besides apoptosis. ASK1 can also regulate E2F activity. It has been shown that ASK1 can
bind and inactivate pRB301. In addition, the downstream target of ASK1, p38, can also phosphorylate
pRB in response to stimulation302, 303. ASK1 is therefore thought to mediate the release and activation
of E2Fs via pRB in two ways. This creates a feedback loop because E2Fs regulate ASK1 expression
(Fig. 24).
Both HIF-1 and E2Fs can regulate the activity of ASK1. ASK1 can also regulate the activity of HIF-1
and E2Fs and thereby possibly create multiple feedback loops. Early during hypoxia, ASK1 is
upregulated which could lead to the activation of more E2Fs. E2Fs could then induce more ASK1
expression. Later during hypoxia, ASK1 can also stabilize HIF-1. HIF-1 can subsequently inactivate
ASK1 which could lead to less HIF-1 stabilization and less activation of E2Fs.
35
6. Discussion
In this literature study a complex interaction was ascertained between HIF and E2F regulatory
pathways under hypoxic conditions. The involvement of HIF and E2F in a multitude of pathways in
diverse fundamental cellular processes underlines the important broad regulatory functions that
these transcription factors have. More importantly, the relationship between HIF and E2F in various
pathways suggests that these transcription factors together could regulate the cellular response
towards hypoxic stress. Much is known about the individual involvement of HIF and E2F in various
cellular processes, however there are not many studies that describe direct or indirect interactions
between these two transcription factors.
Although HIF and E2F are involved in many of the same cellular pathways, there is still not much
evidence of a direct interaction between these transcription factors. One recent study shows that
HIF-1α can associate with E2F7 or E2F8 in the same transcriptional complex9. In addition, also HIF-2α
was shown to bind to E2F7. It is not yet known if other E2Fs can also bind directly to HIF. Another
possibility is that both HIF and E2F could associate to the same transcriptional complexes, but not
directly to each other. Both HIF and E2F transcriptionally regulate several overlapping target genes,
which implicates that these transcription factors could be part of the same transcriptional complex
that regulates these genes. In addition, HIF and E2F could possibly also be found together in other
protein complexes. HIF and E2F both can bind to BRCA1 and it is therefore likely that both
transcription factors are recruited to DNA lesions by BRCA1 (Fig. 13). However, it is not yet known if
HIF and E2F are part of the same BRCA1-complex. Nevertheless, BRCA1 could also function as a
scaffold protein and could recruit kinases, such as ATM174, to activate HIF or E2F (Fig. 14).
It is also thought that, at least under certain conditions, HIF can regulate the expression of E2F
and vice versa. E2Fs possess an E-box consensus sequence (CACGTG) in their promoter regions107, 108,
which is very similar to the HRE sequence (G/ACGTG) to which HIF binds. HIF can also regulate E2F
activity by regulating the E2F/DP heterodimer configuration. HIF-2α inhibits the expression of DP4
and therefore promotes E2F activity (Fig. 20). An E2F/DP4 heterodimer is unable to bind to DNA.
Another important way in which HIF could regulate E2F activity is by direct binding to pRB58 (see also
paragraph 2.1). Accumulation of HIF-1α was shown to induce the release of E2Fs from pRB.
Moreover, pRB regulates the transcriptional activity of HIF58. This suggests that when pRB becomes
more phosphorylated during the cell cycle, it dissociates from E2Fs to regulate the activity of HIF
instead. It is not yet known if other pocket proteins also can bind to HIF-1α and if pocket proteins can
bind to HIF-2α. It has also been shown that HIF1A has E2F-binding sites in its promoter region10. This
implicates a transcriptional role for E2Fs in the regulation of HIF-1-expression (Fig. 5). Transcriptional
regulation of HIF-1 by E2Fs has at least been shown when cyclin E is dysregulated in mammary
epithelial tissue10 (see also paragraph 2.3). It is not yet known if HIF2A also harbors E2F-binding sites
in its promoter region. Thus, HIF and E2F can regulate the expression of each other in various ways.
Another important regulator in both the HIF and E2F pathways is c-Myc (Fig. 7). The activity of HIF
is regulated by c-Myc, as both HIF and c-Myc compete for the same promoter binding site in
overlapping target genes. As mentioned before, E2Fs possess E-box consensus sequences in their
promoter regions. E2Fs are therefore regulated by c-Myc. Moreover, c-Myc activity and expression is
regulated by HIF and E2F respectively. This complex relationship between these three transcription
factors could play an important role in the regulation of a wide range of genes that are involved in
different cellular processes. Important proteins regulated by HIF, E2F, and c-Myc include CKIs, Sp1,
glycolytic enzymes, BRCA1, MMR proteins and hTERT (Fig. 7, 9, 10, 13, 17, 18 and S1). HIF-1 and HIF-2
respectively negatively and positively regulate c-Myc activity. The regulation of the ratio between
these isoforms is therefore of importance during hypoxia and could determine cell fate.
The transcription factor Sp1 is regulated by HIF and E2F (Fig. 9). Sp1 is involved in the regulation
of various pathways involved in several cellular processes. This transcription factor also associates
with HIF, E2F and c-Myc (Fig. 9). HIF, E2F and c-Myc are therefore involved in the regulation of Sp1responsive genes. Conversely, Sp1 could also be involved in the regulation of HIF, E2F or c-Myc36
regulated genes. It is therefore not surprising that Sp1 is involved in the regulation of MMR and
hTERT (Fig. 17, 18 and S1). Sp1 generally represses DNA repair in cooperation with HIF-1α and E2F1.
It is therefore likely that the crosstalk between HIF, E2F and Sp1 leads to cell death.
Also signaling pathways play an important role in the regulation of HIF and E2F. The PI3K-pathway
regulates both HIF activity and expression (Fig. 6). Moreover, the PI3K-pathway both regulates and is
regulated by E2F. HIF can also regulate a downstream target in the PI3K-pathway, mTORC1, but this
regulation is dependent on a specific residue phosphorylation on HIF-1α by ATM (Fig. 14). This
feedback loop is therefore only established upon DNA damage. HIF and E2Fs can also be regulated by
MAPK signal transduction pathways. A well described MAPK-pathway is the Ras-pathway. The
downstream mediators of this pathway, ERK1/2, appear to be important for inducing the
transcriptional activation of both HIFs and E2Fs21, 98, 304-306. In addition, E2F1 can promote the
functional activity of ERKs, creating a positive feedback loop307. Under hypoxia this positive feedback
loop would promote extra transcription of HIFs, resulting in an enhanced cellular hypoxic response.
Another MAPK-pathway that regulates both HIF and E2F is the ASK1-pathway (Fig. 24). The ASK1pathway both regulates and is regulated by HIF and E2F. Thus, various important signaling pathways
can be controlled by HIF and E2F. This could be an important way for the cell to adapt to hypoxic
stress.
HIF and E2Fs can bind to several overlapping transcriptional cofactors. Both HIF and E2Fs can bind
the HATs p300/CBP. Association with p300/CBP promotes the ability of HIFs to regulate gene
expression and is essential in the HIF transcriptional complex26. Furthermore, HIFs can also be
acetylated by p30023. Acetylation on the lysine-709 residue is shown to stabilize HIF23. E2Fs can also
be acetylated by p300/CBP. Acetylation was found to enhance the functions of E2Fs and also
stabilizes the transcription factors308, 309. Acetylation of E2F1 in response to DNA damage also
promotes its apoptotic function. E2F1 acetylation is required to bind to the promoter of the
proapoptotic factor p73 and is important to induce apoptosis in response to DSBs310. The FOXO
transcription factor FOXO3a is involved in the regulation of the binding of p300/CBP to either HIF or
E2F. FOXO3a induces the expression of CITED2 which competes with HIF and E2F for the binding to
p300/CBP (Fig. 21). HIF and E2Fs can also bind the coactivator PARP-1 (Fig. 16). It is therefore likely
that HIF and E2Fs compete for the binding to these coactivators. This competition could affect
transcriptional regulation by these transcription factors.
Hypoxia has a pronounced effect on the cell cycle through HIF. HIF can control the expression of
cyclins that directly regulate E2F-activity and therefore also regulate cell cycle progression. HIF can
induce the expression of the CKIs p21Cip1 and p27Kip1 and thereby promote cell cycle arrest (Fig. 5).
HIF can induce the expression of CKIs through c-Myc, TGF-β and FOXO3a (Fig. 7, 8 and 21). E2Fs can
also promote cell cycle arrest by promoting CKI-activity through the PI3K-pathway (Fig. 6). Thus, HIF
generally appears to promote cell cycle arrest through CKIs. However, HIF can also promote cell cycle
progression under certain circumstances. Both HIF and E2F can induce the expression of cyclin D1,
which promotes cell cycle progression (Fig. 4). Thus, depending on the cellular context, hypoxia can
regulate the cell cycle differently. However, in general, it appears that HIF can cause cell cycle arrest
through inhibiting E2F. This could allow the cell to adapt to hypoxic stress.
Members of the 14-3-3 protein family could also be involved in the cooperation between HIF and
E2F. The 14-3-3 proteins are important regulators of cell cycle control311. HIF-1α downregulates
mTORC1 via REDD1 under hypoxia188 (Fig. 14). Under low oxygen tension REDD1 is thought to
regulate mTORC1 activity via a pathway which involves the TSC1/2 complex. REDD1 mediates the
dissociation of inhibitory 14-3-3 proteins from the TSC1/2 complex which causes mTORC1
inhibition312. Furthermore, the cell cycle inhibitor 14-3-3ε is regulated by VHL70. It is however still
unclear if this regulation is mediated by HIF. E2F1 is shown to be regulated by 14-3-3 proteins190. It is
therefore not unlikely that HIF-1α could dissociate 14-3-3 proteins from the TSC1/2 complex via
REDD1 to stabilize E2F1. However, it is not yet clear which 14-3-3 proteins associate to the TSC1/2
complex. At least 14-3-3γ and 14-3-3ζ have been shown to associate to the TSC1/2 complex313, 314.
However, only 14-3-3τ has been found to bind to E2F1 so far190 (Fig. 14). Nonetheless, further
37
investigation could determine the potential role of the 14-3-3 protein family in the cooperation
between HIF-1α and E2F.
HIF and E2F appear to cooperate in the regulation of cell metabolism. Both HIF and E2F stimulate
anaerobic metabolism by promoting the glycolysis (Fig. 10). HIF and E2F can both induce the
expression of glycolytic enzymes and inhibit the conversion of pyruvate into AcCoA through PDKs.
Moreover, HIF can also inhibit AcCoA production through PPARα (Fig. 11). The intermediates of the
glycolysis can also be used for synthesis of glycine, serine, purines, pyrimidines, and phospholipids.
This is therefore also promoted by HIF and E2F. HIF and E2F can induce the expression of PPARγ, and
therefore also control important metabolic processes such as lipid anabolism and adipogenesis (Fig.
11). Hypoxia therefore can, like E2F, better prepare the cell against stress by adjusting several
metabolic pathways.
HIF and E2F are also involved in a complex pathway that regulates cell metabolism which involves
the PI3K-pathway, c-Myc and FOXO transcription factors. c-Myc is involved in cell metabolism by
regulating glycolytic enzymes (Fig. 10) and c-Myc is regulated by HIF and E2F140 (Fig. 7). Furthermore,
c-Myc is also stabilized by the Ras-pathway and Ras-induced PI3K-pathway315. The PI3K-pathway can
also inhibit FOXO transcription factors and FOXO transcription factors can regulate the PI3Kpathway316 (Fig. S1). Moreover, FOXO3a has been shown to regulate mitochondrial gene expression
by inhibiting c-Myc249 (Fig. S1). As HIF and E2F regulate the expression of FOXO3a (Fig. 21), these
transcription factors can therefore also control cell metabolism through this FOXO transcription
factor. HIF and E2F can therefore also regulate cell metabolism in various indirect ways. However,
FOXO3a was also shown to block HIF-1 transactivation function on the GLUT1 promoter250. FOXO3a
has therefore also a function in inhibiting glycolysis. Thus, HIF and E2F appear to regulate different
steps of the PI3K/c-Myc/FOXO3a pathway that is involved in regulating cell metabolism. Moreover,
HIF and E2F are in turn regulated by this pathway as well.
HIF and E2F also are involved in the same DNA repair pathways. Both HIF and E2F can regulate
DNA DSB repair through BRCA1, ATM and DNA-PK (Fig. 13, 14 and 15). HIF and E2F appear to
promote DSB repair especially through BRCA1 and ATM. Also single stranded DNA lesion repair can
be induced by HIF and E2F through ATR and PARP-1 (Fig. 14 and 16). It is however unknown if PARP-1
in complex with HIF or E2F is also involved in BER. HIF generally inhibits MMR, whereas E2Fs promote
MMR (Fig. 17). Telomere synthesis is also regulated by HIF and E2F (Fig. 18). Thus, hypoxia generally
positively influence DNA repair, except MMR. This suggests that hypoxia can promote cell survival.
However, HIF and E2F phosphorylated by ATM promote apoptosis (Fig. 14). It is therefore possible
that the decision between cell survival and cell death through the DNA repair pathways by hypoxia
and E2F is dependent on the extend of the DNA damage.
Cell death is also regulated by HIF and E2F. The p53-dependent apoptosis pathway is generally
inhibited by HIF, suggesting that hypoxia promotes cell survival (Fig. 19). In contrast, E2F1 promotes
apoptosis via p53, leading to E2F1-mediated apoptosis. p53 can repress the expression of BNIP3 and
therefore specifically inhibit hypoxia-mediated cell death228. Although p53 generally promotes cell
death by apoptosis, p53 inhibits autophagic cell death317 and necrosis caused by hypoxia (Fig. 22).
p53 is also involved in cell survival214. Moreover, p53 promotes DNA repair pathways such as NHEJ,
MMR and BER, similarly to HIF and E2F214. HIF prevents p53 proteasomal degradation and inhibits
p53-mediated apoptosis. It is therefore possible that HIF in this way stimulates the cell survival
function of p53. However, severe hypoxic conditions could lead to enhanced inhibition of p53 by HIF
which could lead to hypoxia-mediated cell death via autophagic cell death or necrosis. HIF and E2F
can also regulate the proapoptotic BH3-only proteins (Fig. 22). Induction or inhibition of these
proteins by HIF likely depends on the cellular context. HIF and E2F also regulate the activity of the
cell survival factor NF-κB. Hypoxia and E2F can both induce and inhibit NF-κB-activity, suggesting that
the regulation of this pathway is also dependent on the cellular context (Fig. 23). Moreover, NF-κB is
thought to regulate the molecular switch that determines whether E2F1 promotes cell cycle
progression or induces apoptosis292. NF-κB was also shown to regulate the E2F1-dependend
transactivation of PDK4156. The control of these functions of E2F1 is therefore also indirectly
38
regulated by hypoxia. Thus, hypoxia can regulate cell death, but how it is regulated is likely
dependent on the severity and length of hypoxia.
Regulation of the different cellular processes by HIF and E2F is very complex (Fig. S1). Not only are
there a multitude of pathways involved that are regulated by HIF and E2F, most of these pathways
are also involved in regulating HIF and E2F expression and function. Furthermore, there is also
crosstalk between multiple pathways independently of either HIF or E2F. Thus, the relationship
between HIF and E2F can be characterized by multiple feedback loops. Regulation by HIF and E2F
also appears to be dependent on the cellular context, the severity and length of hypoxia. Even a
single exposure to hypoxia and intermittent hypoxia have different effects on cellular pathways5, 318.
It is nonetheless likely that hypoxia induces cell cycle arrest by inhibiting E2F. This allows the cell to
adapt to this stressful condition. In addition, DNA damage repair is generally promoted by hypoxia
and the cellular metabolism is altered to provide the necessary energy. If the conditions are too
severe or the cellular damage is too extend, hypoxia can induce cell death.
Despite the relatively small number of publications that describe the interplay between HIF and
E2F-pathways, a clear picture is beginning to form of the relationship between these transcription
factors. HIF and E2F regulate, either directly or indirectly, various overlapping pathways that are
involved in several important cellular processes. To gain a more comprehensive understanding of this
relationship, also the interplay between these pathways needs to be studied further. Elucidation of
the relationship between these cellular pathways and the role of HIF and E2F therein, may provide a
better understanding of the role of hypoxia in development and disease.
39
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Abbreviations
AcCoA – Acetyl coenzyme A
APAF1 – Apoptotic protease activating factor 1
ARF – Alternate reading frame
ARNT – Aryl hydrocarbon nuclear translocator
ASK1 – Apoptosis signal-regulating kinase 1
ATM – Ataxia teleangiectasia mutated kinase
ATP – Adenosine-5'-triphosphate
ATR – Ataxia telangiectasia and Rad3-related protein
BAD – Bcl-2-associated death promoter
Bcl-2 – B-cell lymphoma 2
BER – base excision repair
BH3 – Bcl-2 homology 3
BID – BH3 interacting-domain death agonist
BIK – Bcl-2-interacting killer
BNIP3 –BCL2/adenovirus E1B 19 kDa protein-interacting protein 3
BRCA1 – Breast cancer susceptibility gene 1
BRCT – BRCA1 carboxyl-terminal
CBP – CREB-binding protein
CDK – Cyclin-dependent kinase
CH1 – Cysteine/histidine-rich 1
CHK – Checkpoint kinase
CITED2 – p300/CBP-interacting transactivator 2
CKI – Cyclin-dependent kinase inhibitor
COX – Cytochrome c oxidase
CREB – Cyclic AMP response element binding protein
DNA-PK – DNA-dependent protein kinase
DNA-PKcs – DNA-PK catalytic subunit
DP – Dimerization partner
DSB – Double strand break
E2F – E2 factor
ER – endoplasmic reticulum
ERK – Extracellular signal-regulated kinase
Fbw7 – F-box and WD40 domain protein 7
FIH1 – Factor inhibiting HIF-1
FOXO – Forkhead box O
GLUT-1 – Glucose transporter-1
GSK-3 – Glycogen synthase kinase-3
HAT – Histone acetyltransferase
HDAC – Histone deacetylase
HIF – Hypoxia-inducible factor
HK – Hexokinase
HLH – helix–loop–helix
HRE – hypoxia responsive element
hTERT – Human telomerase reverse transcriptase
hTR – Human telomerase RNA component
HUVEC – human umbilical vein endothelial cell
IKK – IκB kinases
JNK – Jun amino-terminal kinase
LDHA – Lactate dehydrogenase A
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MAPK – Mitogen-activated protein kinases
MAPKK – MAPK kinase
MAPKKK – MAPKK kinase
MCPH1 – Microcephalin 1
MDM2 – Murine double minute 2
MEF – Mouse embryonic fibroblast
miRNA – micro RNA
MKK – MAPK kinase
MMR – mismatch repair
mTOR – Mammalian target of rapamycin
mTORC – mTOR complex
NF-κB – Nuclear factor kappa-light-chain-enhancer of activated B cells
NHEJ – non-homologous end joining
NER – nucleotide excision repair
NPM – Nucleophosmin
PARP-1 – Poly(ADP-ribose) polymerase 1
PAS – PER–ARNT–SIM
PDH – Pyruvate dehydrogenase
PDK – Pyruvate dehydrogenase kinase
PHD – Prolyl-hydroxylases
PI3K – Phosphatidylinositide 3′-OH kinase
PIN1 – Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1
PKB – Protein kinase B
PKM2 – Pyruvate kinase isozymes M2
PPAR – Peroxisome proliferator-activated receptor
PUMA – p53 upregulated modulator of apoptosis
RB – Retinoblastoma
REDD1 – Regulated in DNA damage 1
SIRT1 – Sirtuin 1
SKP2 – S-phase kinase-associated protein 2
Sp – Specificity protein
TCA – Tricarboxylic acid
TFIID – Transcription factor II D
TGF – Transforming growth factor
TNF-α – Tumor necrosis factor-alpha
TRRAP – Transactivation/transformation-domain associated protein
TSC1/2 – Tuberous sclerosis tumor suppressor complex 1/2
VEGF – Vascular endothelial growth factor
VHL – Von Hippel-Lindau
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Supplementary information
Figure S1. Overview of HIF and E2F regulated pathways. HIF and E2F regulate the expression and activity of proteins that
are involved in many different pathways. Many of these pathways also regulate HIF and E2F and are therefore connected
through these transcription factors. In addition, there is also crosstalk between many of these pathways independent of HIF
and E2F. Blue arrow = positive effect, red line = negative effect.
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