A disproportion between oxygen delivery and consumption leads to a restricted
oxygenation of tissues. In a variety of pathologies like ischemia, stroke, inflammation
and cancer this shortage of oxygen is a key feature. It enrolls a unique response that
is based on the transcriptional regulation of hundreds of downstream target genes
that promote eventually the adaption of cell metabolism. The maintenance of the
oxygen homeostasis is centrally governed by hypoxia-inducible transcription factors
(HIFs). Direct target genes of these transcription factors are increasingly expressed
by binding of the HIF-complex to the cis-acting highly conserved consensus
sequence 5‘-RCGTG-3’; also referred to as hypoxia response elements (HRE).
Heterodimeric HIFs consist of a tightly O2-regulated a-subunit (in humans HIF-1a,
HIF-2a or HIF-3a) and a constitutively expressed b-subunit (HIF-1b). In oxygenated
conditions HIF a-subunits are continuously marked for proteasomal degradation
through hydroxylation of two key prolyl-residues by prolyl-4-hydroxylase domain
(PHD) oxygen sensor proteins. In hypoxic conditions the HIF-� subunit is stabilized
and translocates to the nucleus where it forms the heterodimer HIF. Biochemically,
the stabilization of the HIF-� subunit is explained by a reduced hydroxylation that is
required for the interaction with the ubiquitin-ligase von-Hippel-Lindau protein
Two of three PHDs are transcriptionally regulated by HIFs. As a consequence HIF
causes increased expression of PHD2/3 that compensates for their decreased
enzymatic activity in hypoxia. In this negative HIF-PHD2/3 feedback loop we decided
to focus on the oxygen sensor PHD2. PHD2 is widely considered as the main cellular
oxygen sensor since, amongst other evidences, only the knockout of the PHD2
(EGLN1) gene shows prenatal lethality in mice.
We regard the transcriptional regulation of the PHD2 gene as important since the
abundance of the PHD2 enzyme determines the above mentioned negative feedback
loop. Therefore, we aimed to profoundly understand the transcriptional regulation by
studying the PHD2 promoter architecture and to elucidate further regulatory
mechanisms of its activity.
We carried out consecutive truncations of the PHD2 promoter and defined the
minimal promoter region. By chromosome immunoprecipitation we could confirm on
an endogenous level that hypoxic PHD2 expression is predominantly mediated
through HIF-1� rather than HIF-2�. Additionally we identified and cloned a 95 and 55
nucleotide PHD2 promoter region encompassing a single HBS as highly conserved
in several organisms and demonstrated high hypoxia-inducibility. To date, HIF is the
only known transcription factor influencing PHD2 gene transcription. However,
various putative transcription factor binding sites were predicted in this conserved
PHD2 promoter region. By a mutation approach we could exclude the ubiquitous
transcription factor Sp1 to be involved in basal or hypoxia-induced regulation of the
PHD2 gene although numerous predicted Sp1-consensus motifs suggested so.
When motifs located 5' or 3' to the HBS were mutated, total abrogation of the hypoxic
response was observed, but binding of the HIF-1 complex remained unaffected. This
suggests that other transcription factors might contribute to hypoxic activation of the
In order to find out which other (co-) transcription factors might influence the PHD2
promoter activity we established a synthetic transactivation screening where 704
arrayed transcription factors were analyzed for their influence on the PHD2 HBS
(Wollenick et al., Nucleic Acid Res., in press). We found several family members of
the activator protein-1 (AP-1) transcription factors, such as JUN and FOSB, and
three ETS-transcription factors to be involved in the activation of the PHD2 promoter.
Most strikingly, the ETS-transcription factor ETS variant 4 (ETV4) showed, when
overexpressed, not only impact on hypoxic PHD2 expression but also on other wellknown
hypoxic target genes such as PHD3 (EGLN3) and carbonic anhydrase 9
(CA9). We hypothesize that ETV4 potentially increases the hypoxic activation of
those promoters or elements that contain a distinct sequence architecture
surrounding the HBS. HBSs that are similar to the PHD2 HBS seem to be
preferentially super-induced by ETV4.
By mammalian two-hybrid and fluorescence resonance energy transfer (FRET)
analysis we found evidence for formation of a complex between ETV4 and HIF-1/2a.
Chromatin immunoprecipitation confirmed the recruitment of HIF-1� and ETV4 to the
PHD2 locus. Additionally, we could provide evidence that the co-activation of hypoxic
target genes by ETV4 also has relevance for clinical data. In vivo data underlined
that ETV4 expression strongly correlates with PHD2, HIF-1/2� and other hypoxic
marker genes in 282 human tissues of breast cancer patients.
Although FRET data suggest a direct interaction, we hypothesize a trimeric complex
composed of HIF:p300/CBP:ETV4. We carried out a thorough HIF-1a domain
mapping and found that during the hypoxically induced HIF-1�:ETV4 interaction
mainly the C-terminal activation domain is involved. Additionally, overexpression of
CBP/p300-interacting transactivator 2 (CITED2), a competitor of HIF for the
p300/CBP interaction, disrupted the ETV4:HIF complex pointing towards the
involvement of p300/CBP. Factor inhibiting HIF (FIH) depletion provoked unregulated
binding of HIF to p300/CBP and as a result the loss of oxygen-dependent
suppression of the interaction between HIF and ETV4. Taken together, these
experiments provided evidence for the cooperation between HIF-1a and p300/CBP in
Recent data provide indications that ETV4 protein is more abundant in hypoxic and in
PHD2 knockdown cells while ETV4 mRNA levels remain unaffected. ETV4 protein
levels were also increased when cells were treated with a PHD inhibitor. That might
hint to a hydroxylation-dependent regulation of ETV4 through PHDs that is inhibited
when the O2-concentration is low or when PHDs are silenced.
In conclusion, this work demonstrated that a synthetic transactivation screening can
unravel so far unrecognized transcriptional pathway interactions that also have
implications on clinical data of different cancer specimen.