From the Department of Radiation Oncology, Division
of Radiation and Cancer Biology, Stanford University Medical School,
Stanford, California 94305-5152, the ¶ Department of Molecular
Genetics, Biochemistry, and Microbiology, University of Cincinnati
Medical Center, Cincinnati, Ohio 45267-0524, the
Department of
Biochemistry and Molecular Biology, University of Texas M. D. Anderson
Cancer Center, University of Texas Medical Center, Houston, Texas
77030-4009, and the ** Department of Biochemistry, Case
Western Reserve University, Cleveland, Ohio 44106-4935
Received for publication, August 30, 2002, and in revised form, November 6, 2002
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ABSTRACT |
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Hypoxia is a growth inhibitory stress associated
with multiple disease states. We find that hypoxic stress actively
regulates transcription not only by activation of specific genes but
also by selective repression. We reconstituted this bimodal response to
hypoxia in vitro and determined a mechanism for
hypoxia-mediated repression of transcription. Hypoxic cell extracts are
competent for transcript elongation, but cannot assemble a functional
preinitiation complex (PIC) at a subset of promoters. PIC assembly and
RNA polymerase II C-terminal domain (CTD) phosphorylation were blocked
by hypoxic induction and core promoter binding of negative cofactor 2 protein (NC2 Solid tumors generally have a poor vascular supply, which results
in areas of decreased perfusion and hypoxia (1). The hypoxic
microenvironment may increase tumor aggressiveness (2), presumably
through specific induction of transcription factors such as
hypoxia-inducible factor 1 (HIF1)1 (4) and alterations
in gene expression (3). Under normoxic conditions the HIF1 In addition to HIF-mediated gene activation, cells under reduced oxygen
also limit non-essential cellular processes in order to survive (2).
Thus, cells under stress may channel their energy into productive gene
expression, whereas non-productive genes and processes are turned off.
Gene-specific transcription inhibition can be induced by regulated
repressors of transcription (reviewed in Refs. 8 and 9). Certain types
of these repressors modify chromatin structure (10), whereas others
inhibit transcription through core promoter and general transcription
factor interactions (11) (reviewed in Ref. 12). Among this latter group
of transcriptional repressors is negative cofactor 2 (NC2 NC2 Our findings reveal an active mechanism by which mammalian cells can
establish inactive promoters in response to hypoxia. We show that the
physiological stimulus of hypoxia increases NC2 activity in mammalian
cells, concomitant with selective transcription repression. A limited
survey of cellular stress conditions reveals that hypoxia may be unique
in exploiting this fundamental mechanism of gene regulation.
Cells and Treatments--
Hepatoma cells (Hepa 1-6, American
Type Culture Collection) and primary, normal human fibroblasts were
plated overnight in glass dishes in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum at 10,000 cells/cm2,
the media was changed, and the cells were transferred into a hypoxic
chamber (Bactron 2, Sheldon Labs) for the indicated times. These
conditions have been established in order to maintain stable pH and
glucose over the course of the experiment. Cell viability, at the end
of all treatments, was judged by trypan blue exclusion and found to be
>70% even under severe, long term hypoxic conditions. Cells extracts
were prepared as described (18). The drug treatments 150 µM CoCl2 (in H2O, Sigma), 0.5 µg ml RNA Analysis and in Vitro Transcription--
Northern blot
probes were obtained from the Image Consortium (Incyte) and sequenced
prior to use to confirm inserts. Primer extension analyses with
lacZ-specific primer (AFP), luciferase-specific primers (3× HRE and
VEGF), chick Immunodepletion and Protein Add-back--
Protein A beads were
swollen in RNase-free H2O (250 mg/2 ml H2O) and
transferred into 1× phosphate-buffered saline. Antibody (anti-NC2,
gift of T. Oegelschlager; anti-TBP, Santa Cruz Biotechnology; anti-CCAAT/enhancer-binding protein, Santa Cruz Biotechnology) was added to the beads at a ratio of 200 ng of antibody/1 ml of 50%
slurry beads. The beads were washed twice in nuclear dialysis buffer
(NDB), (20 mM Hepes, pH 7.9, 50mM KCl, 0.2 mM EDTA, and 20% glycerol). Nuclear extract was added to
the beads at 1.5× the volume of washed, antibody-bound beads and
incubated for 1 h at 4 °C, rocking. Beads were removed by
centrifugation, and supernatant was used as an immunodepleted extract.
TFIID, PC4, and FLAG-RNA polymerase II were purified and added to
transcription reactions as described previously (21). Recombinant
DR1/NC2 Recapitulation of Hypoxia-mediated Transcription Regulation in
Vitro--
We investigated mechanisms of hypoxia-regulated gene
expression using in vitro transcription. Whole-cell
transcription extracts were prepared from murine hepatoma (Hepa 1-6)
cells that are p53 wild-type and AFP positive. Activation of
AFP is associated with hepatocellular carcinoma and various
malignant tumors. Extracts were prepared after cell exposure to
ionizing radiation, 0.01% oxygen, the hypoxia-mimetic
CoCl2 (150 µM), or the proteosome inhibitor
ALLN (Fig. 1A). In
vitro transcription of AFP in each extract, including control
normoxia and HeLa extracts, revealed complete inhibition of
transcription only in the hypoxic cell extract.
The transcription inhibitory response to hypoxia was neither
tissue-specific nor restricted to transformed cells. We used extracts
of normal human fibroblast cells grown as primary cultures (Fig.
1B) in normoxia, hypoxia, or the hypoxia-mimetic cobalt chloride (150 µM) and found an equally dramatic
inhibition of transcription, specifically in the hypoxic extracts. This
repression in hypoxic extracts is dominant in mixing experiments,
suggesting the function of an inhibitor rather than the loss of an
essential protein. Control and hypoxic hepatoma extracts were mixed
together at the indicated ratios and tested for transcriptional
activity on the AFP template (Fig. 1C). Note that even at
20% input (lane 3) the hypoxic extract inhibited
transcription activity. The dominant function of the hypoxic extract
supports a model of active repression versus simply loss of
energy under hypoxic conditions.
Hypoxic Repression Is Widespread but Selective--
The general
nature of hypoxia-mediated repression was revealed by transcription of
three gene templates that share few if any trans-acting
regulatory elements upstream of the proximal promoter, i.e.
the p21/WAF1 gene (which is p53-activated) (22), AFP (which is repressed by p53) (19), and chick
Though multiple genes were repressed in hypoxic extracts, this
repressive effect is selective. As observed in vivo, the
transcription of gene templates containing hypoxia-inducible HIF1
regulatory elements is activated in vitro rather than
repressed (Fig. 2B). One of these constructs contains three
copies of an HRE from the 3'-untranslated region of the erythropoietin
(EPO) gene (24) fused to a heterologous promoter/luciferase
reporter (3× HRE). The other hypoxia-activated template is a natural
VEGF promoter and upstream regulatory region (from
We determined by Northern blot analysis that hypoxia can result in
repression of specific mRNAs in vivo. Fig. 2C
shows that, in primary human fibroblasts exposed to severe hypoxia,
specific genes show decreased mRNA levels, whereas others are
unchanged or activated by hypoxia. The selective repression of certain
genes with the activation of others suggests an active process rather than a passive loss of macromolecular synthesis under conditions of
reduced energy. This decline of steady-state RNA levels for a variety
of genes, combined with in vitro transcription results, supports a role for hypoxia-regulated transcription repression through
widely conserved regulatory elements.
Hypoxia-induced Repression Acts at the Core Promoter--
Because
a diverse set of templates was repressed (Fig 2A), we
focused on the promoter region and examined PIC assembly in control and
hypoxic cell extracts (Fig.
3A). Under established conditions for in vitro single-round transcription (20),
transcription is dependent solely on protein-DNA interactions (PIC
assembly) established (in the absence of NTPs) before washing the
protein-bound DNA template and adding Sarkosyl detergent, which
prevents subsequent protein binding (see Fig. 3A,
diagram). Addition of Sarkosyl after PIC assembly (Fig.
3A, lanes 4-8) revealed that hypoxic cell
extract is incapable of functional PIC assembly (Fig. 3A, lanes
6 and 8). PICs assembled in hypoxic extract could not
elongate (Fig. 3A, lane 8) nor could transcription be
rescued by control extract when DNA binding of control extract
proteins is precluded (Fig. 3A, lane 6). Control extract
rescue of hypoxic transcription does occur in the absence of Sarkosyl
(Fig. 3A, lane 3). Importantly, we found that hypoxic
extract did not inhibit transcription elongation by PICs preassembled
in control extract (Fig. 3A, lane 5). Thus, any inhibitory
factors present in the hypoxic cell extract must act during PIC
assembly rather than by altering preassembled transcription initiation
complexes or inhibiting transcription elongation.
One direct consequence of complete PIC assembly is phosphorylation of
the RNA polymerase II C-terminal domain (CTD) before initiation (27,
28). Western blot analyses with specific antibodies raised against the
RNA polymerase II amino terminus (N-20), CTD phosphoserine 5 (H14), CTD
phosphoserine 2 (H5), and unmodified CTD (C-19) (29) revealed marked
differences between the hypoxic cell extract and other control or
treated cell extracts (Fig. 3B). All of the cell extract
preparations contained similar levels of unphosphorylated RNA
polymerase II (IIA form) as revealed by the N-20 and C19 antibodies.
However, there was a dramatic absence of RNA polymerase II with its CTD
phosphorylated (IIO form) at serine 5, and very low amounts of IIO
phosphorylated at serine 2 in the hypoxic cell extract. Comparison of
blots probed with phospho-specific antibodies and functional analysis
of transcription in vitro (Fig. 1) revealed a correlation
between loss of RNA polymerase II CTD phosphorylation and repression of
transcription. RNA polymerase II CTD-hypophosphorylation at both
serines 5 and 2 in hypoxic extracts is most consistent with
inhibition of initiation (30). These findings support a model in which
PIC assembly is affected by hypoxia, a dysfunction marked by the
lack of RNA polymerase II CTD phosphorylation.
Assembly of a PIC is regulated at numerous levels and involves the
interactions of many proteins (recently reviewed in Refs. 8 and 31). We
surveyed a number of purified proteins for their potential ability to
restore transcription function to the hypoxic extract. These proteins
included TFIID, TFIIH, mediator complex, RNA polymerase IIA, RNA
polymerase IIO, immunopurified RNA polymerase II (21), and recombinant
TBP (Fig. 3C, and data not shown). Among these factors, only
relatively high concentrations of recombinant TBP could overcome
hypoxia-mediated transcription repression to any degree (to 15% of the
control level; Fig. 3D) when added alone. FLAG-tagged RNA
polymerase II (Fl-RNA Poll II; Ref. 21) alone was also unable to
enhance hypoxic transcription (Fig. 3C, lanes
4-6). However, the combination of both TBP and purified RNA
polymerase II (Fig. 3 C, lanes 7-9) reversed
hypoxia-mediated repression and increased transcription of hypoxic
extract to control activated transcription levels (Fig. 3C,
lane 1). From these data, we hypothesized that high levels
of recombinant TBP partially squelched the repressive effect of hypoxia
to regain a basal level of transcription (compare with transcription
driven by purified RNA polymerase II plus PC4 and TFIID Fig.
3D, lane 7). The addition of both FLAG-RNA
polymerase II, which is not hyperphosphorylated (Fig. 3B),
and TBP could effectively rescue hypoxic transcription to control
activated levels. These data suggest that a hypoxia-induced repressor
interacted with both transcription factors and/or that the RNA
polymerase II preparation contained proteins that augmented the ability
of TBP to squelch inhibition and promote activated transcription.
Hypoxia Induces Accumulation of a Negative Regulator of PIC
Assembly--
One of several negative regulators of PIC assembly is
NC2 NC2 (Dr1/DrAP1) Induction Blocks PIC Assembly--
We
assayed for endogenous NC2 activity by comparing the PIC components
(TBP, TFIIB, and Dr1/NC2
We extended the PIC analysis to RNA polymerase II and its
phosphorylated forms bound to the DNA versus total protein
(Fig. 4D). Again, there were similar levels of
unphosphorylated RNA polymerase IIA in both hypoxic and control
extracts (N-20 antibody) and sharply reduced hypoxic levels of
CTD-phosphorylated RNA polymerase II (H14 antibodies). Parallel
analysis of PICs revealed that no RNA polymerase II was bound in
hypoxic-assembled PICs. Therefore, in the presence of NC2 (Dr1/DrAP1),
TFIIB and RNA polymerase II cannot assemble as part of a functional
PIC. Reported roles for NC2 (Dr1/DrAP1) complex function (reviewed in
Refs. 12, 35, and 37) in the repression of transcription by blocking
entry of TFIIB in PIC assembly is consistent with our results of PIC analysis in hypoxic extracts.
To determine whether NC2 protein complexes were primarily responsible
for transcription repression induced by hypoxia, we immunodepleted
NC2
The NC2 (Dr1/DrAP1) complex could assume multiple roles in both
transcription repression and activation due to either
post-translational modifications or association with specific protein
binding partners. Interpretation of the NC2/TBP/TATAA DNA ternary
complex crystal structure suggests that transcriptional activators or
co-activators could overcome NC2-mediated inhibition of functional PIC
assembly (38). Post-translational modification may be "stressor-"
or target site-specific, as phosphorylation of NC2 by casein kinase II
inhibits binding to DNA in general and increases the specificity of TBP
interaction (39). Genome-wide expression analyses and chromatin
immunoprecipitation of temperature shift-induced NC2 protein in
S. cerevisiae revealed NC2 association with both positively and negatively regulated promoters in response to the stress of heat
shock (13). More recently, a role for NC2 in stabilizing TBP-DNA
binding to promote basal transcription and the displacement of NC2 to
effect activated transcription has been demonstrated both in
vivo and in vitro for S. cerevisiae
(40).
A mechanism for a dual role in positive and negative regulation of
transcription has been proposed for NC2 (15, 16) (reviewed in Ref. 31).
NC2, which represses PIC assembly at TATA-containing core promoters,
activates distal promoter element-regulated promoters in
Drosophila (15). This model presents a potential paradigm for mammalian cells in that one induced protein such as NC2 could act
as a repressor at many TATA-containing promoters but as an activator at
different subsets of genes, e.g. those regulated by
HIF1, evoking a timely and energy-efficient response to stress. Potential interaction(s) between HIF-responsive gene promoters and NC2
protein therefore becomes an important question for future investigations.
/
, Dr1/DrAP1). Immunodepletion of NC2
/Dr1 protein
complexes rescued hypoxic-repressed transcription without alteration of normoxic transcription. Physiological regulation of NC2 activity may
represent an active means of conserving energy in response to hypoxic stress.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunit
is hydroxylated at proline 564, promoting high affinity interaction
with the von Hippel-Lindau (VHL) tumor suppressor protein and
ubiquitin-mediated degradation (5, 6). Upon exposure to low oxygen
tension, HIF1
is not hydroxylated, is stabilized, and interacts with
HIF1
. The HIF1 heterodimer binds to hypoxia-responsive elements and
activates the promoters of HIF-responsive genes (7).
/
,
Dr1/DrAP1), which blocks transcription by association with DNA-bound
TFIID and inhibits PIC assembly in vitro.
and NC2
are essential genes, and
their gene products regulate ~17% of all Saccharomyces
cerevisiae genes either positively or negatively (13). In S. cerevisiae, NC2 is required for specific TATA-containing gene
repression under times of reduced nitrogen availability (14). Recent
work surveying Drosophila promoter elements and studies with
purified proteins reveal that NC2 represses transcription from TATA
element-containing promoters but activates promoters that rely on
downstream promoter elements (DPEs) for regulation (15-17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 doxorubicin (in H2O, Sigma), and 50 µM ALLN (in Me2SO, Calbiochem) were
added to fresh media at the indicated times. Ionizing radiation was
delivered from a 137Cs source at 3.5 gray/min.
-globin-specific primer, and a chloramphenicol acetyl
transferase (CAT)-specific primer (p21) were performed under
standard conditions (19). Single-round transcription conditions were
performed as published (20), except that a 10-min preincubation period
was used as established for supercoiled templates.
protein was purchased from ProteinOne, College Park, MD.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Extracts from hypoxic cells show decreased
transcription in vitro. A, AFP DNA was
transcribed in extracts made from Hepa 1-6 cells as follows: normoxic
cells (lane 1) and cells exposed to ionizing radiation (4 h
post 6 or 8 gray, IR1 and IR2, respectively),
hypoxia (0.01% for 24 h), CoCl2 (150 µM
for 24 h), and a proteasomal inhibitor, ALLN (1 h). HeLa nuclear
extract transcription was an additional control (lane 7).
B, extracts from normal human fibroblasts also show
hypoxia-dependent transcriptional repression. Control,
hypoxic (0.01% for 24 h), and CoCl2-treated (50 µM for 24h) cells were used to make extracts for in
vitro transcription of AFP as indicated. C, hypoxic
repression is dominant in mixed extracts. Control and hypoxic Hepa 1-6
extracts were mixed as indicated (microgram extract protein of each,
total microgram protein held constant) and tested for transcription of
AFP. All newly synthesized transcripts were quantitated by primer
extension. Molecular weight standards (MW) are radiolabeled
X174 DNA digested with HaeIII (Invitrogen).
-globin (which is not regulated by p53) (23)
(Fig. 2A). Transcription of
each gene was strongly inhibited in hypoxic cell extracts. By contrast, treatment of cells with doxorubicin (0.5 µg ml
1 for
24 h) robustly induced p53 in these cells (data not shown) but had
varied effects, including none (AFP), 2-fold inhibition (
-globin), and induction of several
transcripts from aberrant start sites (p21). These data
confirmed hypoxia-dependent repression of transcription
conferred through a widely conserved regulatory element independently
of p53 activation.
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Fig. 2.
Hypoxia effects selective transcription
repression or activation. A, several tested templates
showed hypoxic repression (+); p21, AFP, and
-globin genes were transcribed in Hepa 1-6 extract from
control (lanes 2, 5, and 9), hypoxic
(lanes 3, 6, and 10), or
doxorubicin-treated cells (lanes 4, 7, and
11). Transcription reactions without extract (lane
1) and with HeLa extract (lane 8) were used as
controls. B, hypoxia-induced genes showed
transcriptional activity in vitro. AFP/lacZ, 3× HRE, and
VEGF/luc were transcribed under identical conditions in extracts from
cells under control conditions, CoCl2, 24 h under 2%
oxygen and 12 and 24 h under 0.01% oxygen treatment.
C, Northern blot analysis of total RNA from normal human
fibroblasts exposed to the control, hypoxia, or cobalt chloride. Probes
are indicated (p53R2, ribonucleotide reductase isoform 2;
Hif1, hypoxia inducible factor 1
), and methylene
blue-stained 18 S RNA was used as a loading control.
2275 to +51)
plasmid containing a single HRE and a luciferase reporter (VEGF). This
construct lacks the identified VEGF RNA stability element (25). The 3× HRE and VEGF constructs are transcriptionally induced when transfected into cells and incubated under hypoxic conditions (data not shown). Each of these DNA templates was transcribed in the same extracts under
identical conditions and exhibited a different profile of time- and
"dose"-dependent responses to hypoxia. AFP
showed hypoxic repression and moderate hypoxia (2% oxygen) yielding
moderate repression, whereas severe hypoxia (0.01% oxygen) showed
increasing repression with time, and CoCl2 showed no
effect. The 3× HRE template showed hypoxic activation, and moderate
hypoxia showed induction, whereas severe hypoxia clearly induced
expression at 12 h, and by 24 h expression was reduced to
control levels; CoCl2 treatment also showed significant
induction. In contrast, the VEGF promoter showed induction
that required more extreme hypoxic conditions. The natural
VEGF HRE-containing, TATA-less promoter transcribed poorly
and was slightly activated at moderate hypoxia but was activated in
severe hypoxia slightly at 12 h, increasing at 24 h, and was
not activated by CoCl2. This pattern of hypoxia-induced VEGF expression, including the limited response to
CoCl2, matched that observed for endogenous VEGF
activation (Fig. 2C and Ref. 26).
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Fig. 3.
Hypoxic repression occurs at the core
promoter. A, hypoxic extracts are dysfunctional for PIC
formation. Immobilized AFP templates were preincubated with control
(C; lanes 1, 2, 4,
5, and 7) or hypoxic extract (H;
lanes 3, 6, and 8) without NTPs to
allow PIC assembly. Protein bead-DNA complexes were washed, and
transcript elongation was initiated with an NTP addition to control
(lanes 1, 3, 4, and 6),
hypoxic (lanes 2 and 5), or no extract
(lanes 7 and 8) and plus (+; lanes
4-8) or minus ( ; lanes 1-3) 0.025% Sarkosyl
(Sigma). B, RNA polymerase II CTD is hypophosphorylated
under hypoxia. Fifty micrograms of total extract protein from control,
hypoxia, doxorubicin, CoCl2, ALLN, and ionizing radiation
exposed cells alongside immunopurified epitope-tagged RNA polymerase II
(Fl-RNA Pol II, lane 1) were immunoblotted sequentially with
RNA polymerase II N-terminal antibody N20 and CTD-specific antibodies
C19 and phosphoserine H14 and H5 antibodies (Research Diagnostics).
C, hypoxic repression overcome by recombinant TPB and
immunopurified RNA polymerase II. TBP protein alone (lane 3;
25 ng), increasing amounts of purified FLAG-RNA polymerase II
(lanes 4-6; 75, 150, and 300 ng, respectively) alone or a
combination of both TBP (25 ng) and FLAG-RNA polymerase II (lanes
7-9; 75, 150, and 300 ng, respectively) were added to hypoxic
extract (lanes 2-9). Lane 1 shows equal total
protein levels of control normoxic extract transcription. D,
addition of high levels of TBP squelches hypoxia-mediated repression to
basal transcription levels. TBP (lanes 3-5; 25, 50, and 100 ng) was added to hypoxic extract (lanes 2-5). Transcription
levels in the presence of TBP are comparable with basal levels
generated by purified FLAG-RNA polymerase II (lanes 6 and
7; ~135 ng) and TFIID (lanes 6 and 7; 0.75 ng)
in the presence of factor PC4 (lane 7; 100 ng).
/
(or Dr1/DrAP1 protein) (12, 32, 33). Additionally,
interactions between NC2 and RNA polymerase II, which affect RNA
polymerase II CTD phosphorylation, have been reported previously (34). In vitro experimentation supports a model wherein the NC2
protein associates with TBP bound at TATA boxes, which inhibits further assembly of the PIC. We examined this candidate repressor of hypoxic transcription by Western blot analysis with antibodies specific for the
NC2 subunits Dr1/NC2
and DrAP1/NC2
. We found that both Dr1/NC2
and DrAP1/NC2
protein levels were elevated in extracts of
hypoxia-treated hepatoma cells (Fig.
4A). NC2 is likely regulated post-transcriptionally, as levels of both NC2
and NC2
mRNA
remain unchanged with hypoxic treatment (data not shown). In these same extracts, TBP levels are unchanged, and AFP protein levels are reduced,
reflecting the transcription response of AFP under hypoxic conditions.
The addition of recombinant Dr1/NC2
to control transcription extracts effected repression in a concentration-dependent
manner (Fig. 4B, lanes 1-7) of the control
extract to levels observed in hypoxia-incubated cell extracts
(lanes 6-10). The ability of the single subunit to repress
transcription in vitro has been previously shown with
multiple gene templates (reviewed in Ref. 35).
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Fig. 4.
Hypoxia-induced NC2 inhibits PIC
assembly. A, NC2 is induced in hypoxic extracts. Fifty
micrograms of control and hypoxic Hepa 1-6 extracts were immunoblotted
with both anti-AFP and anti-TBP antibodies (top
panel) and sequentially with anti-Dr1 and anti-DrAP1
monoclonal antibodies (bottom two panels).
B, control (C, lanes 1-5) and hypoxic
(H, lanes 6-9) extracts were supplemented with
recombinant NC2 (Dr1) protein (lanes 2-5 and
7-9; 2, 20, 40, and 100 ng, respectively), and
transcription was performed. C, PIC assembly is incomplete
in hypoxic extracts. Immobilized AFP templates were incubated with
control, hypoxia, or 1:1 control/hypoxia mixture without NTPs for 10 min and processed for immunoblotting of PIC-assembled proteins with
anti-TBP, TFIIB (both from Santa Cruz Biotechnology), and NC2
polyclonal antibodies. Total extracts were immunoblotted as a control.
D, PIC assembly and immunoblotting for the bound RNA Pol IIA
and Pol IIO forms were similarly performed for control, hypoxic, or
mixed extracts. E, transcription levels in control extracts
were unaffected by immunodepletion using nonspecific (NS;
CCAAT/enhancer-binding protein) or NC2
antibodies but were severely
inhibited by anti-TBP depletion. Hypoxic extracts (H) were
reactivated for transcription by depletion with an anti-NC2
but were
not reactivated by immunodepletion with nonspecific or TBP antibodies
(Ab).
) present in control and hypoxic cell
extracts (Fig. 4C) to those bound to promoter DNA, as
described previously (Fig. 4C, diagram, and Ref.
36), under conditions for single-round transcription (Fig.
2A). Similar levels of total TBP (or TFIID) and TFIIB
proteins were present in hypoxic and control extracts, but NC2
/Dr1
was increased by hypoxia (Fig. 4C, lanes 4-6).
Comparison of soluble extract to DNA-bound PIC proteins (lanes
1-3, long exposure) revealed an inverse relationship between NC2
and TFIIB in the PICs (lanes 2 and 3). Analyses
of mixed hypoxic/control (1:1) extracts support a
concentration-dependent profile of proteins specifically
bound at the PIC (TBP and NC2) versus those excluded from
the PIC (TFIIB) rather than effects on protein degradation or modification.
protein complexes from hypoxic and control cellular extracts.
Hypoxic and control extracts were incubated with antibody-coated beads,
the beads were removed, and the depleted extracts were assayed for
transcription function (Fig. 4E). Incubation with
nonspecific antibody did not alter the transcription properties established for hypoxic and control extracts. Depletion of TBP protein
from control extract obliterated transcription and demonstrated the
effectiveness of immunodepletion. Incubation with NC2
antibody-coated beads restored transcription capability to hypoxic cell
extract and did not alter the ability of control extract to transcribe in vitro (lanes 4 and 5). These data
show that either NC2
or an NC2
-associated protein complex is an
essential component of hypoxia-mediated transcription repression, the
removal of which rescues transcription function.
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ACKNOWLEDGEMENTS |
---|
We are grateful to M. Czyzyk-Krzeska, T. Oegelschlager, D. Reinberg, K. Ladaroute, and J. Abraham for providing essential materials and to L. Sang and J. Irish for technical support. We thank M. Czyzyk-Krzeska, A. Giaccia, J.A.K. Harmony, D. Reinberg, and A.J. Crowe for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (to N. D., C.-M. C., and M. C. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 713-794-1161;
Fax: 713-791-9478; E-mail: mbarton@odin.mdacc.tmc.edu.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M212534200
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ABBREVIATIONS |
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The abbreviations used are:
HIF, hypoxia-inducible factor;
HRE, HIF-regulatory element;
NC2, negative
cofactor 2;
PIC, preinitiation complex;
ALLN, N-acetyl-Leu-Leu-norleucinal;
AFP, -fetoprotein;
VEGF, vascular endothelial growth factor;
TBP, TATA-binding protein;
EPO, erythropoietin;
CTD, C-terminal domain.
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