Somatic pairing of chromosome 19 in renal oncocytoma is associated with deregulated EGLN2-mediated [corrected] oxygen-sensing response.
ABSTRACT Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase EGLN2 [corrected] a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of EGLN2 [corrected] in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma.
- SourceAvailable from: pnas.org[show abstract] [hide abstract]
ABSTRACT: Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia.Proceedings of the National Academy of Sciences 07/1995; 92(12):5510-4. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.Nature 06/1999; 399(6733):271-5. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel-Lindau protein (pVHL). Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. This is the first function to be ascribed to the pVHL beta-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.Nature Cell Biology 08/2000; 2(7):423-7. · 20.76 Impact Factor
Somatic Pairing of Chromosome 19 in Renal Oncocytoma
Is Associated with Deregulated ELGN2-Mediated
Julie M. Koeman1., Ryan C. Russell2., Min-Han Tan3,4,5,6., David Petillo3, Michael Westphal3, Katherine
Koelzer1, Julie L. Metcalf2, Zhongfa Zhang3, Daisuke Matsuda3, Karl J. Dykema7, Heather L. Houseman7,
Eric J. Kort8, Laura L. Furge3, Richard J. Kahnoski9, Ste ´phane Richard10,11,12, Annick Vieillefond12,13,
Pamela J. Swiatek1, Bin Tean Teh3,4, Michael Ohh2, Kyle A. Furge7*
1Laboratory of Germline Modification and Cytogenetics, Van Andel Research Institute, Grand Rapids, Michigan, United States of America, 2Department of Laboratory
Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, 3Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, Michigan,
United States of America, 4NCCS-VARI Translational Research Laboratory, National Cancer Centre of Singapore, Singapore, Singapore, 5Department of Medical Oncology,
National Cancer Center of Singapore, Singapore, Singapore, 6Centre for Molecular Epidemiology, Department of Community, Occupational and Family Medicine, National
University of Singapore, Singapore, Singapore, 7Laboratory of Computational Biology, Van Andel Research Institute, Grand Rapids, Michigan, United States of America,
8Laboratory of Molecular Epidemiology, Van Andel Research Institute, Grand Rapids, Michigan, United States of America, 9Department of Urology, Spectrum Health
Hospital, Grand Rapids, Michigan, United States of America, 10Ge ´ne ´tique Oncologique EPHE, French Kidney Cancer Consortium, AP-HP, Service d’Urologie, Le Kremlin-
Bice ˆtre, France, 11CNRS FRE 2939, Institut Gustave Roussy, Villejuif, France, 12French Kidney Cancer Consortium, Paris, France, 13Laboratoire d’Anatomie Pathologique,
AP-HP, Ho ˆpital Cochin, Paris, France
Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close
association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has
been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene
expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in
renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene
expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase ELGN2, a key protein that
regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of ELGN2 in renal
oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-
target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of
chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to
numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as
genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that
maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of
Citation: Koeman JM, Russell RC, Tan M-H, Petillo D, Westphal M, et al. (2008) Somatic Pairing of Chromosome 19 in Renal Oncocytoma Is Associated with
Deregulated ELGN2-Mediated Oxygen-Sensing Response. PLoS Genet 4(9): e1000176. doi:10.1371/journal.pgen.1000176
Editor: Bruce Clurman, Fred Hutchinson Cancer Research Center, United States of America
Received March 27, 2008; Accepted July 18, 2008; Published September 5, 2008
Copyright: ? 2008 Koeman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was supported in part by the Van Andel Research Institute, National Institutes of Health grant R33-CA10113-01 to KAF, Canadian Institutes
of Health Research (CIHR), Canadian Cancer Society (CCS) and the Kidney Foundation of Canada (KFoC) grants to MO, the Gerber Foundation, the Hauenstein
Foundation, the Michigan Economic Development Corporation, and the Michigan Technology Tri-Corridor grants to BT, the Singapore Millenium Foundation and
the Singapore Cancer Society to MHT.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Cellular adaptation to changes in oxygen tension is vital for the
integrity, maintenance and survival of cells. Hypoxia-inducible
factor (HIF), the major transcription factor of the ubiquitous
oxygen-sensing pathway, is a heterodimer composed of a and b
subunits . While HIFb is constitutively expressed and stable,
HIFa is oxygen-labile by the virtue of the oxygen-dependent
degradation (ODD) domain, which undergoes rapid oxygen-
dependent ubiquitin-mediated destruction [2–5]. Thus, the
stability of HIFa dictates the transcriptional activity of HIF .
Critical regulators of HIFa stability are the prolyl-hydroxylase
domain-containing enzymes (PHD/EGLNs) that hydroxylate
HIFa on conserved prolines within the ODD domain in the
presence of oxygen [7,8]. Hydroxylated HIFa is recognized by the
von Hippel-Lindau (VHL) protein. VHL is the substrate-
conferring component of an E3 ubiquitin ligase called ECV
(Elongins/Cul2/VHL) that specifically polyubiquitinates prolyl-
hydroxylated HIFa for subsequent destruction via the 26S
PLoS Genetics | www.plosgenetics.org1September 2008 | Volume 4 | Issue 9 | e1000176
Deregulation of HIFa regulatory proteins has been strongly
associated with cancer development. Germline inheritance of a
faulty VHL allele on chromosome 3p25 is the cause of VHL
disease, characterized by a high frequency of clear cell renal cell
carcinoma (RCC), cerebellar hemangioblastoma, pheochromocy-
toma, and retinal angioma . Inactivation of the remaining wild-
type VHL allele in a susceptible cell leads to tumor formation.
Somatic biallelic inactivation of VHL is also responsible for the
development of sporadic clear-cell RCCs, the predominant form
of adult kidney cancer [10–12]. Cells that are devoid of functional
VHL show elevated expression of numerous hypoxia-inducible
genes due to a failure to degrade HIFa. In addition to VHL,
deregulation of the PHD/EGLN family of prolyl-hydroxylases
have also been associated with abnormal cell growth. Develop-
ment of erythrocytosis, characterized by an excess of erythrocytes,
has been associated with inactivating germline mutations in
PHD2/EGLN1 [13,14]. Pheochromocytoma, a neuroendocrine
tumor of the medulla of the adrenal glands, is linked with
deregulation of PHD3/EGLN3 .
While biallelic inactivation of VHL is found in the majority of
clear cell RCCs, kidney cancer is a heterogeneous disease that can
be divided into several subtypes based on morphological and
cytogenetic features [16,17]. Chromophobe RCC and renal
oncocytoma are two related kidney tumors that together account
for approximately 10% of all renal masses. In contrast to clear cell
RCC, VHL mutations and/or increased expression of hypoxia-
inducible genes are not found in these tumor subtypes and
molecular genetic defects that are associated with tumor
development remain unclear. Identification of molecular genetic
defects in renal oncocytoma is particularly challenging as these
cells are often described as karyotypically normal and the presence
of cytogenetically abnormal regions in which to search for tumor
modifying genes is rare in this tumor subtype.
To identify molecular defects associated with renal tumor
development, we analyzed gene expression data from a variety of
kidney tumors. This analysis revealed that renal oncocytoma and
chromophobe RCC have a striking transcriptional disruption
along chromosome 19. While in chromophobe RCC the
disruption reflected a chromosome 19 amplification, in the renal
oncocytoma cells the disruption reflected the close association, or
pairing, of chromosome 19q in interphase. EGLN2 located within
the paired region was dramatically overexpressed in renal
oncocytoma cells and was associated with the deregulation of
numerous hypoxia-inducible genes including a pro-death BNIP3L.
Thus, chromosome 19q pairing in renal oncocytoma unveils a
unique mechanism of disrupting oxygen homeostasis via altering
the expression of EGLN2.
Gene expression profiling data derived from renal oncocytomas
and chromophobe RCCs was scanned for regional increases or
decreases in RNA production, which often indicate the presence of
chromosomal amplifications or deletions [18–24]. Consistent with
previous cytogenetic studies, the renal oncocytoma cells were
largely devoid of transcriptional abnormalities that would reflect a
DNA amplification or deletion. In contrast, losses of chromosomes
1, 2, 6, 10, and 17 are frequently found in chromophobe RCC. In
our chromophobe RCC samples, these well-established chromo-
somal losses were strongly reflected in the gene expression profiling
data (Figure 1A). In addition, a transcriptional abnormality
involving genes mapping to chr 19 was frequently identified in
both the renal oncocytomas and the chromophobe RCCs but not
other subtypes of RCC (Figure 1A and Figure S1). In renal
oncocytomas, the transcriptional abnormality primarily involved
the q arm of chromosome 19, while in chromophobe RCC the
abnormality involved the entire chromosome (Figure 1A,B).
Regional increases in overall RNA production often indicate the
presence of an underlying DNA amplification. As gain of
chromosome 19 has not been previously reported as a recurrent
abnormality in either renal oncocytoma or chromophobe RCC,
DNA copy number analysis was performed on a subset of these
samples using high-density single nucleotide polymorphism (SNP)
arrays. From the SNP data, an amplification of the entirety of
chromosome 19 was detected in the chromophobe RCC samples
(Figure 1C,D). This whole-chromosome amplification was con-
firmed by fluorescence in-situ hybridization (FISH) using locus-
specific probes that mapped to the p and q arms of chromosome
19 (Table S1). In contrast, no change in DNA copy number was
detected in the renal oncocytoma samples (Figure 1C,D). As a
positive control for the DNA copy number analysis, only
oncocytoma (ON) samples derived from female patients were
examined, and a relative gain of the X chromosome was clearly
detected in these samples (Figure 1C).
To determine the status of chromosome 19 in more detail in the
renal oncocytoma cells, this chromosome was evaluated further
using a panel of FISH probes. Two distinct and well-separated
FISH signals, typical of diploid cells in interphase, were frequently
observed when probes specific to the chr 19p arm were used
(Figure 2 and Table S2). In contrast, a single, large FISH signal
(singlet) or two FISH signals that were in close proximity (proximal
doublet) were frequently observed when probes specific to the chr
19q arm were used. Approximately 35% of cells examined
contained the singlet signal, while an additional 18% of cells
contained proximal doublets (Table S2 and data not shown).
Semi-quantitative image analysis was used to examine the
characteristics of the large FISH singlet (Figure 2B). This analysis
demonstrated that the size of the singlet FISH signal was on
average 1.5-fold larger than the size of two well-separated 19q
FISH signals (P=0.02). This large signal was observed using
multiple probes directed against the q arm of the chromosome,
including centromeric and telomeric probes (Figure 2C,E). The
large FISH singlet had striking similarities to the FISH signals
Together, renal oncocytoma and chromophobe renal cell
carcinoma (RCC) account for approximately 10% of masses
that are resected from the kidney. However, the molecular
defects that are associated with the development of these
neoplasias are not clear. Here, we take advantage of recent
advances in genetics and computational analysis to screen
for chromosomal abnormalities that are present in both
renal oncocytoma and chromophobe RCC. We show that
while chromophobe RCC cells contain an extra copy of
chromosome 19, the renal oncoctyoma cells contain a
rarely reported chromosomal abnormality. Both of these
chromosomal abnormalities result in transcriptional dis-
ruptions of EGLN2, a gene that is located on chromosome
19 and is critical for the cellular response to changes in
oxygen levels. Defects in oxygen sensing are found in
other types of kidney tumors, and the identification of
EGLN2 directly implicates defects in the oxygen-sensing
network in these neoplasias as well. These findings are
important because the chromosomal defect present in
renal oncocytomas may also be present in other tumor
cells. In addition, deregulation of EGLN2 reveals a unique
way in which perturbations in oxygen-sensing are
associated with disease.
Somatic Pairing Mediated Deregulation of EGLN2
PLoS Genetics | www.plosgenetics.org2 September 2008 | Volume 4 | Issue 9 | e1000176
observed in studies of somatically paired chromosomes [25–27].
Somatic pairing refers to the close association of homologous
chromosomes and is typically associated with chromosomes in
meiotic prophase. However, somatic pairing has also been
observed in interphase in normal human cells and some tumor
cells [26,28–32]. The presence of a large FISH singlet reflects the
overlapping FISH signals generated from two chromosomal
regions in very close proximity [26,27]. The lack of evidence for
a DNA copy number change coupled with the presence of large
FISH singlets and proximal doublets using multiple locus-specific
probes, suggested that chr 19q was somatically paired.
renal oncocytoma cells, the p and q arms of chr 19 were visualized
simultaneously using whole-arm chromosome painting (WCP).Using
this approach, two distinct p arms, typical of diploid cells in
interphase, were frequently observed in renal oncocytoma cells
(Figure 2G,H and Table S2). However, the majority of cells
contained a single q-arm signal that was located proximal to the
quantification the fluorescence signal, this pattern is consistent with
the locus-specific FISH analysis and further indicates that the q arms
of the chromosomes are in close proximity or are paired in these cells.
Figure 1. Transcriptional abnormalities in oncocytoma and chromophobe RCC. (A) Genomic regions that have significantly higher (red) or
lower (blue) RNA production in renal oncocytoma (ON, n=10) and chromophobe RCC (CR, n=10) relative to non-diseased kidney (n=10) were
identified using the comparative genomic microarray analysis (CGMA) method as described in the Materials and Methods. Plotted is the resulting t-
statistic obtained for each chromosome arm. Only the most significant results are shown (P,0.005). (B) For each gene on chr 19, the average log2-
transformed expression ratio comparing oncocytoma or chromophobe RCC to non-diseased kidney is plotted relative to genomic location. The red
circle indicates the location of the centromere. A smoothing curve was fit to the log2-transformed data to highlight regions that contain a
disproportionate number of up-regulated genes. (C) Genomic regions that have overall increased (green) or decreased (red) DNA copy number in
renal oncocytoma (ON) and chromophobe RCC (CR). SNP-derived DNA copy numbers were computed as described in the Material and Methods. All
tumor samples were obtained from female patients except of the single male sample indicated (M). (D) For each SNP on chr 19, the average log2-
transformed DNA copy number ratio comparing oncocytoma (n=4) or chromophobe RCC (n=3) to a pooled normal reference is plotted relative to
genomic location as described in (C).
Somatic Pairing Mediated Deregulation of EGLN2
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The changes in gene expression that accompanied the somatic
pairing suggested that deregulation of a gene, or multiple genes,
associated with tumor development mapped within the paired chr
19q region. As deregulation of the oxygen-sensing network is a
common event in other types of sporadic renal cell carcinomas,
genes associated with HIF regulation and that mapped to chr 19q
were identified from the Entrez Gene database and tested for
expression defects (see Materials and Methods). We also identified
additional genes that were related to kidney-cancer via additional
literature searching (Table S3). Both analyses identified EGLN2/
PHD1 as a possible candidate gene in this region. To verify that
the prolyl-hydroxylase EGLN2/PHD1 was significantly deregu-
lated in renal oncocytoma cells, the level of EGLN2 protein was
evaluated in these tumors (Figure 3A,B). Analysis of matched
oncocytoma-normal tissue pairs revealed a dramatic increase in
the level of EGLN2 in the oncocytoma tumors versus the level
observed in corresponding normal tissue. Higher expression of
EGLN2 was also observed in 2 of 3 chromophobe RCCs
examined (Figure S2). These results are in contrast to the EGLN2
levels found in clear cell RCC. Consistent with the gene expression
data, virtually no EGLN2 protein was detected in patient-derived
clear cell RCC samples, while low basal amounts of EGLN2 were
visualized by Western blot analysis in the matched normal samples
(Figure 3 A,C).
EGLN2 is one of three prolyl-hydroxylases known to post-
translationally modify HIFa, which is required for VHL-mediated
destruction of HIFa. To address whether increased expression of
EGLN2 influenced the binding and ubiquitination of HIF-
1aODD via VHL, in vitro translated35S-labeled HA-VHL and in
vitro translated unlabeled Gal4-HA-HIF-1aODD were mixed in
extracts in which EGLN2 was enriched (see Materials and
Methods). Enrichment of EGLN2 led to an increased association
of VHL to the wild-type ODD, but not to a mutated ODD in
which a proline residue critical for VHL binding was changed to
an alanine (P546A) (Figure 3D). In addition, an in vitro HIF-
1aODD ubiquitination assay was performed to determine whether
the increased VHL-HIF-1aODD association led to increased HIF-
1aODD ubiquitination. Increased levels of EGLN2 resulted in a
dose-dependent increase in VHL-mediated HIF-1aODD ubiqui-
tination (Figure 3E). These results suggest that overexpression of
EGLN2 in oncocytoma could further decrease the level of HIFa
below the level observed in normal tissue.
In clear cell RCC, an increase in HIFa due to functional
inactivation of VHL induces a transcriptional program that
mimics cellular exposure to hypoxic conditions. In contrast, in the
renal oncocytoma, the functional effects of increased expression of
EGLN2 would be to decrease HIFa levels. To examine the
cellular effects of decreased HIFa, we re-evaluated previously
published data that measured HIF-1 DNA-binding activity, HIF-
1a protein levels, and HIF-1b protein levels in cells exposed to
hypo- and hyper-oxygenated conditions . Normoxic conditions
in the kidney cortex is estimated to be 3–5% oxygen . Induction
of a hypo-oxygenated condition was associated with a significant
increase in HIFa and HIF activity levels (Figure 4A). Specifically, a
six-fold decrease in oxygen concentration (3% to 0.5% oxygen)
resulted in approximately a four-fold increase in HIF-1a levels (2.5
to 9.8 densitometry units). Further, we noted that HIF-1a levels
change in an analogous manner upon induction of hyper-
oxygenated conditions: a six-fold increase in oxygen concentration
(3% to 18% oxygen) results in greater than a three-fold decrease in
HIF-1a levels (2.5 to 0.75 densitometry units). The association
between decreased HIF-1a and hyper-oxygenated conditions is
easier to evaluate if the HIF dose-response data is plotted on a log-
log scale rather than a linear-linear scale (Figure 4B). The log-log
Figure 2. Somatic chromosome pairing in renal oncocytoma.
Representative photomicrographs of tri-color interphase FISH on renal
oncocytoma touch preparations. White arrows indicate large singlet or
proximal doublet signals. In all images DAPI counterstaining is shown in
blue. (A,B) Labeling with 19p13.3 (green) and 19q13.31 (red) probes.
Image area of the 19q13.31 signal was quantified across multiple cells
(n=25) in the same image plane. Area mean and standard error are
shown. (C,E,F) Labeling with 19p telomere (green) and 19q telomere
(red) probes or 19p12 (green) and an alpha satellite probe for chr 19
that also cross-hybridizes to chromosomes 1 and 5 (red). Inset
highlights centromeric pattern. (D) Schematic representation of
frequently observed FISH patterns. (G,H) Whole-arm chromosome paint
(WCP) for the chr 19 p-arm (red) and chr 19 q-arm (green). The inset
shows a normal cell. Schematic representations of the paired
chromosomes are shown below. Dashed lines represent chromosomal
regions perpendicular to the plane of the image.
Somatic Pairing Mediated Deregulation of EGLN2
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transformed data follow a straight line, indicating that HIFa level
and oxygen concentration follow a power-law relationship (i.e.,
f(x)=axk), rather than an exponential relationship (i.e., f(x)=kax).
The biological implications of the power-law relationship is that an
n-fold change in oxygen concentration leads to a proportional n-
fold change in HIF-1a levels and HIF activity (Figure S3).
Figure 3. Overexpression of EGLN2 in renal oncocytoma. (A) Relative expression of EGLN2 as determined by gene expression microarray. (B)
Anti-EGLN2 immunoblot analysis of whole-cell extracts prepared from oncocytoma (T) or patient-matched normal tissue (N) samples (lanes 3–8, top
panel) and exogenously expressed EGLN2 controls (lanes 1–2, top panel). U2OS were transfected with plasmid containing human EGLN2 or empty
vector alone (MOCK); EGLN2 appears as a single band of 45 kDa. Anti-vinculin immunoblot of whole-cell extracts was performed as a loading control.
Asterisk (*) denotes background band. (C) Anti-EGLN2 immunoblot analysis of whole-cell extracts prepared from renal clear cell carcinoma (T) or
patient-matched normal tissue (N) samples as described in (B) with the exception that a longer film exposure was required to reveal the EGLN2
protein in normal tissue. (D) In vitro translated Gal4-HA-HIF-1aODD(WT) and Gal4-HA-HIF-1aODD(PA) were treated with or without cellular extracts
enriched for EGLN2 and mixed with in vitro translated35S-labelled HA-VHL. Reaction mixtures were immunoprecipitated with anti-Gal4 antibody,
bound proteins resolved on SDS-PAGE and visualized by autoradiography (lanes 3–6). In vitro translated
immunoprecipitated with anti-HA or anti-Gal4 antibody as input controls (lanes 1 and 2) and visualized by autoradiography. (E) In vitro ubiquitylation
of35S-labelled Gal4-HA-HIF-1aODD(WT) treated with increasing amounts of exogenous EGLN2 was performed in S100 cellular extracts devoid of VHL
or reconstituted with VHL. All reaction mixtures except in lane 6 received purified ubiquitin. Reaction mixtures were immunoprecipitated with anti-
Gal4 antibody, resolved on SDS-PAGE and visualized by autoradiography. * denotes uncharacterized modified Gal4-HA-HIF-1aODD; IP:
immunoprecipitation; AR: autoradiography.
35S-labelled HA-VHL was also
Somatic Pairing Mediated Deregulation of EGLN2
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Moreover, these results demonstrate that while increases in HIF-
1a are associated with hypo-oxygenated conditions, decreases in
HIF-1a are associated with hyper-oxygenated conditions.
To determine whether EGLN2 overexpression is inducing a
HIF-mediated hyperoxic cell response in the renal oncocytoma
cells, the expression pattern of several known HIF target genes
were examined in the renal oncocytoma cells and, for comparison,
in clear cell RCC . Consistent with VHL defects present in the
clear cell RCC, gene set enrichment analysis revealed a significant
up-regulation of the HIF-1 target genes in clear cell RCC
Figure 4. Decreased HIF levels associated with hyperoxic cell state. (A) Normalized densitometry of HIF-1a protein levels (N),HIF-1b protein
levels (m), and HIF-1 DNA-binding activity (X) as presented in Figure 5B of the Jiang et al. article. The large (N) highlights the 3% oxygen HIF-1a
protein levels in both figures. (B) The densitometry data and oxygen-concentration data presented in (A) were log2-transformed and re-plotted. The
summary statistics of the best-fit line are also shown. (C,D) Relative gene expression levels of HIF target genes in clear cell RCC (n=10) and renal
oncocytoma (n=10) compared with non-diseased kidney (n=12). For each gene, red indicates increased expression, blue decreased expression. (E)
Relative expression of BNI3PL as determined by gene expression profiling.
Somatic Pairing Mediated Deregulation of EGLN2
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(P=0.0001; Figure 4C). Notable up-regulated genes included
carbonic anhydrase IX (CA9), ferroxidase (CP), vascular endothe-
lial growth factor A (VEGFA), and glucose transporter (GLUT1).
However, in the renal oncocytoma cells, a distinct population of
HIF-target genes were significantly down-regulated (P=0.01;
Figure 4D). Specifically, the HIF-target genes heme oxygenase 1
(HMOX1), enolase 1 (ENO1), and Cbp/p300-interacting transacti-
vator (CITED2) were significantly down-regulated, but genes such
as CA9, VEGFA, and GLUT1 were not. In addition, the recently
identified tumor suppressor BNIP3L is downregulated three-fold in
the renal oncocytoma cells (Figure 4E). BNIP3L is an oxygen-
regulated member of the Bcl-2 family (Figure S4). BNIP3L is a
pro-death gene (induces features of apoptosis, necrosis and
autophagy) and knockdown of this gene is sufficient to convert
non-tumorigenic cell lines into tumorigenic lines in xenograft
studies [34–36]. In support, while hypoxia mimetic treatment
significantly induced the expression of BNIP3L, HMOX1, ENO1,
and CITED2 (Figure 5A, right panel and Figure S5), ectopic
transient expression of EGLN2 under physiologic hypoxia (cyclical
0–7% oxygen exposure ) was associated with reduced level of
expression of these genes in comparison to cells transfected with
empty plasmid (Figure 5 and Figure S5). These results demonstrate
that over expression of EGLN2 can downregulate HIF1
responsive factors, such as BNIP3L. Moreover, while up-
regulation of HIF-target genes such as VEGFA are associated with
the development of clear cell RCC, these results suggest that
down-regulation of distinct subset of HIF-target genes are
associated with the development of renal oncocytomas.
A proper oxygen-sensing response is vital to the maintenance of
normal cellular functions. Deregulation of HIF, the principal
driver of the adaptive response to hypoxia, is associated with the
pathogenesis of several diseases, including cancer. While the
hypoxic tumor microenvironment - by the virtue of the ubiquitous
oxygen-sensing pathway - results in modulation of HIF activity,
loss-of-function mutations in a growing list of tumor suppressor
genes also can affect HIF function. Mutations in PTEN, PML,
TSC, and VHL have been identified in tumor cells that result in
the deregulation of HIF via multiple distinct mechanisms involving
Akt/PI3K, mTOR and the ubiquitin pathway. Emerging evidence
now implicates cancer-causing mutations that directly impinge on
EGLNs. For example, mutations in succinate dehydrogenase (SDH)
result in the cytosolic accumulation of succinate, which inhibits
EGLNs, leading to the stabilization and activation of HIF-1a
[38,39]. Inactivating germline mutations in EGLN1 have been
identified to cause erythrocytosis [13,14] and deregulation of
EGLN3 has been linked to the development of pheochromocy-
toma, a neuroendocrine tumor of the adrenal glands .
In this study, we reveal somatic pairing of chr 19q as a recurrent
cytogenetic abnormality in renal oncocytoma that results in
dramatic changes in transcription within the paired region. The
functional consequence of chromosome joining is formally
unknown but it is may disrupt chromatin structure causing the
juxtaposition of cis and trans regulatory regions that modulate the
transcription of a large set of genes. The identification of EGLN2 as
a significantly deregulated gene that maps within the paired chr
19q region directly implicates defects in the oxygen-sensing
network to the pathobiology of renal oncocytoma. These results
suggest that in addition to numerical and structural chromosomal
abnormalities, somatic pairing should be considered as a
chromosomal event that associates with tumorigenesis.
Although the loss of EGLN2 does not lead to decreased HIF1a
accumulation, perhaps due to the compensatory activity of
EGLN3, the data from this study suggest that overexpression of
ELGN2 leads to decreased HIF1 levels. More recently, an E3
ubiquitin ligase called Siah2 was identified to target EGLN2 for
ubiquitin-mediated destruction and thereby revealing another
level of HIF regulation . The activity of Siah2 is induced under
physiologic hypoxia (,10% oxygen), resulting in reduced levels of
EGLN2 and stabilization of HIF-1a. The present findings suggest
that the overexpression of EGLN2 via somatic pairing is sufficient
to counteract the suppressive activity of Siah2 under physiologic
Figure 5. BNI3PL is regulated by an EGLN2 mediated oxygen-sensitive response. (A) BNIP3L expression level was measured by qRT-PCR in
U2OS cells transfected with plasmids encoding EGLN2 or empty plasmid (MOCK) under physiologic hypoxia (cyclical 0–7% oxygen) or hyper-
oxygenated condition (21% oxygen) (left panel). BNIP3L expression level was measured by qRT-PCR in U2OS cells maintained under hyper-
oxygenated condition with or without CoCl2(right panel). BNIP3L expression was normalized to beta-actin and expression in the MOCK transfected
cells was arbitrarily set to 1. Error bars represent the standard deviation between the normalized value versus the MOCK performed in triplicate. (B)
The experiment was performed in CAKI cells that contain detectable levels of BNIP3L via Western blot analysis. CAKI cells were transfected with
plasmids encoding EGLN2 or empty plasmid (MOCK), lysed, equal amounts of cell lysates separated on SDS-PAGE and immunoblotted with the
indicated antibodies (left panel). CAKI cells grown in hyper-oxygenated conditions were treated with or without CoCl2. Equal amounts of cell lysates
were resolved by SDS-PAGE and immunoblotted with the indicated antibodies (right panel).
Somatic Pairing Mediated Deregulation of EGLN2
PLoS Genetics | www.plosgenetics.org7September 2008 | Volume 4 | Issue 9 | e1000176
hypoxia. Under hyper-oxygenated conditions (21% oxygen;
frequently used as experimental normoxia), Siah2 activity is
attenuated via a yet-defined mechanism, resulting in the increased
abundance of EGLN2 and concomitant reduction in the level of
HIF-1a . The ectopic expression of EGLN2 under 21%
oxygen did not result in further diminution of HIF-target gene
expression (data not shown), which is likely due to the fact that
endogenous EGLN2 is highly abundant or that every available
EGLN2 is already activated under hyper-oxygenated conditions.
HIF-regulated genes are involved in many physiological
processes including angiogenesis, metabolism, cell proliferation,
survival, and apoptosis. As such, disruption in the regulation of
HIF may affect several regulatory pathways that contribute to the
transformation of normal cells into cancer cells. Evasion of
apoptosis is one of the hallmark features of cancer cells and
represents a key oncogenic event. BNIP3L is a regulator of p53-
dependent apoptosis and silencing of BNIP3L has been associated
with enhanced tumorgenicity and reduced apoptotic response
. We show here that BNIP3L is one of several HIF-responsive
genes governed, in part, by EGLN2. Therefore, we propose that
the downregulation of BNIP3L is the result of chromosome-
pairing induced upregulation of EGLN2 and that downregulation
of BNIP3L contributes to the inhibition of apoptosis to facilitate
oncocytoma cell survival and growth.
The disruption of HIF activity has been associated with kidney
cancer related to VHL disease, sporadic clear cell RCC, and
hereditary papillary RCC [38,41,42]. The present study reveals
deregulation of the oxygen-sensing response in renal oncocytoma,
as well as chromophobe RCCs (which display DNA amplification
mediated up-regulation of EGLN2) and thereby supporting the
dysfunction of HIF pathway as a common and perhaps central
theme in the pathogenesis of kidney cancer.
Materials and Methods
Gene Expression Profiling and Analysis
Single-color expression profiles were generated using the HG-
U133 Plus 2.0TMchipset (Affymetrix, Santa Clara, CA) from renal
oncocytoma (n=10), chromophobe RCC (n=10), and nondi-
seased kidney (n=12) samples as described . The gene
expression data can be obtained at the Gene Expression Omnibus
(GSE8271 and GSE7023). Analysis was performed using BioCon-
ductor version 2.0 software. Data preprocessing was performed
using the RMA method as implemented in the affy package and
using updated probe set mappings such that a single probe set
describes each gene [44,45,46]. Chromosomal abnormalities were
predicted using the comparative genomic microarray analysis
(CGMA) method as implemented in the reb package . Briefly,
for each measured gene, the gene expression value was normalized
such that the average gene expression value in the nondiseased
samples was subtracted from the tumor-derived gene expression
value. A Welsh’s t-test was applied to the relative gene expression
values that mapped to each chromosome arm. For the smoothing
curve, the normalized expression values derived from genes
mapping to chromosome 19 were replaced by a summary score
that comprised a running two-sided t-test statistic using window
sizes of 61, 245, and 611 (representing 5%, 20%, and 50% of the
length of the chromosome). The results of the three smoothing
curves were averaged. To identify HIF-interacting genes, the
EntrezGene database (http://www.ncbi.nlm.nih.gov/sites/
entrez) was searched using the search string ‘(‘‘HIF’’ or ‘‘VHL’’)
and ‘‘19’’[chr] and ‘‘homo sapiens’’[orgn]’. Differentially ex-
pressed genes were identified using a two-sided t-test. For HIF
target gene analysis, 36 known HIF-responsive genes identified in
Maynard et al. were isolated . Enrichment of up- and down-
regulated genes in the HIF target gene set was performed by
comparing differences in the expression level ranks between HIF
target gene set to the results of 10,000 randomly generated 36-
gene sets. Ranks were based on tumor versus normal expression
comparisons as implemented in the limma package .
DNA Copy Number Profiling and Analysis
SNP allele calls were generated using the GeneChip Mapping
100 K SetTM(Affymetrix, Santa Clara, CA) according to the
manufacturer’s supplied protocol. Image quantification was
performed with a GeneChip Scanner 3000 and the resulting data
was processed using GCOS 1.4 (Affymetrix, Santa Clara, CA) with
default analysis settings. Allele calls were generated using GTYPE
4.0 (Affymetrix, Santa Clara, CA) with a confidence threshold set
at 0.25. Raw copy numbers in log2-transformed format (non-
paired reference and test samples) were exported from the CNAG
version 2.0 (Affymetrix, Santa Clara, CA) software using normal
references downloaded from Affymetrix (http://www.affymetrix.
com;ccnt_reference_data). DNA copy number changes were
visualized by data smoothing in which raw copy number values
were replaced by a summary score that comprised a running 1-
sided t-test statistic with window size set to 31, where each SNP
probe along with 15 59 SNPs and 15 39 SNPs were included in the
window. DNA copy number data can be obtained at the Gene
Expression Omnibus (GSE8271).
FISH and WCP
Bacterial artificial chromosomes (BACs) RP11-157B13 (19p12),
RP11-1137G4 (19p13.3), RP11-15A1 (19q13.31) were obtained
from the Children’s Hospital Oakland Research Institute (http://
bacpac.chori.org) and BAC CTC-429C10 (19q13.41) was pur-
chased from Invitrogen (Invitrogen Corporation, Carlsbad, CA).
These clones were labeled with either SpectrumGreen or
SpectrumOrange (Abbott Molecular Inc, Des Plaines, IL) by nick
translation and applied to tissue touch preps of oncocytoma samples
as described , with the exception that slides were counterstained
with VECTASHIELD (Vector Laboratories, Inc. Burlingame, CA)
anti-fade 49,6-diamidino-2-phenylindole (DAPI). Telomere-specific
DNA probes, the chr 1,5,19 alpha satellite probe, and the arm-
specific paints were purchased from Q-BIOgene (MP Biomedicals,
Solon, OH). FISH was performed using these probes according to
the manufacturer’s supplied protocol. As the alpha satellite probe
cross-hybridizes to chromosome 1 and chromosome 5, in all studies
chromosome 19 was co-labeled with a probe that maps distal to the
centromere, RP11-157B13 (19p12). In addition, analysis of the
centromeric probe on the metaphase spreads of control cells
revealed that hybridization to chromosome 1 resulted in a
significantly brighter signal (data not shown). These hybridization
characteristics allowed the discrimination between chr 1 and 5
For image quantification, three separate photomicrographs
containing five, six, and three cells, respectively, in which the
19q31.31 FISH signals were in the same image plane were
obtained. Photomicrographs were processed using the rtiff package
for the R environment . The fluorescent FISH signals were
automatically segmented from background using the method of
Ridler and Calvard , individual spots were identified using the
connected component algorithm , and the number of pixels
per feature were calculated. Twelve doublet FISH signals and
eight singlet FISH signals were compared. Differences in size were
evaluated using a one-sided Student’s t-test.
Somatic Pairing Mediated Deregulation of EGLN2
PLoS Genetics | www.plosgenetics.org8 September 2008 | Volume 4 | Issue 9 | e1000176
U2OS osteosarcoma cell and CAKI renal clear-cell carcinoma
cell lines were obtained from the American Type Culture
Collection (Rockville, MD) and maintained in Dulbecco’s
modified Eagle’s medium supplemented with 10% heat-inactivat-
ed fetal bovine serum (Sigma, Milwaukee, WI) at 37uC in a
humidified 5% CO2atmosphere. Cyclic hypoxia treatment of cells
were performed in humidified chambers at 37uC and flushed with
5% CO2balance N2for 30 min, followed by 5% CO2and 7% O2
balance N2for 30 min as one cycle. Cells were grown in these
chambers for 16 hours .
Polyclonal anti-EGLN2 and anti-BNIP3L antibodies were
obtained from Bethyl Laboratories (Montgomery, TX) and Sigma
(Milwaukee, WI), respectively. Polyclonal HIF1a and monoclonal
HIF2a antibodies were obtained from BD Biosciences (San Jose,
CA) and Novus (Littleton, CO), respectively. Monoclonal anti-
vinculin antibody was obtained from Abcam (Cambridge, MA).
Mammalian expression plasmids pcEglN2 was generated by PCR
from Flag-EglN2, a kind gift from Dr. Mircea Ivan, using primers 59-
GACGACGGATCCATGGACAGCCCGTGCCAGC-39 and 59-
GACGACGAATTCCTAGGTGGGCGTAGGCGGC -39. The
PCR product was then ligated into the BamHI and EcoRI sites in
pcDNA3(+). Plasmid was confirmed by direct DNA sequencing.
Western blotting were performed as described previously .
Quantitative Real-Time PCR
For first-strand cDNA synthesis, 1 ml of oligo(dT)23 primer
(Sigma) was incubated with 5 mg of RNA and distilled H2O (total
reaction volume of 20 ml) for 10 min at 70uC in a thermal cycler
(MJ Research, Boston, MA). The mixture was cooled to 4uC, at
which time 4 ml of 56 first-strand reaction buffer, 2 ml of 0.1 M
DTT, 1 ml of a 10 mM concentration of each deoxynucleoside
triphosphate, and 1 ml of Superscript II reverse transcriptase
(Invitrogen) were added. cDNA synthesis was performed for 1.5 h
at 42uC, followed by 15 min at 70uC in the thermal cycler. Human
genomic DNA standards (human genomic DNA was obtained from
Roche, Mannheim, Germany) or cDNA equivalent to 20 ng of total
RNA were added to the quantitative PCR (qPCR) reaction mixture
in a final volume of 10 ml containing 16 PCR buffer (without
MgCl2), 3 mM MgCl2, 0.25 units of Platinum Taq DNA polymer-
ase, a 0.2 mM concentration of each deoxynucleoside triphosphate,
0.3 ml of SYBR Green I, 0.2 ml of ROX reference dye, and a
0.5 mM concentration of each primer (Invitrogen). Amplification
conditions were as follows: 95uC (3 min), 40 cycles of 95uC (10 s),
65uC (15 s), 72uC (20 s), and 95uC (15 s). qPCR was performed
using the ABI Prism 7900HT Sequence Detection System (Applied
Biosystems, Foster City, CA). Gene-specific oligonucleotide primers
designed using Primer Express (Applied Biosystems) were as follows:
BNIP3L primer set (59- CTGCACAAACTTGCACATTG-39 and
59- TAATTTCCACAACGGGTTCA-39), HMOX1 primer set (59-
GAATTCTCTTGGCTGGCTTC-39 and 59- TCCTTCCTCC-
TTTCCAGAGA-39), ENO1 primer set (59- CAGCTCTAGCT-
TTGCAGTCG-39 and 59-GACACGAGGCTCACATGACT-39),
CITED2 primer set (59-ACTGCACAAACTGCCATCTC-39 and
59-CAGCCAACTTGAAAGTGAACA-39), beta-actin primer set
(59- GGATCGGCGGCTCCAT-39 and 59- CATACTCCTGC-
TTGCTGATCCA-39), GLUT-1 primer set (59- CACCACCT-
CATGTTTCTA-39). SYBR Green I fluoresces during each cycle
of the qPCR by an amount proportional tothe quantity ofamplified
cDNA (the amplicon) present at that time. The point at which the
fluorescent signal is statistically significant above background is
defined as the cycle threshold (CT). Expression levels of the various
transcripts were determined by taking the average CTvalue for each
cDNA sample performed in triplicate and measured against a
standard plot of CTvalues from amplification of serially diluted
human genomic DNA standards. Since the CTvalue is inversely
proportional to the log of the initial copy number, the copy number
ofanexperimentalmRNAcanbeobtained fromlinearregression of
the standard curve. A measure of the relative difference in copy
number was determined for each mRNA. Values were normalized
to expression of beta-actin mRNA and represented as the mean value
experiments performed in triplicate6standard deviations.
Purification of HIF Prolyl Hydroxylase 1 (EGLN2/PHD1)
Extracts containing enriched EGLN2 were purified from rabbit
reticulocyte lysate as previously described . Briefly, approxi-
mately 1 L of rabbit reticulocyte lysate (Green Hectares, Oregon,
WI) was diluted to 5 L in 50 mM Tris-HCl (pH 7.4), 0.1 M KCl,
and 5% (vol/vol) glycerol and then was precipitated with 0.213 g/
ml (NH4)2SO4. After centrifugation at 16,0006g for 45 min at
4uC, the resulting supernatant was precipitated with an additional
0.153 g/ml (NH4)2SO4. After centrifugation at 16,0006g for
45 min at 4uC, the pellet was resuspended in Buffer A (40 mM
HEPES-NaOH [pH 7.4] and 5% (vol/vol) glycerol), dialyzed
against Buffer A to a conductivity equivalent to Buffer A
containing 0.2 M KCl, and applied at 0.5 L/h to a 0.5 L
phosphocellulose (Whatman, P11) column equilibrated in Buffer A
containing 0.2 M KCl. The phosphocellulose column was eluted
stepwise at 1 L/h with Buffer A containing 0.5 M KCl, and 100-
ml fractions were collected. Proteins eluting in the phosphocellu-
lose 0.5 KCl step were pooled and precipitated with 0.4 g/ml
(NH4)2SO4. After centrifugation at 16,0006g for 45 min at 4uC,
the pellet was resuspended in 4 ml of Buffer A. Following
centrifugation at 35,0006g for 30 min at 4uC, the resulting
supernatant was applied at 2 ml/min to a TSK SW3000 HPLC
column (Toso-Haas, Montgomeryville, PA; 21.56600 mm) equil-
ibrated in Buffer A containing 0.15 M KCl. The SW3000 column
was eluted at 2 ml/min, and 4 ml fractions containing enriched
EGLN2 were collected.
In Vitro Binding Assay
An in vitro binding assay was performed as described previously
. TNT reticulocyte lysate (Promega) translation products were
synthesized in the presence or absence of35S-methionine. HIF1a-
(ODD) translation products were incubated with cellular extract
fractions containing enriched EGLN2, where indicated, for
30 min at 37uC. Gal4-HA-HIF-1a (10 ml) and HA-VHL (10 ml)
translation products were incubated with the indicated antibodies
and protein A-Sepharose in 750 ml of EBC buffer (50 mM Tris
[pH 8], 120 mM NaCl, 0.5% Nonidet P-40). After five washes
with NETN buffer (20 mM Tris (pH 8), 100 mM NaCl, 0.5%
Nonidet P-40, 1 mM EDTA), the bound proteins were resolved on
SDS-PAGE and detected by autoradiography.
In Vitro Ubiquitylation Assay
An in vitro ubiquitylation assay was performed as described
HA-HIF1a(ODD) (4 ml) were incubated in RCC 786-O S100
extracts (100–150 mg). Reactions were supplemented with an
increasing titration of EGLN2-enriched cellular fraction where
[35S]Methionine-labeled reticulocyte lysate Gal4-
Somatic Pairing Mediated Deregulation of EGLN2
PLoS Genetics | www.plosgenetics.org9September 2008 | Volume 4 | Issue 9 | e1000176
indicated. Additional reaction supplements include 8 mg/ml
ubiquitin (Sigma), 100 ng/ml ubiquitin-aldehyde (BostonBiochem,
Inc., Cambridge, MA), and an ATP-regenerating system (20 mM
Tris [pH 7.4], 2 mM ATP, 5 mM MgCl2, 40 mM creatine
phosphate, 0.5 mg/ml of creatine kinase) in a reaction volume of
20–30 ml for 1.5 h at 30uC.
HIF Dose Response
Figure 5B from the Jiang et al. article  was obtained in
Portable Document Format (PDF, Adobe Systems), imported into
Canvas 9 (ACD Systems), and the x- and y-graphic device
coordinates of each data point, the x-axis ticks (oxygen
concentration), and the y-axis ticks (densitometry) were extracted.
Linear interpolation was used to convert the graphic device
coordinates to protein densitometry measurements and oxygen
concentrations. Based on comparisons between the extracted
oxygen concentrations (0.5, 1.9, 2.9, 3.9, 4.8, 5.8, 7.9, 9.9, 11.9,
13.9, 19.9) and the actual oxygen concentrations (0.5, 2, 3, 4, 5, 6,
8, 10, 12, 14, 20), the extracted data varied on average less than
2% from the original data. The densitometry and oxygen
concentration data were log2-transformed and linear model fit to
the transformed data. The best-fit power-law equation is
HIF1a=22.61O20.85, where HIF1a represents HIF-1a protein
levels and O represent oxygen concentration.
Found at: doi:10.1371/journal.pgen.1000176.s001 (0.06 MB PDF)
Regional transcriptional abnormalities in renal
Found at: doi:10.1371/journal.pgen.1000176.s002 (0.22 MB PDF)
Expression of EGLN2 in Chromophobe RCC.
in response to changing oxygen concentration.
Found at: doi:10.1371/journal.pgen.1000176.s003 (0.22 MB PDF)
HIF1 protein and HIF1 DNA-binding activity levels
Found at: doi:10.1371/journal.pgen.1000176.s004 (0.15 MB PDF)
BNIP3L expression associates with HIF expression.
are suppressed by EGLN2.
Found at: doi:10.1371/journal.pgen.1000176.s005 (0.01 MB PDF)
Hypoxia-responsive genes repressed in oncocytoma
Found at: doi:10.1371/journal.pgen.1000176.s006 (0.04 MB PDF)
Chromosome 19 FISH patterns in chromophobe
Found at: doi:10.1371/journal.pgen.1000176.s007 (0.04 MB PDF)
Chromosome 19 FISH patterns in oncocytoma.
Found at: doi:10.1371/journal.pgen.1000176.s008 (0.07 MB PDF)
Cancer related genes mapping to chromosome 19q.
We would also like to thank the Cooperative Human Tissue Network of the
National Cancer Institute and the French Kidney Cancer Consortium
(Sophie Giraud, Sophie Ferlicot, Philippe Vielh, Delphine Amsellem-
Ouazana, Bernard Debre ´, Thierry Flam, Nicolas Thiounn, Marc Zerbib,
Ge ´rard Benoı ˆt, Ste ´phane Droupy, Vincent Molinie ´) for providing samples
Conceived and designed the experiments: BTT MO KAF. Performed the
experiments: JK RR DP MW KK DM LF. Analyzed the data: JK RR JM
ZZ HH EK KAF. Contributed reagents/materials/analysis tools: MHT
KD EK RK SR AV PS BTT MO KAF. Wrote the paper: PS BTT MO
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PLoS Genetics | www.plosgenetics.org11September 2008 | Volume 4 | Issue 9 | e1000176