Differential expression of a novel ankyrin
containing E3 ubiquitin-protein ligase, Hace1,
in sporadic Wilms’ tumor versus normal kidney
Michael S. Anglesio1,2, Valentina Evdokimova1,2, Nataliya Melnyk1,2, Liyong Zhang3,4,
Conrad V. Fernandez5, Paul E. Grundy6, Stephen Leach7, Marco A. Marra7,
Angela R. Brooks-Wilson7, Josef Penninger8and Poul H.B. Sorensen1,2,*
1Department of Pathology and2Department of Pediatrics, British Columbia Research Institute for Children’s and
Women’s Health, University of British Columbia, Vancouver, BC, Canada V5Z 4H4,3Department of Medical
Biophysics and4Department of Immunology, University of Toronto, Toronto, Ontario, Canada,5Department of
Pediatrics, IWK Grace Health Centre, Halifax, Nova Scotia, Canada B3L 3G9,6Cross Cancer Institute, Edmonton AB,
Canada T6G 1Z2,7Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4S6
and8Institute for Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria
Received May 7, 2004; Revised and Accepted July 6, 2004
We have analyzed the chromosome 6q21 breakpoint of a non-constitutional t(6;15)(q21;q21) rearrangement in
sporadic Wilms’ tumor. This identified a novel gene encoding a protein with six N-terminal ankyrin repeats
linked to a C-terminal HECT ubiquitin-protein ligase domain. We therefore designated this gene HACE1
(HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1). HACE1 is widely expressed in
human tissues, including mature and fetal kidney. We show that Hace1 protein possesses intrinsic ubiquitin
ligase activity, utilizes UbcH7 as a candidate partner E2 enzyme and localizes predominantly to the endoplas-
mic reticulum. Although the HACE1 locus was not directly interrupted by the translocation in the index
Wilms’ case, its expression was markedly lower in tumor tissue compared with adjacent normal kidney.
Moreover, HACE1 expression was virtually undetectable in the SK-NEP-1 Wilms’ tumor cell line and in four
of five additional primary Wilms’ tumor cases compared with patient-matched normal kidney. We found no
evidence of HACE1 mutations or deletions, but hypermethylation of two upstream CpG islands correlates
with low HACE1 expression in tumor samples. Our findings implicate Hace1 as a novel ubiquitin-protein
ligase and demonstrate that its expression is very low in primary Wilms’ tumors.
Analysis of chromosomal translocations in human malig-
nancies has led to the characterization of numerous genes
involved in oncogenesis. For example, numerous transloca-
tions have been found to either activate proto-oncogenes or
to generate gene fusions encoding dominantly acting chimeric
oncoproteins (1). Less commonly, mapping of translocations
has highlighted the positions of novel tumor suppressor
genes (TSGs) within deletion hotspots of tumors, including
the von Hippel Lindau (VHL) gene of renal carcinoma (2), the
NF2 gene in neurofibromatosis type 2 (3), the hSNF5/INI1
gene in malignant rhabdoid tumors (4,5) and the BCSC-1
locus in breast and other cancers (6).
We recently identified a balanced non-constitutional
t(6;15)(q21;q21) translocation in a sporadic Wilms’ tumor
occurring in a 5-month-old male (7). Although Wilms’
tumor is the most common renal neoplasm in children,
accounting for ?90% of pediatric kidney tumors and 6% of
all childhood cancers, the genetics of sporadic Wilms’ tumor
remain largely unknown (8). Rearrangements of the 6q21
region have been previously reported in this tumor, including
t(5;6)(q21;q21) and t(2;6)(q35;q21) translocations (9–11).
LOH of this region appears to be rare in Wilms’ tumor (9),
Human Molecular Genetics, Vol. 13, No. 18 # Oxford University Press 2004; all rights reserved
*To whom correspondence should be addressed. Tel: þ1 6048752936; Fax: þ1 6048753417; Email: email@example.com
Human Molecular Genetics, 2004, Vol. 13, No. 18
Advance Access published on July 14, 2004
and there is currently little evidence for a 6q21 Wilms’ tumor
suppressor locus. However, deletions of 6q21 have been
widely reported in human malignancies, including carcinomas
of the breast, ovary and prostate, as well as in leukemias and
lymphomas (12–15), and this region is hypothesized to harbor
one or more TSGs (16). These observations highlight chromo-
some 6q21 as a region frequently targeted for genomic altera-
tions in human cancer. We therefore postulated that the t(6;15)
translocation of the index case might be contributing to
Wilms’ tumor oncogenesis, either through oncogene acti-
vation or by targeting a 6q21 TSGs. This prompted us to
search for the 6q21 gene or genes potentially altered by the
In this study, we have characterized the 6q21 region inter-
rupted by the Wilms’ tumor t(6;15) translocation. We report
that the 6q21 breakpoint maps to a non-coding region with
.200 kb from the nearest known gene, but ?50 kb upstream
of a novel open reading frame (ORF). This ORF encodes a
protein with a previously unreported domain architecture con-
sisting of six ankyrin repeats linked to a HECT (homologous
to E6-AP C-terminus) domain. HECT domains have thus far
only been described in E3 ubiquitin-protein ligases (17,18).
E3 ligases are essential components of a highly conserved
pathway involving conjugation of one or more ubiquitin
(Ub) polypeptides to specific substrate proteins, leading in
most cases to substrate proteasomal degradation (17,19). We
therefore designated this gene HACE1 (HECT domain and
Ankyrin repeat Containing E3 ubiquitin-protein ligase 1).
Hace1 protein has in vitro and in vivo ubiquitin ligase activity
and represents, to our knowledge, the first documented
example of a HECT domain containing ubiquitin ligase pos-
sessing N-terminal ankyrin repeats. We found that although
HACE1 is ubiquitously expressed in normal tissues, including
both adult and fetal kidneys, mRNA and protein levels were
almost undetectable in the index case and in four of five insti-
tutional Wilms’ tumor cases compared with matching adjacent
normal kidney, and in the SK-NEP-1 Wilms’ tumor cell line.
Lower expression was strongly associated with hypermethyla-
tion of two CpG islands located upstream of the HACE1 locus.
Our findings implicate Hace1 as a novel HECT E3 ubiquitin-
protein ligase whose lack of expression is associated with
sporadic Wilms’ tumor.
Mapping the 6q21 breakpoint in Wilms’ tumor
To assess candidate genes potentially affected by the
t(6;15)(q21;q21) translocation in the index case, we focused
on the 6q21 breakpoint as this region has previously been
implicated in Wilms’ tumors and other human malignancies.
Using 6q21 bacterial artificial chromosomes (BACs) for fluor-
escence in situ hybridization (FISH), we identified several
overlapping BACs that spanned the translocation breakpoint
(i.e. that showed three signals as opposed to the normal two;
Fig. 1A). On the basis of sequence information from NCBI
(http://www.ncbi.nlm.nih.gov) and UCSC (http://genome.
ucsc.edu) genome databases, we assembled a precise map of
the involved 6q21 region and narrowed the breakpoint to an
?12 kb region between the D6S2097 and STSG6084
polymorphic markers (Fig. 1C). We next determined the
restriction enzyme distribution within this region from pub-
licly available genomic sequences. Southern blotting using
genomic DNA from the index case and the SB1 probe (Fig.
1C) allowed us to confirm the position of the 6q21 breakpoint,
as rearrangements were detected in the tumor sample but not
in matched normal tissue or control samples (shown for PstI
digests in Fig. 1B).
Identification of the HACE1 gene
Using public database sequences, we next inspected the region
surrounding the 6q21 breakpoint for candidate genes poten-
tially affected by the rearrangement. No previously character-
ized genes were present in this region. We therefore used
GenScan gene prediction software to search for novel ORFs.
No coding sequences were identified spanning the breakpoint,
indicating that the translocation does not directly disrupt a
gene in the 6q21 region. However, a novel 2727 bp ORF
located ?50 kb downstream was identified by this strategy,
a portion of which matched to the 50end of a non-annotated,
IMAGE Consortium expressed sequence tag (EST; GenBank
accession no. BC034982). We obtained this cDNA clone
and sequenced it in its entirety, revealing 100% sequence iden-
tity with the predicted full-length ORF from 6q21. Alignment
to publicly available 6q21 genomic sequences using the UCSC
genome browser BLAT tool predicts that this ORF is orga-
nized into 24 exons (Fig. 1C). The nearest known gene,
BVES (blood vessel epicardial substance), lies ?200 kb
upstream of the breakpoint (Fig. 1C).
Protein domain analysis predicted that the 6q21 ORF
encodes a 909 amino acid protein (?103 kDa) possessing
six N-terminal ankyrin repeats and a C-terminal HECT
domain (Fig. 1D). Ankyrin repeats are well-documented to
mediate protein–protein interactions (20), whereas HECT is
a catalytic domain possessing ubiquitin-protein ligase activity
(17,18,21). We therefore designated this gene HACE1. Com-
parison of the Hace1 protein sequence to public databases
using NCBI BLAST and DART tools (http://www.ncbi.nlm.
nih/BLAST) highlights Hace1 as the first documented
example of an E3 ligase in which a HECT domain is linked
to ankyrin repeats. Interestingly, when the HACE1 coding
sequence was aligned with those of other genomes using the
BLAT tool at the UCSC genome database (http://genome.
ucsc.edu), orthologues of HACE1 appear to be present only
in vertebrate species (data not shown). Hace1 ankyrin
repeats show .47% sequence similarity with those of the
p19INK4D (Fig. 1D), both of which are known to be inacti-
vated in human malignancies including Wilms’ tumor (22).
The Hace1 HECT domain is also highly conserved, with
more than 53% sequence similarity to the HECT domains of
well-characterized ubiquitin ligases including E6-AP, which
destabilizes p53 in HPV infected cervical carcinoma cells
(23), Nedd4, which regulates stability of the epithelial Naþ
channel (ENaC) and insulin-like growth factor 1 receptor
(21), and Smurf 1 and 2, which are involved in ubiquitination
of TGF-b receptors (24). HECT family E3 ligases are known
to contain an invariant C-terminal cysteine residue necessary
2062 Human Molecular Genetics, 2004, Vol. 13, No. 18
Figure 1. Identification of a novel gene, HACE1, within the t(6;15)(q21;q21) breakpoint region in the Wilms’ tumor index case. (A) FISH using 6q21 BAC probe
809N15 (green) and a telomeric probe for 6q (red). Normal Chr 6 (white arrow), derivative Chr 15 (red arrow) and derivative Chr 6 (yellow arrow) are indicated.
(B) Southern blotting using SB1 probe indicated in (B) demonstrates re-arrangement in PstI digested genomic DNA from the index tumor (T) case but not in
peripheral blood (N) or control samples (C1and C2). (C) Mapping of the 6q21 breakpoint region. Twenty-four exon structure of HACE1 as predicted by the
UCSC genome browser BLAT tool is shown. BACs used for mapping of the breakpoint region were identified using Finger printed contigs (FPC) software
and sequence information from the NCBI and UCSC genome databases and are indicated as follows: BACs spanning the t(6;15)(q21;q21) breakpoint
(double green lines); BACs that remain on the derivative Chr 6 (red single lines); BACs detected on the derivative 15 (blue single line), green boxes denote
CpG islands. (D) Hace1 (GenBank accession number AAH34982) consists of six N-terminal ankyrin repeats and a C-terminal HECT domain as predicted
by Pfam and SMART database tools. An invariant Cys residue in the HECT domain is indicated by asterisk. Alignment to a number of proteins containing
ankyrin repeats or HECT domains are shown below; hashed region corresponds to the region of significant similarity. Percentage similarity was calculated
using BLAST tools.
Human Molecular Genetics, 2004, Vol. 13, No. 182063
for thioester bond formation with ubiquitin (18); in Hace1 this
appears to be represented by Cys-876 (Fig. 1D).
Normal HACE1 expression patterns
We next examined HACE1 expression profiles in normal
human tissues by northern analysis. Using a full-length
cDNA probe, we found that human HACE1 is expressed as
a single mRNA species of ?4.6 kb in multiple tissues, includ-
ing strong expression in heart, brain and kidney (Fig. 2A). As
Wilms’ tumor is hypothesized to derive from early metane-
phrogenic stem cells (25–28), we also assessed HACE1
expression in three (day 54–122) human fetal kidney tissue
samples. We found that, although variable, mRNA transcripts
are expressed in these cells at levels that are similar to or
greater than in mature pediatric kidney (Fig. 2B). There was
no obvious trend in expression patterns with the age of the
human fetal kidney samples, although this requires a larger
sample size for rigorous evaluation. To examine expression
of Hace1 protein, we generated polyclonal a-Hace1 antibodies
directed towards either the second ankyrin repeat or to full-
length recombinant protein. Both antibodies detect a protein
with the expected size of 103 kDa identical to that of recom-
binant Hace1 in both fetal and pediatric kidney cells, as well
as in the HEK293 human embryonic kidney cell line (Fig. 2C).
Hace1 possesses ubiquitin ligase activity in vitro and in vivo
To determine whether Hace1 possesses a ubiquitin ligase
activity, we tested the ability of this protein to form a thioester
E3 ligases (17). In vitro [35S]-labeled Hace1 was incubated in
the presence of GST–Ub and E1 Ub-activating enzyme,
along with a panel of E2 Ub-conjugating enzymes (Fig. 3A).
As HECT domains contain a conserved Cys residue which is
crucial for Ub transfer to substrate proteins, we mutated Cys-
876 to Ser and tested this Hace1-C876S mutant in thioester
bond formation. As seen in Figure 3A (top panel), an additional
higher molecular mass band corresponding to the expected size
presence of the UbcH7 E2 enzyme (lane 8). Thioester bond for-
mation was completely abolished by the C876S substitution in
Hace1 (Fig. 3A, bottom panel) or by disulphide bond reducing
agent b-mercaptoethanol (Fig. 3B; compare lanes 1 and 4),
indicating specificity of the reaction. These data confirm
Hace1 ubiquitin ligase activity in vitro and identify UbcH7 as
a candidate partner E2.
To demonstrate the involvement of Hace1 in the ubiqui-
tination of proteins in vivo, HA-tagged Hace1 was stably
expressed in NIH3T3 fibroblasts (Fig. 3C). Cytoplasmic
extracts from these cells were then subjected to immunopreci-
pitation with a-HA antibodies followed by western blotting
with a-Ub antibodies to assess whether Hace1 associates
with ubiquitinated proteins. As shown in Figure 3D, overall
levels of protein ubiquitination were similar in HA–Hace1
and vector alone cells. However, high molecular weight
ubiquitinated proteins could only be detected in a-HA
immunoprecipitates from the HA–Hace1 expressing cells
(compare lanes 3 and 4). The level of ubiquitinated proteins
in these immunoprecipitates was increased in the presence of
proteasome inhibitors such as lactacystin or MG132 (data
not shown), indicating that at least some of the proteins tar-
geted for ubiquitination by Hace1 are normally degraded by
the proteasome. Moreover, we found that Hace1 directly inter-
acts with the 26S proteasomal complex as Hace1 could be
immunoprecipitated using antibodies against the 20S core pro-
teasomal subunits (Fig. 3E, lane 3). Although the identity of
Hace1 target proteins in the earlier mentioned immunoprecipi-
tates remains to be established, our data strongly support the
involvement of Hace1 in ubiquitination and degradation of
Subcellular localization of Hace1
To further characterize Hace1, we analyzed its localization
western blotting demonstrated that in exponentially growing
NIH3T3fibroblasts both endogenous
expressed Hace1 are found predominantly in the endoplasmic
reticulum (ER) and the cytoplasm, although a small amount of
endogenous protein is also present in other fractions (Fig. 4A).
ER localization was confirmed by co-immunostaining of
Figure 2. Hace1 expression in normal tissues. (A) Northern blotting demon-
strating HACE1 mRNA expression in a panel of normal tissues. Membrane
was probed with the full-length Hace1 cDNA. (B) qRT–PCR showing
HACE1 expression in three normal fetal kidney samples (fk1–3), pediatric
(ped.) kidney and HEK293 (human embryonic kidney) are shown for compari-
son. Expression of HACE1 is normalized to the ped. kidney sample. (C)
Western blotting showing Hace1 protein expression fetal kidney samples
[from (B)]. Actin serves as a loading control (lower panel).
2064 Human Molecular Genetics, 2004, Vol. 13, No. 18
Hace1 with the ER resident, BiP (Grp78). As seen in
Figure 4B, a predominantly perinuclear reticular staining
pattern of the ER and of the nuclear envelop was observed
for both Hace1 and BiP in NIH3T3 cells using immunofluor-
escence microscopy. This suggests that a significant portion of
Hace1 localizes to the ER.
Altered expression of HACE1 in sporadic Wilms’ tumor
As the 6q21 breakpoint in the index case occurred ?50 kb
upstream of the HACE1 locus, we hypothesized that HACE1
expression might be affected by the t(6;15) translocation. More-
over, we wondered whether altered HACE1 expression might
represent a recurrent molecular alteration in sporadic Wilms’
tumor. To begin to address this possibility, we assessed
HACE1 mRNA levels in the index case and in five additional
institutional Wilms’ tumor cases with patient-matched adjacent
normal kidney by quantitative RT–PCR (qRT–PCR). The his-
tology of each tumor sample confirmed the presence of at least
80% tumor cells (data not shown). Of the six paired samples,
five tumor specimens showed markedly lower HACE1 mRNA
expression compared with their matching normal kidney
counterparts, including the index case (Fig. 5A). Although
there was variability in the absolute degree to which HACE1
expression was lower, the average decrease in transcript levels
was ?5-fold. Low expression was confirmed at the protein
level by western blotting using a-Hace1 antibodies (Fig. 5B),
as expression of Hace1 protein was very low or undetectable
in the index case and in the other four cases with low mRNA
levels, compared with matching normal kidney. We next
assessed HACE1 expression levels and the presence of variant
transcripts in the SK-NEP-1 Wilms’ tumor cell line by northern
analysis. Transcript levels are virtually undetectable in these
cells compared with HEK293 human embryonic kidney cells
or Ewing tumor and rhabdomyosarcoma cell lines (Fig. 5C).
Hace1 mRNA expression was also low to non-detectable in
neuroblastoma cell lines. In agreement with these findings,
Hace1 protein levels are extremely low in SK-NEP-1 and neuro-
blastoma cell lines compared with HEK293, Ewing tumor and
rhabdomyosarcoma cells (Fig. 5D).
Absence of HACE1 mutations or deletions in sporadic
Current views hold that Wilms’ tumor most likely derives
from embryonic kidney (26–28). As we observed strong
Figure 3. Hace1 exhibits E3 ubiquitin ligaseactivity in vitro and in vivo. (A) Thioester bond formation assay was performedusing the [35S]-methionine labeled wild-
exclusively UbcH7 has been used as a source of E2. Reactions were stopped with or without b-mercaptoethanol. Note that addition of b-mercaptoethanol (lane 1) or
mutation of the invariant Cys residue (lane 8) completely abolished thioester formation. (C and D) Cytosolic extracts from NIH3T3 cells ectopically expressing HA-
Hace1 or vector alone were used for immunoprecipitation (IP) with a-HA antibodies. Of the IP fractions 20% were then subjected to SDS–7% PAGE and western
blotting with a-HA (C) or a-Ub (D) antibodies. Input fraction lanes represent 5% of cytosolic cell extract used for IP. (E) Cytosolic extracts obtained from NIH3T3
cells expressing HA–Hace1 were used for IP with either rabbit pre-immune, a-Ub, a-Hace1 or a-20Sa and b proteasomal subunits antibodies, as indicated. Immu-
noprecipitated proteins were then separated by SDS–7% PAGE and analyzed by western blotting using a-HA antibodies.
Human Molecular Genetics, 2004, Vol. 13, No. 182065
Hace1 expression in both mature pediatric and fetal kidneys
but not in most of the Wilms’ tumors analyzed, one possibility
is that Hace1 down regulation or inactivation is a common and
potentially etiologic event in this disease. However, it is
equally plausible that the specific (as yet unknown) blastemal
cell giving rise to Wilms’ tumor does not express HACE1 and
that the observed lack of expression simply reflects the normal
expression profile of the putative neoplastic precursor cell.
We therefore assessed the Wilms’ tumor cohort for potential
genetic mechanisms of HACE1 down regulation as a strategy
for distinguishing between these possibilities, as loss of TSGs
in malignancies typically involves inactivation of both the
alleles (29). Current literature indicates that chromosomal
rearrangements, large scale deletions or LOH of the 6q21
region are rare in Wilms’ tumor (8–11). Unfortunately,
aside from the index case, karyotypes were not available for
the Wilms’ tumors analyzed in this study. To search for
alternative mechanisms that might underlie altered HACE1
expression in Wilms’ tumor, we screened the cohort of cases
and matched normal kidney as well as the SK-NEP-1 cell
line for HACE1 mutations. All exons and intron–exon bound-
ary regions were sequenced from genomic DNA of each
sample. No sequence discrepancies between tumor and
normal DNAs were found for any of the six cases. Further-
more, no mutations were found in the SK-NEP-1 cell line.
Sequence data were compared with the UCSC human
genome database and a total of eight HACE1 polymorphisms
were identified in the earlier mentioned cohort (Supplementary
Material, Table S1). Two of the polymorphisms were within
the coding sequences, but only one represents an amino acid
change. However, the latter (Asp to Gly at amino acid 399)
does not affect any identifiable functional domains within
Hace1 protein, and when a non-disease control cohort of 95
individuals was tested for this polymorphism it was present
in 1% of samples. Therefore, it most likely represents a low-
frequency polymorphism in the general population. Of the
five Wilms’ tumor cases with low HACE1 expression, at
least one heterozygous polymorphism was found in four of
the tumor (and normal) DNAs as well as in the SK-NEP-1
cell line, suggesting that LOH of the HACE1 locus is unlikely
in this cohort. However, our sequencing strategy cannot rule
out micro-deletions of exons or other genomic sequences
located between these polymorphisms. Therefore, our data
indicate that a genetic mechanism involving point mutations
or deletions is unlikely to explain the low HACE1 expression
observed in Wilms’ tumor, although larger numbers of cases
must be assessed to rigorously test this assumption.
Upstream CpG island methylation is associated with low
Recent studies indicate that promoter hypermethylation may
be as frequent as inactivating mutations for reducing TSG
expression in human malignancies (30). Methylation involves
the addition of methyl groups to cytosine residues within CpG
islands within coding or non-coding sequences of genetic loci,
generally resulting in gene silencing (31,32). We therefore
investigated the methylation status of CpG islands at the
HACE1 locus in the primary Wilms’ tumor cohort and in
the SK-NEP-1 cell line. There are three CpG islands located
within or near the HACE1 locus, one at the promoter extend-
ing into exon 1 which contains 88 CG dinucleotides (CpG-88),
and two located ?50 kb upstream of the Hace1 transcriptional
start site containing 177 (CpG-177) and 29 (CpG-29)
CG dinucleotides repeats, respectively (Fig. 1C). To assess
expression, we analyzed methylation status at each of these
CpG islands by digestion with the methylation sensitive
restriction enzymes HaeII, HpaII or BssHII, followed by
semi-quantitative PCR (Fig. 6A). No evidence of methylation
at CpG-88 was found in any of the tumor or normal kidney
samples (Fig. 6A). However, hypermethylation of either
CpG-177 and CpG-29, or both, was observed in four out of
five Wilms’ tumors with low HACE1 mRNA expression
(Fig. 6A) as well as in SK-NEP-1 cells (Fig. 6B). It was
never observed in any of the normal kidney samples (data
If hypermethylation of CpG-29/CpG-177 is mechanistically
related to low HACE1 expression, then pharmacological
demethylation of these islands would be expected to restore
Figure 4. Hace1 is mainly localized to the ER. (A) Subcellular fractionation of NIH3T3 cell extract. Equal amounts (50 mg) of nuclear (N), mitochondrial (M),
endoplasmic reticulum/polyribosome (ER) and cytosolic (S) fractions were analyzed by western blotting using the respective antibodies, as indicated. Histone
H3, BiP and Grb2 were used as markers of nuclear, ER and cytosolic fractions, respectively. (B) Indirect immunofluorescence microscopy of NIH3T3 cells using
a-Hace1 (red) and BiP (green) antibodies, as indicated. Nuclear staining by propidium iodide is in blue.
2066 Human Molecular Genetics, 2004, Vol. 13, No. 18
expression levels. As SK-NEP-1 Wilms’ tumor cells express
extremely low HACE1 levels and both CpG-29 and CpG-
177 are methylated in this line, we treated cells with the
methylation inhibitor 5-aza-2-deoxycytidine (5AZ) (33,34)
and assessed effects on HACE1 expression. As shown in
Fig. 6B, treatment with this agent reduced methylation at
both CpG-29 and CpG-177, particularly at 5 mM 5AZ. More-
over, this was associated with at least a 4-fold increase in
HACE1 mRNA expression in the presence of 5 mM 5AZ
(Fig. 6C). These findings strongly suggest that methylation
status of CpG-29 and CpG-177 influences HACE1 expression.
To examine the functional consequences of this observation,
we next performed chromatin immunoprecipitation (ChIP)
to analyze whether the HACE1 locus exists in an active or
inactive chromatin conformation in SK-NEP-1 cells. This
was assessed by comparing the relative levels of acetylated-
histone H3 versus dimethyl-(Lys79)-histone H3 bound to the
HACE1 promoter. Acetylated-histone H3 is known to asso-
ciate with DNA of active chromatin, whereas dimethyl-
(Lys79)-histone H3 correlates with chromatin present in the
inactive conformation of heterochromatin or silenced genes
(35–37). As shown in Figure 6D, the promoter region of
the HACE1 gene exists predominantly in an inactive chroma-
tin conformation in normally growing SK-NEP-1 cells
(i.e. associated with dimethyl-(Lys79)-histone H3 by ChIP).
However, treatment with 5 mM 5AZ reverses this pattern,
switching the HACE1 promoter to an active chromatin confor-
mation associated predominantly with the acetylated-histone
H3. As a control, we performed identical studies with
HEK293 cells (Fig. 6D, left panel) which abundantly
express HACE1 (Fig. 4). Consistent with its high expression,
the HACE1 locus exists in an active chromatin organization
in these cells, and no change in chromatin structure is
evident after treatment with 5AZ. Taken together, our data
are consistent with a role for CpG-29 and CpG-177 hyper-
methylations in influencing HACE1 expression, although
additional studies are required to confirm this possibility and
whether it represents an acquired alteration in tumor cells
versus the normal pattern of methylation in the Wilms’
tumor neoplastic precursor cell.
In this study, we demonstrate that a previously uncharacteri-
zed E3 ubiquitin-protein ligase gene is located ?50 kb down-
stream of the 6q21 breakpoint of a t(6;15) translocation in
sporadic Wilms’ tumor. This gene, which we designated
HACE1, encodes a 103 kDa protein containing six N-terminal
ankyrin repeats connected via a linker region to a C-terminal
HECT domain. The latter has to date only been described in
E3 ligases (17), implicating the Hace1 protein as a new
member of the HECT family of E3 ubiquitin-protein ligases.
The protein ubiquitination process involves a highly conserved
pathway in which one or more Ub polypeptides become con-
jugated to specific substrate proteins (17,19). This multistep
process involves a cascade of three different classes of
enzymes. An ATP-dependent Ub-activating E1 enzyme first
forms a thioester bond with Ub, and then transfers the
activated Ub moiety to one of a number of different E2 Ub-
conjugating (Ubc) enzymes. Finally, E3 ubiquitin ligases
catalyze the transfer of Ub from the cognate E2 to a lysine
on the substrate protein. Binding of substrate proteins
appears to be mediated by the N-terminal protein–protein
interaction domains of E3 ligases (17,18,21).
Figure 5. Hace1 expression in Wilms’ tumors and pediatric cancer cell lines.
(A) qRT–PCR showing expression of HACE1 mRNA in Wilms’ tumor
samples relative to patient-matched normal kidney. HACE1 expression relative
to a normal pediatric kidney sample is shown for SK-NEP-1 and HEK293
(human embryonic kidney) cells. (B) Western blotting for the Hace1 protein
showing the tumor (T) samples [from (A)] patient-matched normal kidney
(N). (C) Northern blotting demonstrating HACE1 mRNA expression in a
panel of pediatric tumor cell lines including neuroblastomas (SAN-2, IMR-
32, SK-N-SH), Ewing’s sarcoma (SK-N-MC), rhabdomyosarcomas (Birch,
CT-10), Wilms’ tumor (SK-NEP-1) and HEK293 cells. Membranes were
probed with the full-length Hace1 cDNA. (D) Western blotting showing
Hace1 protein expression in pediatric tumor cell lines [from (C), and an
additional neuroblastoma line, KCNR]. Westerns were done using the N-
terminal a-Hace1 antibody. Recombinant Hace1 protein (lane 10) is used to
confirm the specificity of the antibody. Grb2 serves as a loading control.
Human Molecular Genetics, 2004, Vol. 13, No. 182067
HACE1 is widely expressed in human tissues, including
both fetal and mature kidneys. The Hace1 protein forms thio-
ester bonds with ubiquitin as expected for a HECT family E3
ligase (17,18), and is associated with high molecular weight
ubiquitinated proteins within cells. Among a panel of E2
ubiquitin carrier proteins, thioester bond formation in vitro
occurred in the presence the UbcH7 E2 enzyme, and weakly
with UbcH6 and UbcH5b, all of which are common partners
of other HECT domain containing E3 ubiquitin-protein
ligases. We also found that Hace1 associates with ubiquiti-
nated proteins and components of the 26S proteasomal
complex, indicating that at least some of the proteins targeted
for ubiquitination by Hace1 are likely to be degraded by the
proteasome. Using cell fractionation and immunofluorescence
microscopy, we found that Hace1 localizes predominantly to
the ER indicating its possible involvement in ER-associated
protein degradation (ERAD). ERAD is a quality control
process that selectively directs degradation of misfolded
and aberrant proteins through ubiquitination within the ER
followed by transport to the proteasome (17,38). More
studies are necessary to demonstrate whether Hace1 contri-
butes to this process.
Most HECT E3 ligases characterized to date contain
N-terminal WW domains for substrate interaction and target-
ing (17,18,21). In fact, the presence of ankyrin repeats
within an E3 ligase as in Hace1 has not been previously
described. Given this modular structure, Hace1 may represent
a novel sub-family of HECT E3 ubiquitin-protein ligases.
Figure 6. Methylation analysis of the HACE1 gene in Wilms’ tumor patient’s samples and SK-NEP-1 cell line. (A) Representative sample: Wilms’ tumor and
patient-matched normal kidney DNA was digested using EcoRI in combination with HaeII, HpaII or BssHI methylation sensitive restriction enzymes (upper
panel illustrates restriction map of each PCR amplicon), methylation of three CpG islands surrounding the HACE1 locus was then assessed by semi-quatitative
PCR. Note that hypermethylation was restricted exclusively to tumor samples in CpG-29 and CpG-177 islands, no methylation was observed at CpG-88. (B) SK-
NEP-1 Wilms’ tumor cells were treated with 5AZ to inhibit methylation. Semi-quantitative PCR was then used to assess methylation status of CpG islands
associated with HACE1 as described in (A). After treatment with 5AZ, methylation of CpG-29 and CpG-177 islands is decreased, whereas there is no evidence
of methylation at CpG-88. Similar results were observed in three independent experiments. (C) qRT–PCR showing an increase in HACE1 mRNA transcripts
upon treatment of SK-NEP-1 cells with increasing doses of 5AZ. A maximal response was observed at 5 mM 5AZ. (D) ChIP assays to detect active or inactive
chromatin were performed using antibodies against either acetyl-histone H3 (acetyl H3) or dimethyl-(Lys79)-histone H3 (methyl H3), respectively. SK-NEP-1
and HEK293 cells treated with or without 5 mM 5AZ prior to ChIP studies. PCR products in the top two panels for each cell line correspond to the transcriptional
start of HACE1 and is overlapped by CpG-88. Identical results were observed in four independent experiments.
2068Human Molecular Genetics, 2004, Vol. 13, No. 18
Ankyrin motifs are well known to mediate protein–protein
interactions (20), and therefore likely serve the same function
in Hace1. Alternatively, the ankyrin repeats may be necessary
for Hace1 to link with non-substrate proteins in higher order
signaling complexes, a function that has been described
previously for ankyrin motifs (20,39). Interestingly, the
Hace1 ankyrin repeats show high sequence similarity to
those of the cyclin-dependent kinase inhibitors p16INK4A
(CDKN2A) and p19INK4D (CDKN2D), which inhibit the
cell cycle by binding to and inhibiting CDK4 and CDK6.
HACE1 expression was very low both at the mRNA and
protein levels in five of the six sporadic Wilms’ tumors com-
pared with patient-matched normal kidney, including the
index case. Moreover, expression of this gene is virtually
undetectable in SK-NEP-1 Wilms’ tumor cells. This raises
the possibility that loss of HACE1 expression may be a recur-
rent alteration in Wilms’ tumor, and that HACE1 inactivation
plays a role in the pathogenesis of Wilms’ tumor. On the other
hand, this may reflect the normal expression profile of HACE1
in the Wilms’ tumor neoplastic precursor cell; i.e. HACE1
may not normally be expressed in the putative blastemal cell
of origin of this disease. Typically, inactivation of TSGs in
human malignancies involves targeting of both alleles by
genetic or epigenetic mechanisms (29). Therefore, we
searched for mechanistic evidence of HACE1 inactivation in
Wilms’ tumor. As chromosomal translocations or other cyto-
genetic abnormalities involving 6q21 have only infrequently
been reported in Wilms’ tumors (7,9–11), this is unlikely as
a mechanism of HACE1 loss. Moreover, in the index case of
the present study the 6q21 breakpoint did not directly
disrupt the HACE1 gene, but occurred outside of the coding
region. We also failed to detect HACE1 mutations in any of
the cases with low HACE1 expression, and LOH of this
gene was not evident by analysis of several informative
markers identified within the HACE1 locus. The latter is con-
sistent with other studies indicating that LOH of 6q21 is rare
in Wilms’ tumor (9). Although further studies using larger
case cohorts to definitively rule out that mutations or deletions
of HACE1 occur in sporadic Wilms’ tumor, our results to date
do not support a role for genetic mechanisms in HACE1 loss
in Wilms’ tumor.
Gene inactivation by methylation appears to be at least
as frequent as inactivating mutations for disrupting TSG
expression in sporadic tumors (30). For example, in sporadic
breast and ovarian carcinomas, BRCA1 mutations are highly
infrequent and epigenetic silencing by promoter methylation
is the only apparent mechanism for loss of function in most
cases (40). We therefore examined whether the CpG islands
associated with the HACE1 locus were differentially methyl-
ated in tumor versus normal kidney samples. Although the
HACE1 promoter CpG island was not methylated, we detected
hypermethylation of CpG-29 and CpG-177, located ?50 kb
upstream of HACE1 exon 1, in four of the five Wilms’
tumors with reduced mRNA expression, and also in the SK-
NEP-1 cell line. Moreover, treatment with the methyltrans-
ferase inhibitor 5AZ not only blocked methylation of these
islands but also restored HACE1 mRNA expression in SK-
NEP-1 cells. One obvious caveat of these experiments is
that 5AZ as a pharmacological inhibitor has a global effect
on CpG methylation. We therefore performed ChIP analysis
of the HACE1 promoter region using antibodies to acetylated-
histone H3 or dimethyl-(Lys79)-histone H3, which associate
with actively transcribed versus inactive, silenced chromatin,
respectively (36,37). These experiments indicated that 5AZ
treatment of SK-NEP-1 cells shifts the HACE1 promoter
from an inactive chromatin conformation to that of active
chromatin, in keeping with the increased expression of this
gene in response to 5AZ. In contrast, the HACE1 promoter
of HEK293 human embryonic kidney cells, which express
high levels of HACE1, exists in an active conformation
under normal growth conditions and there is no change in
chromatin structure upon treatment with 5AZ. These findings
provide compelling evidence that HACE1 expression is influ-
enced by upstream CpG island methylation. It is important to
note, however, that our findings do not establish whether the
correlation between CpG island hypermethylation and low
HACE1 expression is specific to transformed cells. That is,
they do not distinguish between acquired hypermethylation
leading to HACE1 inactivation as an etiologic event in
Wilms’ tumor oncogenesis, versus hypermethylation leading
to low HACE1 expression as part of the normal transcriptional
regulation of this gene in the Wilms’ tumor precursor cell.
Functional studies are necessary to determine whether the
Hace1 protein has suppressive effects on cell growth, and
whether its loss contributes to Wilms’ tumor oncogenesis.
Although methylation of CpG dinucleotides outside of pro-
moters has been documented to influence gene transcription
(41), most studies which describe epigenetic silencing of
genes have focused on CpG islands located within promoters
or coding regions of genes (30). Recently, distance effects
on insulator methylation were reported for the H19 gene,
whereby imprinting by CpG methylation at more distant
sites regulated mono-allelic expression of both H19 and Igf2
(42,43). In addition, reduced expression of Sonic hedgehog
(SHH) has been associated with chromosomal rearrangements
occurring 15–250 kb from the SHH locus in holoprosence-
phaly (44,45). It is possible that long distance effects of
CpG island methylation on transcription may be a more
general mechanism for gene silencing in human cancers.
This might explain how chromosomal translocation break-
points located at long distances from promoters of potential
TSGs could influence gene silencing. It is well-documented
that heterochromatin has a silencing effect on adjacent euchro-
matin, likely through spreading of histone methylation (46–48).
It is possible that by juxtaposing heterochromatin in the vicin-
ity of potential TSGs, a chromosomal translocation can exert
long distance transcriptional suppression on that locus by
effecting local methylation changes. For the index case in
this study, the 6q21 breakpoint mapped very closely to if
not within CpG-29 or CpG-177. Moreover, both of these
islands were methylated in tumor tissue but not in adjacent
normal kidney tissue. Therefore, the direct effects of the
t(6;15) in this case might have been to trigger methylation
of these CpG islands, resulting in HACE1 silencing. Alterna-
tively, effects on methylation due to translocation breakpoints
occurring in this region may not be limited to HACE1. It is
possible that long distance effects on expression may also
affect other genes in the region, and that targeting of these
genes for activation or inactivation may actually be contri-
buting to Wilms’ tumor oncogenesis rather than HACE1.
Human Molecular Genetics, 2004, Vol. 13, No. 18 2069
However, the only known genes mapping to within ?500 kb
of the breakpoint include the telomeric genes BVES, Popeye
domain containing protein 3 (POPDC3) and prolyl endo-
peptidase (PREP) (http://genome.ucsc.edu/). None of these
genes have been implicated as oncogenes or TSGs, although
this remains to be rigorously determined. No known genes
are located within at least 1 Mb centromeric to the breakpoint.
Interestingly, a recent reported case of Wilms’ tumor with a
6q21 breakpoint maps to a 1.3 Mb region we now know
includes the HACE1 locus (9), suggesting that other rearrange-
ments of this region may similarly occur near HACE1 and
adjacent loci in Wilms’ tumor.
The 6q21 region is commonly deleted in several other
human neoplasms. Examples include malignancies of the
prostate, breast and ovaries, as well as leukemias and lympho-
mas (12–15). For example, a recent study found that ?50% of
prostate cancers had deletions of the 6q21 region (15). A
number of candidate metastasis suppressor genes have also
been mapped to chromosome 6q21 (49). There is emerging
interest in the role of protein degradation in neoplasia, and
E3 ligases have been implicated in both tumor formation
and suppression (50). In fact, the E6-AP HECT E3 ligase
was originally defined by its ability to ubiquitinate and
promote p53 degradation after recruitment by the E6 onco-
protein in HPV infected cervical carcinoma cells (23,51,52).
It will be important to further assess the possibility that the
Hace1 E3 ligase plays a role in human cancer.
MATERIALS AND METHODS
FISH and Southern blotting
Single color FISH was performed on metaphase and inter-
phase nuclei from the index Wilms’ tumor case. BAC
probes used for mapping were obtained from the RPCI-11
human BAC library, labeled with Spectrum Green or Spec-
trum Red kits (Vysis) and hybridized to denatured slides as
described previously (53). For Southern blotting, genomic
DNA (?15 mg) extracted from frozen tissue sections was
first digested with respective restriction enzymes, and then
genomic probe generated by PCR from a human DNA tem-
plate SB1 (primers: forward, GGAAA CAAAA GCAAA
GCGAC CCAAC TAT; reverse, GGCGG CCGAG ACCTG
The HACE1 coding region from the IMAGE cDNA clone no.
4838835 (ATCC; GenBank accession no. BC034982) was
excised using AscI and BsaAI, blunted with T4 DNA polymer-
ase and ligated in frame into blunted XhoI site of the mamma-
lian expression vector pcDNA3–HA (Invitrogen) or pET-15b
(Novagen). The HA-tagged (hemagglutinin peptide tag,
YPYDVPDYA) construct was excised from pcDNA3–HA
with SacII, blunted with T4 DNA polymerase, and subcloned
into the HpaI site of pMSCVhygro(Clontech). To construct the
plasmid encoding the Cys to Ser mutant (C876S), the 30XbaI
fragment of HACE1 was subcloned from pcDNA3-HA-Hace1
into the XbaI site of pBluescript-II. Site directed mutagenesis
was done using the QuickChange kit (Stratagene) (primers:
forward, TCAAG CACAT CCATC AACAT G; reverse,
CATGT TGATG GATGT GCTTGA). The mutation was
sequence verified and the XbaI fragment was than replaced
into the original pcDNA3–HA–Hace1 construct. The N-term-
inal Hace1 fragment, p75, was generated by removal of a 30
XbaI fragment of HACE1 from the pET-15b–Hace1.
NIH3T3 cells (ATCC) were grown in Dulbecco’s modified
Eagle’s medium (DMEM) (Invitrogen) containing 10% calf
serum (CS) (Invitrogen). SK-NEP-1, SK-N-SH, SK-N-MC,
HEK293 and Bosc23 cells were grown in DMEM sup-
plemented with 10% fetal bovine serum (FBS) (Invitrogen).
KCNR, SAN-2, IMR-32, Birch and CT-10 were maintained
in RPMI 1640 (Invitrogen) with 15% FBS. NIH3T3 cell
lines stably expressing HA–Hace1 were generated using a ret-
roviral system as described previously (54). Briefly, ecotropic
virus packaging Bosc23 cells were transfected with empty
vector (pMSCVhygro) or pMSCVhygro–HA–Hace1 plasmid
using calcium phosphate. Retrovirus-containing supernatants
were collected 48 h after transfection, filtered and incubated
with NIH3T3 cells for 24 h. Infected cells were selected for
4–8 days with hygromycin B (600–800 mg/ml) (Invitrogen).
Methylation sensitive restriction enzyme digests followed by
semi-quantitative PCR have been performed as described
(55). Briefly, 100 ng of DNA, extracted from tumor and
normal kidneys frozen tissue sections, was digested to com-
pletion (overnight) with EcoRI + HaeII, EcoRI + BssHII
or EcoRI + HpaII (NEB) as indicated in Figure 6. Ten nano-
grams of digested DNA was used for semi-quantitative PCR
with amplicons surrounding CpG islands near the HACE1
locus (Fig. 1). The 50region of the MIC2 gene, previously
shown to be unmethylated (56), was used as a control for com-
plete digestion. All reactions were performed using Platinum
Taq (Invitrogen) with the included buffer supplemented with
10% DMSO and carried out with the following conditions:
CpG-29 (794 bpforward:
GCGAC CCAAC TAT, reverse: GGCGG CCGAG ACCTG
AGACC, 100 nM each), 50 mM dNTPs, 1.0 mM MgCl2, 948C
30 s, 568C 10 s, 728C 60 s, 30 cycles. CpG-177 (804 bp,
CTAGC CTGGG TGTGA GAGGG, 100 nM each), 100 mM
dNTPs, 1.5 mM MgCl2, 948C 45 s, 588C 20 s, 728C 120 s,
five cycles, 948C 30 s, 568C 10 s, 728C 90 s, 30 cycles.
CpG-88(939 bp, forward:
100 nM each), 50 mM dNTPs, 1.0 mM MgCl2, 948C 45 s,
588C 10 s, 728C 90 s, 30 cycles. MIC2 (512 bp, forward:
AGTAT CTGTC CTGCC GCC, reverse: TTTGC AACTC
CGACA ACAAA CGC, 100 nM each), 50 mM dNTPs,
1.0 mM MgCl2, 948C 45 s, 558C 15 s, 728C 45 s, 35 cycles.
The optimal number of cycles for each reaction was deter-
mined empirically by testing a range of 25–40 cycles.
Above conditions are those in which the most reproducible
differences in amplification within the exponential phase of
GGAAA CAAAA GCAAA
2070 Human Molecular Genetics, 2004, Vol. 13, No. 18
amplification were achieved. All reactions were performed in
To assess the effect of CpG island methylation on HACE1
expression efficiency, SK-NEP-1 cells were treated with
methylation inhibitor 5-AZ (Sigma). Treatment was done
twice for 24 h over a 7-day period (on days 2 and 5) as pre-
viously described (57). DNA and RNA were isolated on day
6 and HACE1 expression and methylation of the upstream
CpG islands were assayed by qRT–PCR and semi-quantitative
PCR, respectively. Each experiment was repeated four times.
Total RNA was extracted using Trizol reagent (Invitrogen)
and converted to cDNA using 2 mg RNA in a random
primed synthesis with the Superscript II Reverse transcriptase
kit (Invitrogen). qRT–PCR was done using the TaqMan 50
exonuclease assay. All primer/probe sets were designed to
span exon boundaries to eliminate the risk of template
contamination by genomic DNA and the need for DNase1
pre-treatment of RNA. HACE1 gene specific primer/probe
set (forward: TCTTA CAGTT TGTTA CGGGC AGTT,
probe: [6FAM]CAAAC CCACC ATGTG GGACC CTG
[TAMRA], reverse: CAATC CACTT CCACC CATGAT)
was multiplexed with VIC-MGB labeled b-actin endogenous
control primer/probekit (Applied
TaqMan universal PCR master mix (Applied Biosystems)
and standard conditions. An ABI 7000 sequence detection
system (Applied Biosystems) was used to run the PCR reac-
tions and measure fluorescence at each cycle. Generated
data were further analyzed using Microsoft Excel. Each PCR
reaction was performed in quadruplet and each sample was
analyzed independently at least twice.
SK-NEP-1 or HEK293 cells were treated with or without 5 mM
5AZ as described earlier. Immunoprecipitation of DNA repre-
senting active versus inactive chromatin was performed using
a ChIP assay kit (Upstate) according to the manufacturer’s
protocol. Briefly, DNA and protein were cross-linked in
culture with 1% formaldehyde. For each reaction 1 ? 106
cells were collected in 200 ml SDS lysis buffer (1% SDS,
10 mM EDTA, 50 mM Tris–HCl, pH 8.1, 1 mM PMSF,
1 mg/ml aprotinin, 1 mg/ml pepstatin A) and sonicated to
shear genomic DNA to a size range of ?300–1000 bp.
Lysates were diluted 1:10 with ChIP buffer (0.01% SDS,
1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH
8.1, 167 mM NaCl) and immunoprecipitated with the appropri-
ate antibody overnight at 48C with agitation. a-Acetyl-histone
H3 (Lys9/Lys14) antibodies (Upstate) were used to immuno-
a-dimethyl-histone H3 (Lys79) antibodies (Upstate) were
used to immunoprecipitate inactive chromatin (35) with
protein A beads. Protein A agarose/Salmon sperm DNA
slurry (Upstate) added to lysates without antibody was used
as a negative control (data not shown). DNA was recovered
by proteinase K digestion and phenol/chloroform extraction
after washing and reversal of cross-links. Equal volumes
were utilized to maintain the quantitative nature of the
fragments (58), whereas
assay. The recovered DNA pellet was air dried and resus-
pended in 25 ml of TE (10 mM Tris–HCl, 1 mM EDTA, pH
8.0). Immunoprecipitated HACE1 levels were determined by
using PCR amplifying the 50end of the gene, overlapping
the CpG-88 island. PCR was carried out with Platinum Taq
(Invitrogen) with the included buffer, supplemented with
10% DMSO and the following conditions: 2.5 ml recovered
ChIP DNA, primers (297 bp product, forward: CGGCT
CACCC TCGGG CAACT CC, reverse: CGGCG GCGGG
TGTAC TGTAG GTGGT C, 200 nM each), 100 mM dNTPs,
2 mM MgCl2, 948C 30 s, 558C 30 s, 728C 45 s, 32 cycles.
Input DNA amounts were verified by PCR using the same con-
ditions as mentioned using DNA extracted from a 100 ml
aliquot of ‘ChIP buffer’ diluted cell lysate taken prior to
addition of antibodies, as recommended by the ChIP assay
kit (Upstate) (shown as ‘input fraction’ in Fig. 6D). Input
was also verified with 2.5 ml of recovered immunoprecipitated
DNA using the a PCR assay for 50end of the unmethylated
MIC2 gene (55,56), as described earlier (data not shown).
The optimal number of cycles for each reaction was deter-
mined empirically by testing a range of 25–40 cycles. The
conditions mentioned earlier are those in which the most
reproducible differences in amplification within the exponen-
tial phase of amplification were achieved. The entire ChIP
experiment was repeated four times for each cell line.
Genomic DNA from Wilms’ tumors, patient-matched normal
kidney (same cohort used for expression analysis) and the
SK-NEP-1 cell line were used as templates for sequencing.
Reference sequence for primer design, exon layout and assem-
bly was obtained from the UCSC human genome database
(http://genome.ucsc.edu/). Forward and reverse primers for
each of the 24 HACE1 exons contained the 21M13F
CAGCTATGAC) sequences at their 50ends, respectively.
After PCR of each exon from genomic DNA samples auto-
mated sequencing was performed by standard methods.
Sequence reads were base-called using Phred software and
subsequently assembled with reference sequences using
Phrap software (59,60). Contigs of sequence traces corres-
ponding to each exon were examined using PolyPhred soft-
ware (61) for detection of heterozygotes and visualized in
Consed software (62) to facilitate verification of sequence
variants by examination of individual traces.
Thioester bond formation assay
L-[35S]-Methionine labeled Hace1 proteins were synthesized
(Promega) using pcDNA3-HA–Hace1 plasmids coding for
the wild-type and mutant Hace1 proteins. Reaction mixtures
contained 2 ml of the translation reaction, GST–Ub (1 mg),
50 mM KCl, 5 mM MgCl2, 2 mM ATP, 10 mM creatine phos-
phate, 0.5 U of creatine phosphokinase, 1 mM DTT, and
20 mM Tris–HCl (pH 7.8). Reactions were also supplemented
with E1 (?0.5 mg) and the respective E2s (?0.4 mg) (Boston-
Biochem). After incubation for 30 min at 308C, reactions
were stopped with 2 ? SDS–PAGE sample buffer at the
Human Molecular Genetics, 2004, Vol. 13, No. 18 2071
absence or presence of 300 mM b-mercaptoethanol. Reactions
containing b-mercaptoethanol were then boiled for 3 min, and
those lacking b-mercaptoethanol were incubated at room
temperature for 20 min before loading. Reaction products
were resolved by SDS–7% PAGE and visualized by
Purification of the recombinant proteins from bacteria
and antibody preparation
Hace1 or p75, lacking the HECT domain, were expressed as
His-tagged fusion proteins in Escherichia coli BL21(DE3).
After 4 h of IPTG induction, bacteria were lysed by sonication
in buffer containing 2 M NaCl, 10 mM Tris–HCl (pH 7.6) and
0.5 mM PMSF. Cell debris was removed by centrifugation at
10 000g for 20 min at 48C. The supernatant was diluted
4-fold with 10 mM Tris–HCl (pH 7.6), 0.5 mM PMSF and
passed through a Niþ–Sepharose column (Qiagen). After
washing the column with loading buffer [500 mM NaCl,
10 mM Tris–HCl (pH 7.6)], bound proteins were eluted with
the same buffer containing 300 mM imidazole and dialyzed
against 200 mM KCl, 10 mM Tris–HCl (pH 7.6). a-Hace1
antibodies were generated by subcutaneous immunization of
rabbits with the full-length Hace1 recombinant protein or
with the N-terminal Hace1 peptide (CLVLL LKKGA
NPNYQ DISG) and used for western blotting at 1:2000
Immunoprecipitation and western blotting
Immunoprecipitation and western blotting were done essen-
tially as described previously (53,63). Briefly, immunoprecipi-
tations were performed using 500 ml of cytoplasmic cell
extracts (?500 mg–1 mg of total protein) and 3 ml (?3 mg)
of the appropriate antibodies immobilized on 20 ml of
protein (A þ G)–Sepharose beads (Qiagen) for 2–3 h at 48C
with rotation. For immunoprecipitation of 26S proteasome,
the mixture of antibodies directed against a and b subunits
[Calbiochem; 5 ml of each antibody per 30 ml of protein(A)–
Sepharose]has been used.
(Babco), a-BiP, a-Grb2 and a-UbcH7 (Transduction Labora-
tories), a-p97 (VCP; Research Diagnostics Inc.), goat a-actin,
rabbit a-Ub (Santa Cruz) and a-acetyl-histone H3 (Upstate)
antibodies were used at 1:1000 dilution.
To prepare cell extracts, NIH3T3 cells expressing HA-Hace1
were trypsinized, washed three times with PBS and lysed
with buffer containing 50 mM KCl, 2 mM MgCl2, 2 mM
DTT, 0.25% NP-40, 20 mM Hepes–KOH (pH 7.8) and the
(10 mM). Nuclear, mitochondrial and post-mitochondrial
fractions were separated by sequential centrifugation at
1000g for 5 min followed by centrifugation at 10 000g
for 15 min. Nuclei were then additionally purified from the
re-suspended 1000g pellet fraction by spinning
through a 50% glycerol cushion. The post-ribosomal super-
natant was obtained by a further 20 min centrifugation of the
post-mitochondrial fraction at 100 000 rpm in a TLA-100
(20 mM) orlactacystin
centrifuge (Beckman). Cell fractions were normalized for
protein concentration using Protein Assay kit (BioRad).
NIH3T3 cells exponentially growing on coverslips were rinsed
with PBS and fixed with 2208C cold methanol for 10 min. To
detect Hace1 and BiP, coverslips were incubated overnight
with a mixture of rabbit a-Hace1 (1:2000 dilution) and
mouse a-BiP antibodies (1:1000 dilution) followed by the
secondary antibodies Rhodamine Red-X-conjugated goat
a-rabbit and Oregon Green 514-conjugated goat a-mouse
(Molecular Probes). Slides were counterstained with DAPI
and analyzed using a Zeiss Axioplan epifluorescent micro-
scope equipped with a COHU-CCD camera. Control staining
with pre-immune antibodies or no primary antibodies
showed no signal (data not shown).
Supplementary Material is available at HMG Online.
We would like to thank Seong-Jin Kim for reagents, Joan
Mathers and Heather Wildgrove for technical assistance,
Francis Ouellette, Yaron Butterfield, Jacqueline Schein,
Stefanie Butlandand Sohrab
support, as well as Fred Barr, Catherine Anderson, Lola
Maksumova, Gregor Reid and Fan Zhang for helpful discus-
sions. Normal fetal kidney samples were provided by Alan
G. Fantel, Birth Defects Research Laboratory, University of
Washington. This study was supported by the National
Cancer Institute of Canada and by a Translational Research
Grant from the Children’s Oncology Group (to P.H.B.S.).
This work was also funded by the Johal Program in Pediatric
Oncology Basic and Translational Research at the BC
Research Institute for Children’s and Women’s Health.
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