Defective binding of transcriptional repressor ZEB via DNA
methylation contributes to increased constitutive levels
of p73 in Fanconi anemia cells
Carlos Pipaon, Pedro J. Real, Jose L. Fernandez-Luna*
Unidad de Gene ´tica Molecular, Hospital Universitario Marques de Valdecilla, Edificio Escuela Universitaria de Enfermeria,
Servicio Cantabro de Salud, Av. Valdecilla s/n, 39008 Santander, Spain
Received 24 May 2005; revised 12 July 2005; accepted 18 July 2005
Available online 28 July 2005
Edited by Varda Rotter
Fanconi anemia (FA) pathway involved in the machinery that
maintains genomic integrity. Here, we report that the levels of
p73 and its target genes, are increased in cells derived from
FA patients belonging to complementation group A (FA-A).
Moreover, functional correction of FA-A cells by gene transfer
reduces the expression of p73. We also demonstrate that DNA
methylation contributes to increased levels of p73 in FA-A cells
by hampering the binding of the transcriptional repressor ZEB to
an intronic regulatory region of the p73 gene. Together, our data
may help explain the susceptibility of these cells to DNA damag-
? ? 2005 Published by Elsevier B.V. on behalf of the Federation of
European Biochemical Societies.
Little is known about the molecular mediators of the
Keywords: p73; ZEB; Fanconi; DNA methylation
Failure in the proper coordination of DNA damage-respon-
sive pathways leads to inadequate proliferation, genome insta-
bility, and altered apoptotic response. There are some
pathologies showing these features, globally known as chro-
mosome instability syndromes . Fanconi anemia (FA) is
one of these syndromes characterized by congenital defects,
progressive bone marrow aplasia and cancer predisposition.
Cells derived from FA patients show a characteristic hypersen-
sitivity to alkylating agents such as mitomycin C (MMC) ,
and this feature has been used in cell fusion studies to identify
at least 11 genes involved in the development of the FA pheno-
Fanconi anemia proteins (FANC) are thought to play a cen-
tral role in the coordination of DNA repair, cell cycle control
and apoptosis pathways in response to DNA damage [3–5].
One of the genes with increased expression levels in FA cells
is the cell cycle inhibitor p21, which may account for the low
proliferation rates of these cells . This gene is a major target
of the tumor suppressor p53, which is mutated in many human
cancers. Other p53 protein family members have been recently
involved in apoptosis of p53-deficient cells . One of these
members, p73, shares high sequence similarity with p53 within
the DNA binding domain, thus allowing it to bind to and acti-
vate some of the p53 target genes. Moreover, p73 is also in-
duced by DNAdamaging
degradation of p53 through activation of its target gene
In the present article, we report that Fanconi anemia com-
plementation group A (FA-A) cells exhibit increased expres-
sion of p73 and its target genes. We also show that impaired
binding of transcriptional repressor ZEB contributes to p73
overexpression, which may help explain the effect of FANC
proteins on cell cycle and apoptosis.
2. Materials and methods
2.1. Cell culture
Peripheral blood mononuclear cells were obtained from healthy do-
nors and FA patients after informed consent. All patients studied so
far belong to FA complementation group A. Epstein-Barr virus –
transformed lymphoblasts from FA-A patients and healthy donors
were maintained in RPMI media (Biochrom, Berlin, Germany) supple-
mented with 15% heat-inactivated fetal calf serum and antibiotics.
Human fibroblasts derived from FA-A patients were maintained in
Dulbecco?s modified Eagle medium supplemented with 20% fetal calf
2.2. Reverse transcriptase (RT)-PCR analysis
Total RNA was prepared using TRIZOL reagent (Invitrogen, San
Diego, CA). To assess mRNA expression, a semiquantitative RT-PCR
method was used as previously described. . The generated cDNA
was amplified by using primers for human full-length p73 (TA)
(50-TCTGGAACCAGACAGCACCT and 50-GTGCTGGACTGCT-
GGAAAGT), N-terminal truncated p73 (DN) (50-CGCCTACCA-
TGCTGTACGTC and 50-GTGCTGGACTGCTGGAAAGT), p73
(amplifies all isoforms) (50-TCAACGAAGGACAGTCTGCTCC and
GAGTCAGATGC and 50-CTTACTGCTTATGTGTGAGC), p53
GTGAGGATGG), Hdm2 (50-AGTGAAGACTATTCTCAGCC and
GGCA and 50-AGAAGATGTAGAGCGGGC), Bax (50-TGGAGCT-
Noxa (50-AGATGCCTGGGAAGAAGG and 50-GTATTCCATC-
(GAPDH). . After 20 (GAPDH), 25 (p53, ZEB, Hdm2, Bax, Noxa),
Abbreviations: FA, Fanconi anemia; FA-A, Fanconi anemia comple-
mentation group A; FANC, Fanconi protein, gene or cDNA; Luc,
luciferase; MMC, mitomycin C; EMSA, electrophoretic mobility shift
assay; RT, reverse transcriptase; Egr1, early growth-response factor 1
*Corresponding author. Fax: +34 942 200952.
E-mail address: firstname.lastname@example.org (J.L. Fernandez-Luna).
0014-5793/$30.00 ? 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
FEBS Letters 579 (2005) 4610–4614FEBS 29824
28 (p21), and 33 (p73) amplification cycles, the expected PCR products
were size fractionated onto a 2% agarose gel and stained with ethidium
2.3. Western blot analysis
Cell lysates were prepared as previously described . Proteins
(100 lg) were resolved on a 12% polyacrylamide gel and transferred
to nitrocellulose filters. Blots were blocked with 3% bovine serum
albumin and incubated with rabbit anti-p53, anti-p21, anti-E2F1 or
anti-ZEB (Santa Cruz Biotechnology, Santa Cruz, CA) or mouse
anti-p73 (Neomarkers, Freemont, CA) or anti-b-tubulin antibodies
(SIGMA, St. Louis, MO) and then incubated with goat anti-rabbit
or anti-mouse antibodies conjugated with peroxidase (SIGMA).
Bound antibodies were detected by a chemiluminescence system
(Applied Biosystems, Foster City, CA).
2.4. Protein–DNA binding assays
Nuclear extracts (5–10 lg of total protein) were incubated with a
32p-labeled double stranded DNA probe from the promoter region
of the p73 gene containing early growth-response factor 1 (Egr1)
(50-GCTTCGGCCTTGCGTGGGCGGCCTCG) or E2F1 (50-GTT-
CTTTCGCGCCGC) sites. Samples were analyzed by electrophoretic
mobility shift assay (EMSA) as previously described . Supershifts
were performed using anti-HA or irrelevant anti-p52 rabbit poly-
clonal antibodies (Santa Cruz).
Human ZEB cDNA was obtained by RT-PCR and cloned into the
pCR2.1 vector (Invitrogen). pCR2.1-ZEB and pRc-CMV-E2F1 
were used to generate full-length polypeptides by the TNT T7
Quick-Coupled Transcription/Translation System (Promega, Madi-
son, WI). The in vitro translated proteins were immunoprecipitated
with anti-ZEB or anti-E2F1 specific antibodies and protein G Sephar-
ose beads. Following a 3-h incubation and extensive washing, the
immunocomplexes were incubated with mechanically sheared genomic
DNA. Bound DNA fragments were eluted and the presence of p73
intron 1 sequences was analyzed by PCR with specific primers
(50-GGGACCGGAGCCACCTCCAGGTCCCGG and 50-CCGGC-
2.5. Gene reporter assay
A genomic PCR fragment of 767 base pair (bp) (?745 to +22, re-
ferred to the transcription start site) from the promoter region of
p73 was cloned into the pGL2-basic luciferase reporter vector (p73-
Luc). Fibroblasts were cotransfected with 1 lg p73-Luc and 0.2 lg
pRSV-b-gal in the presence or in the absence of pRc-CMV-E2F1, in
triplicate by using Superfect (Qiagen, Hilden, Germany). After 24–
36 h of transfection, cell extracts were analyzed for luciferase activity
by a dual-light reporter gene assay system (Applied Biosystems). Re-
sults were normalized for transfection efficiency with values obtained
2.6. Methylation analyses
Genomic DNA was digested with 10 U of HpaII or MspI. After 16 h
by using primers specific for the first intron of p73 (50-CTGGGGTT-
TTAGCCCAGCCCAGGATC and 50-AGAGCTCCAGAGGTGCT-
CAAACGTG). When indicated, cells were cultured in the presence of
different concentrations of 5-azacytidine (SIGMA) for 60 h and then
total RNA was analyzed for the expression of p73 target genes by semi-
3.1. p73 is over-expressed in FA-A cells
It has been described a high constitutive expression of p21 in
FA lymphoblasts  which may account for the restrained
growth of these cells. Because p21 is a downstream target of
p53, we analyzed the expression of p53 mRNA in FA-A
lymphoblast cell lines and found no significant differences with
respect to normal control cells (Fig. 1A). However, the p53
protein levels decreased in FA-A cells indicating a reduced
stability of this protein (Fig. 1B). Consistent with previous
data, the mRNA (Fig. 1A) and protein (Fig. 1B) expression
of p21 was higher in FA-A than in control cells, which suggest
that upstream regulatory proteins other than p53 must be
Another protein of the p53 family, p73, has been described
to share at least some of the activities of p53. As shown in
Fig. 2A, the mRNA levels of both full-length (transcriptionally
active) and amino-terminus truncated (dominant negative)
isoforms of p73 were strikingly increased in lymphoblasts
derived from FA-A patients. Similar results were obtained in
SV40-transformed fibroblasts (Fig. 2A).
Moreover, the protein levels of p73 a and b isoforms were
also increased in FA-A fibroblasts with respect to control
cells (Fig. 2B). Then, we studied whether expression of the
correct FANC gene could revert the levels of p73. A repre-
sentative experiment is shown in Fig. 2C. We found that
cells transduced with FANCA but not FANCC or FANCG
partially reverted the mRNA levels of p73 (Fig. 2C). Anal-
ysis of transduced cells revealed also a partial correction
of the sensitivity to MMC. By five days of treatment with
33 nM MMC, more than 75% of control vector-transduced
cells were death as assessed by the uptake of propidium
iodide, whereas 78% of FANCA-transduced cells were viable
(data not shown).
Evidence to date suggests that signaling pathways mediated
by p73 cross-talk partially with that of p53 . We showed
that Bax and Hdm2 had higher expression in FA-A than in
Fig. 1. Constitutive levels of p53 protein are reduced in FA-A cells.
(A) Total RNA was purified from control and FA-A lymphoblast cell
lines (represented by numbers) and analyzed for p53 and p21 mRNA
levels by semiquantitative RT-PCR. GAPDH mRNA was used as an
amplification control. (B) Cell lysates were obtained from lympho-
blasts and subjected to western blotting with anti-p53 and anti-p21
antibodies. The levels of b-tubulin were analyzed to assure equal
C. Pipaon et al. / FEBS Letters 579 (2005) 4610–4614
control cells, whereas the p53 apoptotic target Noxa did not
modify its mRNA levels (Fig. 3).
3.2. E2F1 and Egr1 are not constitutively activated in FA-A cells
Transcriptional activation of p73 is mediated at least in
part by Egr1 and E2F1 proteins [13,14]. We previously de-
scribed that constitutive expression of Egr1 is reduced in
FA-A cells . Here, we confirmed and extended this find-
ing by analyzing the capacity of Egr1 to bind to the p73
promoter in FA-A cells. As shown in Fig. 4A, both lympho-
blasts and fibroblasts derived form FA-A patients displayed
a decreased capacity to form DNA–protein complexes as
compared to control cells. Additionally, nuclear extracts
from FA-A and control fibroblasts appeared to have the
same capacity for binding the E2F1 sequence contained in
the p73 promoter (Fig. 4B). Furthermore, we transiently
co-transfected cells with a 767 bp DNA fragment from the
p73 promoter cloned into a luciferase reporter vector (p73-
Luc) along with a plasmid encoding E2F1. As shown in
Fig. 4C the luciferase activity was similar in both FA-A
and control fibroblasts. These data suggest that Egr1 and
E2F1 are by themselves insufficient to explain the regulation
of p73 in this cell system.
3.3. DNA methylation impedes ZEB binding to a regulatory
region of p73 gene
Recently, it has been described that the transcriptional
repressor ZEB regulates the expression of p73 through bind-
ing to consensus sites within the first intronic region .
Thus, we tried to elucidate the contribution of ZEB to the
expression of p73. A representative experiment showed that
the mRNA levels of ZEB are similar in both FA-A and con-
trol fibroblasts (Fig. 5A). Moreover, we determined the
capacity of ZEB to bind to the intronic regulatory region
of p73. For this purpose, we incubated in vitro translated
ZEB protein (Fig. 5B) with fragmented genomic DNA from
FA-A or control fibroblasts and the protein–DNA complex
was analyzed in anti-ZEB immunoprecipitate via intron-spe-
cific PCR. As shown in Fig. 5C, ZEB binding to the p73
DNA fragment obtained from FA-A cells was drastically de-
creased as compared with control cells. When a similar anal-
ysis was performed in anti-E2F1 immunoprecipitate, a faint
intron-specific PCR product was equally amplified in both
FA-A and control cells, most likely due to overamplification
of residual DNA (Fig. 5C).
Then, we explored the mechanism that underlay this bind-
ing blockade. Because some members of the FA-signaling
pathway, including FANCF and BRCA1 are downregulated
by promoter hypermethylation [17,18], we studied the epige-
netic changes in the ZEB binding sites of the p73 gene using
methylation-sensitive restriction enzymes. Notably, no diges-
tion of the intronic sequence was detected in DNA isolated
from FA-A cells (Fig. 5D). On the contrary, the same DNA
fragments obtained from FA-A or control cells were equally
Fig. 3. Analysis of p53-family target genes. Total RNA was extracted
from control and FA-A lymphoblast cell lines (represented by
numbers) and analyzed for the expression of different p53-family
target genes by semiquantitative RT-PCR.
Fig. 2. p73 is upregulated in FA-A cells. (A) Total RNA from control
and FA-A lymphoblast and fibroblast cell lines (represented by
numbers) were analyzed for the mRNA levels of TA and DN isoforms
of p73. (B) Cell lysates were obtained from control and FA-A
fibroblasts and analyzed for p73 protein by western blot. Two splicing
variants of p73, a and b, are shown. Endogenous levels of p73 in
HEK293T cells as well as the expression of p73 in cells transfected with
each variant are included as positive controls. The levels of
b-tubulin were analyzed to assure equal loading. (C) T lymphocytes
derived from FA-A patients were transduced with different FANC
cDNAs or with an empty control vector, and then total RNA was
extracted and analyzed for the expression of p73 mRNA. First lane
shows the expression of p73 in control lymphocytes.
C. Pipaon et al. / FEBS Letters 579 (2005) 4610–4614
If methylation disregulates the expression of p73 in FA-A
cells, then a reduction of this epigenetic change would re-
store the mRNA levels of this gene. Treatment of fibroblasts
with 5-azacytidine, an inhibitor of DNA methyltransferase,
significantly reduced the expression levels of p73 but not
ZEB in a dose-dependent manner (Fig. 5E), indicating that
Fig. 5. ZEBbindingtothefirstintronofp73isimpairedbymethylation.
(A) Total RNA from control and FA-A fibroblasts was analyzed for
ZEB mRNA expression levels. (B) In vitro transcribed/translated
proteins were detected by western blotting with anti-ZEB and anti-
E2F1 antibodies. An irrelevant protein obtained by the same in vitro
system was used as a specificity control (middle lane). (C) The in vitro
translated proteins ZEB and E2F1 were immunoprecipitated with
specific antibodies and incubated with mechanically sheared genomic
DNA from control and FA-A fibroblasts. Bound DNA fragments were
PCR-amplified to reveal the presence of p73 intron 1 sequences. The
isolated from control and FA-A fibroblasts and then incubated with
HpaII (H) and MspI (M) or left untreated (U). Digested and undigested
DNA was amplified by using primers specific for the first intron of p73.
(E) Semiquantitative RT-PCR analysis for expression of the p73 and
ZEB genes in control and FA-A cells cultured in the presence or in the
that treatment with Aza did not affect the expression of ZEB.
Fig. 4. DNA binding activity of Egr1 and E2F1 is not upregulated in
FA-A cells. (A) Nuclear extracts from control or FA-A cell lines
(represented by numbers) were analyzed for the formation of Egr1-
DNA complexes by an EMSA using a32P-radiolabeled probe from the
p73 promoter. (B) Nuclear extracts from control or FA-A fibroblasts
were analyzed for the formation of E2F1-DNA binding complexes as
described in (A). HEK293T cells transfected or not transfected with
HA-tagged E2F1 were used as a positive control. Anti-HA and
irrelevant anti-p52 antibodies were used to demonstrate the specific
binding of E2F1. (C) Control and FA-A fibroblasts were transfected
with a luciferase reporter vector containing a 767 bp fragment from the
p73 promoter (p73-Luc) either in the presence or in the absence of an
E2F1-containing vector. Following 24 h of transfection, cell extracts
were analyzed for the relative luciferase activity. Results were
normalized for transfection efficiency with values obtained with
pRSV-b-gal. The relative induction levels are given as fold induction
with respect to the control transfection with the reporter vector alone.
All data points represent the means ± S.D. of triplicate analyses.
C. Pipaon et al. / FEBS Letters 579 (2005) 4610–4614
methylation of the ZEB binding site contributes to the tran-
scriptional upregulation of p73 in FA-A cells.
The discovery of new members of the p53 family has
added complexity to the response to DNA damage. One
of these members, p73 was identified as a structural and
functional homolog of p53. We showed that both full-length
(TA) and amino-terminus truncated (DN) isoforms of p73
were elevated in all the studied cell lines derived from
FA-A patients. DN-p73 functions as a trans-repressor of
p53 family members and thus plays an antiapoptotic role
in a number of cell systems . Although we cannot estab-
lish a reliable ratio between TA and DN isoforms, FA-A
cells are highly responsive to DNA damaging agents, which
promote apoptosis via transcriptionally active p53 and p73.
These data suggest that TA-p73 should be the prevalent iso-
form in FA-A cells.
ing Hdm2, which promotes its ubiquitination and degradation
at the proteasome . Consistently, the increased levels of p73
in FA-A cells correlated with overexpression of Hdm2 mRNA
and low levels of p53 protein, although the mRNA expression
of p53 remained unchanged with respect to control cells. These
results suggest that p73 but not p53 may account for the slow
growth and increased apoptotic response in FA-A cells.
Although E2F1 and Egr1 are transcriptional activators of
p73, we found that they are unlikely to account for the
upregulated expression of this gene, which suggest that other
transactivators might be involved. Consistent with this, Sp1
and AP-2 binding sites have been described in the promoter
region of p73 , although their contribution to the expres-
sion of p73 needs to be elucidated. Alternatively, loss of a
repressor mechanism could result in constitutively higher
p73 levels. In line with this, it has been described that the
first intron of p73 contains consensus binding sites for the
transcriptional repressor ZEB . These authors show that
ectopic expression of a ZEB dominant negative restored p73
expression in proliferating C2C12 and P19 cells. Consis-
tently, binding of ZEB to the intronic regulatory sequence
of p73 was impaired in FA-A cells. Furthermore, we demon-
strated that methylation of this region provides the mecha-
inhibition of the p73 gene by hypermethylation has been de-
scribed in lymphoid neoplasms . However, methylation
studies of p73 in cancer cells have been mostly focussed
on the promoter sequences and a CpG island in the 50
untranslated region. Thus, promoter hypermethylation of
p73 may be a feature of hematologic malignancies, whereas
methylation of other regulatory regions, including the first
intronic sequence, may promote the expression of p73 
In conclusion, we found that methylation of the first in-
tron of p73 avoids binding of the repressor protein ZEB
in FA-A cells, which accounts, at least in part, for the over-
expression of this gene. Methylation-dependent upregulation
of p73 provides also the rationale for therapeutic strategies
with demethylating agents aimed to block the constitutive
expression of this apotosis promoter gene in Fanconi
Acknowledgments: This work was supported by Grants PI02/3030 to
C.P., and G03/073 and C03/10 (programa RTICCC) to J.L.F.-L., from
‘‘Fondo de Investigacion Sanitaria’’. We thank C. Marin and J.A.
Casado for technical advice.
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