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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3737-3747
A Novel BTB/POZ Transcriptional Repressor Protein Interacts With the
Fanconi Anemia Group C Protein and PLZF
By
Maureen E. Hoatlin,
Yu Zhi,
Helen Ball,
Kirsten Silvey,
Ari Melnick,
Stacie Stone,
Sally Arai,
Nicola Hawe,
Gareth Owen,
Arthur Zelent, and
Jonathan D. Licht
From Division of Hematology and Medical Oncology, Oregon Health
Sciences University, Portland, OR; Derald H. Ruttenberg
Cancer Center and Department of Medicine, Mount Sinai School of
Medicine, New York, NY; and The Leukemia Research Fund Center,
Institute of Cancer Research, London, UK.
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ABSTRACT |
Fanconi anemia (FA) is an autosomal recessive cancer
susceptibility syndrome. The phenotype includes developmental defects, bone marrow failure, and cell cycle abnormalities. At least eight complementation groups (A-H) exist, and although three of the corresponding complementation group genes have been cloned, they lack
recognizable motifs, and their functions are unknown. We have isolated
a binding partner for the Fanconi anemia group C protein (FANCC) by
yeast two-hybrid screening. We show that the novel gene, FAZF, encodes
a 486 amino acid protein containing a conserved amino terminal BTB/POZ
protein interaction domain and three C-terminal Krüppel-like zinc
fingers. FAZF is homologous to the promyelocytic leukemia zinc finger
(PLZF) protein, which has been shown to act as a transcriptional
repressor by recruitment of nuclear corepressors (N-CoR, Sin3, and
HDAC1 complex). Consistent with a role in FA, BTB/POZ-containing
proteins have been implicated in oncogenesis, limb morphogenesis,
hematopoiesis, and proliferation. We show that FAZF is a
transcriptional repressor that is able to bind to the same DNA target
sequences as PLZF. Our data suggest that the FAZF/FANCC interaction
maps to a region of FANCC deleted in FA patients with a severe disease
phenotype. We also show that FAZF and wild-type FANCC can colocalize in
nuclear foci, whereas a patient-derived mutant FANCC that is
compromised for nuclear localization cannot. These results suggest that
the function of FANCC may be linked to a transcriptional repression
pathway involved in chromatin remodeling.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
FANCONI ANEMIA (FA) is an autosomal
recessive disorder characterized by progressive pancytopenia, diverse
congenital anomalies, and predisposition to cancer, particularly acute
myeloid leukemia (AML) and squamous cell
carcinoma.1-5 The diagnostic hallmark of FA cells is a
unique hypersensitivity to DNA crosslinking agents such as mitomycin C
(MMC) and diepoxybutane (DEB). In addition, FA cells have an abnormal
cell cycle profile, described as an elongation or arrest at the G2
phase, and this abnormality is exacerbated by treatment with
MMC.6,7 Because of associated genomic instability and
cancer predisposition, FA has been classified along with xeroderma
pigmentosum, Cockayne's syndrome, trichothiodystrophy, ataxia
telangiectasia, Bloom syndrome, Werner's syndrome, and hereditary
nonpolyposis colorectal cancer as one of the "caretaker gene
diseases."8 However, the basic defect in FA is unknown.
FA is genetically heterogeneous, comprising at least eight different
complementation groups (A-H) identified by somatic cell hybrid
analysis.9,10 Genes mutated in three of the FA
complementation groups have been identified (FANCA,
FANCC, and FANCG).11-13 In addition,
FANCD has been mapped to chromosome 3p22-26,14 and FANCE has recently been mapped to chromosome
6p21-22.15 The proteins predicted to be encoded by the
known FA genes lack recognizable motifs, and their functions are
unknown. Because FA patients from different complementation groups have
similar clinical and cellular phenotypes, a common molecular pathway in
which the FA gene products participate has been hypothesized.
Although FA appears to have the characteristics of a DNA repair
disorder, early work with the first known gene product, Fanconi anemia
group C protein (FANCC), indicated that FANCC was
located primarily in the cytoplasm, suggesting an indirect role in DNA repair.16,17 However, FANCC was subsequently also detected in the nuclear compartment, confirming that at least some FANCC is
available in the nucleus for DNA-related activities.18,19 Attempts to investigate even these most basic issues regarding subcellular localization of FANCC have been confounded by low endogenous expression levels, changes in expression during the cell
cycle, and presence in both nuclear and cytoplasmic
compartments.16-20
It is likely that the activities of FA proteins are regulated or
mediated by other cellular proteins. In support of this notion, evidence based on coimmunoprecipitation and in vitro binding
experiments suggest that FANCC binds to several cytosolic proteins,
including the molecular chaperone GRP94, nicotinamide adenine
dinucleotide phosphate (NADPH) cytochrome P450
reductase, the cyclin dependent kinase cdc-2, and Fanconi anemia
complementation group A (FANCA).18,20-22 Indeed, there is
growing awareness that the FA proteins may interact with each other
within a large protein complex.18,23,24
We report here that FANCC interacts with a new human BTB/POZ (for
Broad Complex, tramtrack, and Bric á Brac/pox virus
and zinc finger) domain protein (for reviews see Albagli et
al25 and Bardwell et al26), which we have named
FAZF. The FAZF protein (for Fanconi anemia zinc
finger) is similar to the promyelocytic zinc finger protein,
PLZF, a Krüppel-like transcription factor involved chromosomal translocations with the retinoic
acid receptor alpha (RAR ) leading to acute promyelocytic leukemia
(APL; for recent review, see Melnick et al27). In
hematopoetic cells, PLZF is localized in nuclear speckles, which become
delocalized in APL. PLZF represses transcription of specific targets by
recruitment of histone deacetylase through the SMRT-mSin3-HDAC
corepressor complex.28-32 In mammalian cells, FAZF is also
located in nuclear speckles, which colocalize with wild-type FANCC, but
not an inactivated FANCC protein. In addition, we show that FAZF is a
transcriptional repressor and it readily forms heterodimers with PLZF.
BTB/POZ proteins are implicated in oncogenesis,29,33-37
hematopoiesis,38-40 and limb development,41-43
suggesting that FANCC/FAZF interaction and the implied connection to
transcriptional repression may be involved in the FA pathway.
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MATERIALS AND METHODS |
Plasmids, cell lines, antibodies, and chemicals.
The bacterial expression plasmids for GST-FAN2 and GST-FAC1 have been
described.19 To make the two-hybrid bait expressing FANCC
amino acid #1-168 (pAS-FAN), polymerase chain reaction
(PCR) primers (159-192 [5'-TTCGCTTTTTCCACCATGGCTCAAGATTCAGTAG-3'] and 660-704 [5'-GGGAGCCATTCGCCTTGGATCCTTCTATCCATTAAGATGATTCT-3']) were used to amplify the FANCC sequence from a full-length cDNA contained in pLFACXN.19 The primers incorporated
NcoI and BamHI restriction sites in the amplified
fragment for use in ligation. For p2HA-FANCC, full-length FANCC was
obtained by PCR from pLFACXN with primers FAC5' BamHI
(5'-CGGGATCCGATGGCTCAAGATTCAGTAGATCTTTCT-3') and
FACW3' BamHI
(5'-CCGGATCCTAGACTTGAGTTCGCAGCTCTTTAAGGA-3') and ligated
into BamHI-digested/calf intestinal phosphatase (CIP)-treated pCEPHA2.RIG.G, a plasmid that encodes two tandem hemagglutinin (HA)
epitopes (kindly provided by Dr Hans Joenje, Free University, Amsterdam, The Netherlands). 2HA-L554P was constructed similarly except
that primer FACM3' BamHI
(5'-CCGGATCCTAGACTTGAGTTCGCGGCTCTTTAAGGA-3') was used
instead of FACW3 BamHI. For construction of the expression vector encoding the epitope-tagged pFlag-FAZF, total RNA was extracted from normal human peripheral blood, and primers FAZF5 BGL2ATC (5'-TACCCAAGCCAAGGCAAGATCTCAATGTCCCTGCCCCCCAT-3') and FAZF3
BGL2 (5'-GCTACCGACACCCCGTAGATCTCAGGTGGTGGAGGAAGAA-3') were
used to obtain the cDNA by reverse transcription
(RT)-PCR. The amplified fragment was digested with
BglII, and ligated into BamHI-digested/CIP-treated vector pCEP4-Flag (kindly provided by Dr Naumovski, Stanford
University, Stanford, CA). All PCR-derived sequences and ligation
joints were verified by DNA dideoxy sequencing using an ABI PRISM Dye
Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Foster
City, CA). Anti-HA was obtained from Roche Molecular Biochemicals
(Indianapolis, IN). The monoclonal antibody (MoAb)
specific for FANCC was produced by standard methods against the keyhole
limpet hemacyanin-conjugated peptide C-ARELLKELRTQV, corresponding to
FANCC aa #547-558.44 A detailed description of the antibody
will be published elsewhere (M.E.H., in preparation).
All chemicals were obtained from Sigma (St Louis, MO), unless otherwise indicated.
Two-hybrid library screen.
The two-hybrid screen was performed essentially as described
previously.45 Briefly, Saccharomyces
cerevisiae Y190 was transformed with the pAS-FAN
bait (binding domain [BD] fusion) plasmid and selected for Trp
prototrophy. After ensuring that the resulting strain expressed the
FANCC bait and did not autoactivate the GAL4 reporter, it was
transformed with a human B lymphocyte library encoding the activation
domain (AD)-library hybrids. Approximately 300,000 His+ primary clones were obtained, and 59 of these
were positive for  -galactosidase (gal) activity.
Plasmid DNA prepared from several of the positive strains was
transformed into Escherichia coli strain HB101, and
transformants were selected. Candidate plasmids that did not repeat
with the original bait, that autoactivated the reporter, or that
interacted with unrelated baits were eliminated. The remaining
interactors were further analyzed. A second screen was also performed
with the same library using a BD expression vector encoding FANCC aa
#116-558. For confirmation of two-hybrid interactions of PLZF and FAZF,
the PJ69-4A strain of S cerevisiae46 was
transformed with BD fusion constructs encoding full-length FAZF, PLZF,
or the PLZF POZ domain. The transformed strains were selected on media
lacking leucine, tryptophan, and adenine. Yeast colonies were counted
and then selected in duplicate for liquid -galactosidase assays as
directed (Clontech, Palo Alto, CA). Results were normalized relative to
the dimerization of PLZF. A full-length GAL4 plasmid was used as a
positive control (Clontech). Other two-hybrid positive and negative
control AD and BD domain plasmids were purchased from Clontech.
In vitro protein binding assays.
Expression and purification of Glutathione S-transferase (GST)-fusion
proteins used in these assays have been described
previously.19 Radiolabeled protein encoded by the library
plasmid pAct8-1 was made by amplifying the insert using T7-promoter
containing primers specific for flanking sequences in the pACT vector
(Clontech). In vitro translations were performed using a commercial
transcription-translation system (TnT, Promega, Madison, WI). Equal
amounts of radiolabeled protein were added to GST fusion proteins
immobilized on glutathione-Sepharose beads (GST, GST-FAC2, and
GST-FAC1) and allowed to bind for 4 hours at 4°C in binding buffer
(10 mmol/L Tris HCl/150 mmol/L NaCl/1 mmol/L EDTA/1% Nonidet P-40/1%
deoxycholic acid). After extensive washing with the buffer, the beads
were boiled in sample buffer and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and autoradiography.
Northern blotting.
A multiple tissue blot of human poly (A+) RNA (Clontech) was probed
with a radiolabeled 525-bp (BamHI/XhoI) restriction
fragment from the library plasmid pAct8-1.
Immunoprecipitation and immunoblotting.
Cells were washed twice with phosphate-buffered saline
(PBS), and cell lysates were solubilized in lysis
buffer 1 (20 mmol/L Tris-Cl [pH 7.5], 150 mmol/L NaCl, 1% Triton
X-100, 10 mmol/L EDTA, 1% deoxycholate, 1.5% aprotinin, 1 mmol/L
phenylmethylsulfonyl fluoride [PMSF]). Anti-Flag
M2-affinity gel (Sigma) or anti-HA (Roche Molecular Biochemicals)
followed by Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology,
Santa Cruz, CA) was added as indicated in the text, and the sample was
rocked overnight at 4°C. The immune complexes were sedimented by
centrifugation, washed in lysis buffer, mixed with Laemmli sample
buffer, boiled, and separated by SDS-PAGE. Proteins were electroblotted
onto Bio-Blot nitrocellulose (Costar, Cambridge, MA) and probed with
anti-HA, anti-Flag M2 (Sigma), or anti-FANCC MoAb 3A11 as previously
described.19 For the PLZF-FAZF interaction, cells were
lysed in lysis buffer 2 (1% NP40, 50 mmol/L NaCl, 50 mmol/L Tris pH
8.0, 1 mmol/L MgCl2, 10 mmol/L ZnCl2, 4%
glycerol and Complet protease inhibitors [Roche Molecular
Biochemicals]). Immune complexes were obtained essentially as
described above using anti-Flag MoAb (Kodak, Rochester, NY) and Protein
A agarose (Roche Molecular Biochemicals). The electrophoresed proteins
were transferred to Immobilon PVDF membrane (Millipore, Bedford, MA)
and were processed as described above using anti-PLZF polyclonal
antibody.47
Transient transfection and immunofluorescence microscopy.
293 Epstein-Barr nuclear antigen (EBNA) cells
(Invitrogen, Carlsbad, CA) were grown on chamber slides and transfected
using lipofectamine as directed by the manufacturer (GIBCO, Grand
Island, NY). Plasmids encoding epitope-tagged FAZF, FANCC, L554P, or
parental vectors as negative controls for antibody specificity were
used as indicated in text. Cells were processed as described
previously19 using monoclonal anti-HA 12CA5 (Roche
Molecular Biochemicals) and/or rabbit polyclonal anti-Flag (Zymed,
South San Francisco, CA) and Oregon Green-conjugated goat antimouse
and/or Texas Red-conjugated goat antirabbit (Molecular Probes, Eugene,
OR) as secondary antibodies. Double-labeled images were acquired with a
Bio-Rad MRC 1024 ES laser scanning confocal imaging system (Bio-Rad
Laboratories, Richmond, CA) attached to an inverted Nikon Eclipse TE300
microscope (Nikon, Melville, NY). The acquisition system (LaserSharp,
Hercules, CA) uses a krypton/argon laser with excitation lines at 488 and 564 nm, and sequential detection using three 8-bit photomultiplier tubes (PMTs). Single-labeled images were acquired with a Leica 900 confocal laser-scanning microscope (Leica Inc, Deerfield, IL) equipped
with a krypton/argon detector. Collected images were imported into
Adobe Photoshop 4.0 (Adobe, San Jose, CA) and overlapped to produce
merged images. Localization study data (see Table 2) was gathered with
a Leitz Orthoplan 2 fluorescence microscope (Ernst Leitz GMBH, Wetzlar,
Germany) using green (488 nm) or red (568 nm) filters and a ×100
oil lens. One hundred cells expressing the protein of interest were
counted for each cell line. Cells were scored as positive if they
contained two or more nuclear foci. Standard error was computed using
StatView 5.0 (SAS Institute, Cary, NC).48
Electrophorectic mobility shift assay (EMSA).
293T (ATCC No. CRL1573) cells were transfected with 20 µg of DNA
using the Ca PO4 method.49 Nuclear extracts
were prepared 48 hours after transfection, frozen in liquid nitrogen,
and stored at  70°C.50 Synthetic duplexes
(Table 1) were end-labeled using the large
Klenow fragment of E coli DNA polymerase and
[-P32] dCTP (3,000 Ci/mmol), and purified by spin
column or PAGE. Each binding reaction contained approximately 2 mg
nuclear extract protein in 20 mmol/L HEPES pH 7.5, 1 mmol/L
MgCl2, 10 mmol/L ZnCl2, 4% glycerol, 100 mg/mL
bovine serum albumin (BSA), and 1 mg dIdC, and was
incubated on ice for 30 minutes. Unlabeled oligonucleotide competitors
or anti-Flag antibody (Kodak) was added at least 20 minutes before the
addition of 10 femtomoles of labeled duplex as indicated in the text.
After a further incubation of 20 minutes, protein-DNA complexes were
separated by electrophoresis on a 4% nondenaturing polyacrylamide
(30:1, acrylamide:bis-acrylamide) gel.
Transcriptional repression assays.
Plasmids encoding PLZF in the pSG5 expression vector were previously
described.51 The Flag-epitope tagged version of FAZF was
constructed in the pSG5 vector by standard methods. Four copies of a
PLZF binding site found within the interleukin (IL)-3
receptor promoter
(5'-TCGAGGATGACTGCGAGTACAGTTGCAACGG-3')51a were
cloned 5' of the thymidine kinase promoter driving transcription
of a luciferase reporter gene (IL-3R-Luc). For transcription assays, 2 × 105 293T cells per well of a 12-well dish were
transfected with 1 µg of DNA, or as indicated in text, and 5 µL of
Superfect (Qiagen, Valencia, CA). Luciferase levels were measured 48 hours after transfection using a Dual Luciferase kit (Promega).
| |
RESULTS |
Identification of a FANCC-interacting protein by yeast two-hybrid
screening.
FANCC has been shown to bind to other cellular proteins and may be part
of a large intracellular complex.18,20-22,24,52 We used a
yeast two-hybrid assay to screen for proteins that bind to FANCC in
vivo in an effort to identify binding partners that might shed light on
the function of the FA pathway.45 The bait was designed
(Fig 1A) to
contain a region of FANCC deleted in patients with a severe phenotype
(IVS4-4A-T), reasoning that this may be an area for functionally
important protein-protein contact.14,53 Related strategies
to compare strains expressing wild-type and mutant proteins (eg,
full-length FANCC baits, and the inactive L554P protein) were
unsuccessful because the strains were unstable and could not be used
for screening (data not shown). The bait strain containing N-terminal
sequences (FAN, aa #1-168) was used to screen a human B-cell
library.45 Transformants were selected that were HIS+, and
-gal+, that did not autoactivate the reporter, interacted with the
FAN bait on retesting, and did not have reporter activity with
unrelated baits. Figure 1B shows the two-hybrid interaction of a
representative interacting clone encoded by library plasmid pACT8-1.
Southern blot analysis of candidates from the screen using an internal
fragment from clone 8-1 indicated that at least three clones of
different sizes contained hybridizing sequences, suggesting that the
same candidate had been selected in the screen several times.
Additional screening performed with a FANCC bait containing aa #116-558
also resulted in capturing sequences contained in clone 8-1, although
this bait proved to be too unstable for further analysis (data not
shown). DNA sequence analysis of the hybridizing clones indicated that
the clones contained overlapping regions of the same sequence (data not
shown).
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| Fig 1.
Identification of a FANCC-interacting protein by
two-hybrid screening. (A) FANCC two-hybrid bait. The upper open
rectangle represents wild-type FANCC. Asterisks indicate location of
the R548X and L554P inactivating mutations.11,53,82 The
lower open rectangle represents the region of FANCC expressed as a
two-hybrid bait. The numbers indicate the corresponding amino acid
residue of FANCC (the binding domain of the two-hybrid fusion is not
shown for simplicity). The overlap with wild-type is indicated by the
arrangement of the boxes in which the shaded area corresponds to the
deletion in the IVS4 4A-T inactivating mutation that leads to a severe
disease phenotype.14,53 (B) (see page 3740)
Two-hybrid interaction. The Y190 yeast strain was cotransformed with
the FAN bait plasmid and a representative prey plasmid
(8-1) isolated by two-hybrid screening. Controls are shown
for strains containing the prey plasmid 8-1 and an unrelated
bait, and strains known to be positive and negative for interaction.
The cotransformed strains are shown growing on media selective for both
bait and prey plasmids (-Trp, -Leu), and under HIS selection. The lower
panel shows results of the -gal assay. (C) The protein encoded by
clone 8-1 interacts with an amino terminal region of FANCC in vitro.
Radiolabeled protein made by coupled in vitro transcription and
translation from 8-1 cDNA bound specifically to immobilized GST-FANC1,
but not to immobilized GST or GST-FAN2.
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To confirm the in vivo interactions we found in yeast, we tested a
radiolabeled in vitro translated protein encoded by clone 8-1 for
binding to bacterially expressed GST-FANCC fusion proteins immobilized
on glutathione Sepharose. In these protein affinity experiments,
radiolabeled 8-1 protein bound to immobilized GST-FAC1 (aa #106-558),
but not to GST-FAN2 (aa #7-106) or to GST alone (Fig 1C), indicating
that the binding between the 8-1 protein and FANCC is direct and
specific for the amino terminal region of FANCC. Because the FAN yeast
bait contains aa #1-168, the in vitro and in vivo binding data suggest
that the region of FANCC participating in the interaction is
approximately between aa #106-168. Thus, binding between protein
encoded by clone 8-1 and FANCC corresponds roughly to the deletion
beginning at aa #116, resulting from the IVS4-4A-T
mutation.54
Characterization of FANCC interacting clone 8-1.
PCR primers were used to amplify the inserts from the library plasmid
pAct8-1. DNA sequence analysis revealed an open reading frame of 300 amino acids in-frame with two-hybrid activation domain sequences. A
BLASTX database search
(http://www.ncbi.nlm.nih.gov/BLAST/)55,56 with the 8-1 sequence indicated similarities to multiple zinc finger-containing
proteins, and near identity with sequences contained within exons of a
genomic clone from human chromosome 19q13.1 (cosmid F24109, GenBank
accession No. AD000671). Thus, FAZF does not map to the location of a
mapped FA gene. This cosmid clone contained additional 5'
sequences, suggesting that clone 8-1 was not complete. Therefore, the
1,461-bp full-length FAZF was cloned by RT-PCR using total RNA from
normal human peripheral blood. Recently, another group has also
deposited sequences in agreement with our data (accession no. AF130255, unpublished).
Examination of the predicted FAZF protein sequence indicated a
significant degree of homology with the PLZF protein of t(11;17) (q21;q23)-associated APL (reviewed in Melnick et al27;
Fig 2A). FAZF contains an amino terminal
POZ/BTB domain25,57 sharing highly conserved residues
appearing in this subfamily that includes LRF,41
LAZ-3/BCL-6,33,36,37 and the recently cloned
Kaiso.58 The POZ domain can mediate
protein dimerization59,60 and transcriptional repression by
PLZF.61 PLZF binds to specific DNA sequences and can do so
using only the last five to seven of nine zinc finger motifs.61,62 FAZF's three zinc fingers are closest in
homology (91% similar) to the last in the series of nine at the
C-terminus of PLZF (Fig 2B), and the POZ/BTB domain is nearly 60%
similar. The degree of similarity between PLZF and FAZF suggested that these proteins might have common transcriptional functions. Examination of the genomic database also indicated a possible evolutionary relationship between PLZF and FAZF. A search of the EST database (expressed sequence tags; NCBI/NLM, Bethesda, MD) showed that the FAZF
sequence (UniGene Hs. 99430;
http://www.ncbi.nlm.nih.gov/UniGene/) is contained in a clone that also
includes the gene HRX2, a gene very similar to the
MLL/HRX (mixed lymphoid or mixed lineage leukemia/homologue of
trithorax) gene,63-66 located adjacent to the PLZF gene on
chromosome 11. This finding suggests that the simpler FAZF gene with
its three zinc fingers might be an evolutionary antecedent to PLZF. The
central portion of FAZF has no significant homology to PLZF or any
other sequences in the database. The lack of homology in sequences
between the BTB/POZ domain and the zinc fingers is a common feature in
other BTB/POZ and Krüppel-like proteins. The FAZF sequences
between aa #1-98, including part of the BTB/POZ domain (aa #1-109), are
apparently not required for FANCC interaction, because we obtained only
clones missing the amino terminal portion of the protein in the
two-hybrid screens.
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| Fig 2.
FAZF is similar to PLZF. (A) Comparison of the amino acid
sequences of FAZF and PLZF showing BTB/POZ domains (underlined at the
amino termini) and zinc fingers (underlined at the C-termini) in
CLUSTAL W format. The FAZF sequence has been submitted
to GenBank (accession no. AF165097). (B) Schematic representation of
the PLZF and FAZF proteins indicating regions of homology and
similarity.
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A Northern blot survey of RNA from multiple tissues probed with a
radiolabeled restriction fragment from clone 8-1 revealed a predominant
weak transcript of 1.4 kb in most tissues, except for testis, in which
multiple strong transcripts were observed (2.0, 2.7, and 4.4 kb) and
the 1.4-kb transcript was absent. Transcripts present in testis,
particularly the 4.4-kb band, were also present in other tissues as
minor transcripts. A strong signal was observed in peripheral blood
leukocytes, consistent with the presence of FAZF hybridizing sequences
in the mRNA from cells similar to that used for construction of the
two-hybrid expression library (Fig 3).
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| Fig 3.
FAZF is ubiquitously expressed. Multiple human tissue
northern blot (Clontech) was probed using radiolabeled restriction
fragment from clone 8-1. Lane 1, spleen; lane 2, thymus; lane 3, prostate; lane 4, testis; lane 5, ovary; lane 6, small intestine; lane
7, colon; lane 8, peripheral blood mononuclear leukocyte. Longer
exposure of this blot showed a 1.4-kb FAZF band in all tissues except
testis.
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FAZF coimmunoprecipitates with FANCC.
To determine whether FANCC associates with FAZF in vivo, expression
vectors for epitope-tagged FANCC and full-length FAZF were
cotransfected into 293 EBNA cells. Immobilized anti-Flag MoAb was added
to lysates prepared from an asynchronous population of transfected
cells, and the resulting complexes were separated by SDS-PAGE and
analyzed by immunoblotting with anti-Flag. Whole cell extracts (WCE)
were analyzed as controls for protein expression. As shown in
Fig 4B, Flag-FAZF is detected as an Mr = 56,000 protein in whole cell extracts (lanes 2 and 5) and in lysates
first immunoprecipitated with anti-Flag (lanes 3 and 4). The Flag-FAZF
signal is absent in lysates from cells transfected with the parental
vector (lane 7), or in lysates from cells expressing HA-FANCC (lane 1).
We conclude that FAZF is expressed, is approximately the size expected, and is specifically detected by immunoprecipitation and by simple blotting in the whole cell lysates of cells transfected with the Flag-FAZF expression vector.
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| Fig 4.
FANCC and full-length FAZF interact in vivo. 293 EBNA
cells were transiently transfected with epitope-tagged expression
vectors for HA-FANCC and/or Flag-FAZF. Lysates were electrophoresed
directly as WCE or after immunoprecipitation with anti-Flag epitope
tag. Proteins were detected by immunoblotting with anti-Flag or
anti-FANCC. (A) Lane 1, FANCC WCE; lane 2, FANCC + FAZF WCE; lane 3, FANCC + FAZF IP; lane 4, FAZF IP; lane 5, FAZF WCE; lane 6, FANCC IP;
lane 7, pCEP-Flag WCE. (B) The same loading order as
in (A), probed with anti-Flag.
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To determine if FANCC was coimmunoprecipitated with Flag-FAZF, the blot
was stripped and reprobed with an antibody specific for FANCC (Fig 4A).
In cell lysates containing Flag-FAZF, FANCC was detected (lane 3), and
this band comigrated with the HA-FANCC in lanes 1 and 2 (whole cell
lysates containing HA-FANCC and both HA-FANCC and Flag-FAZF,
respectively). We conclude that HA-FANCC coimmunoprecipitates with
Flag-FAZF. The reverse experiment was performed on the same lysates by
first immunoprecipitating HA-FANCC with anti-HA serum followed by
immunoblotting with either anti-FANCC or anti-Flag. Although the
background was much higher using the HA antibody, we found that
Flag-FAZF was specifically coimmunoprecipitated with HA-FANCC (data not shown).
FAZF is a nuclear protein with a punctate pattern.
To determine the subcellular localization of FAZF, a Flag
epitope-tagged protein was expressed in 293 EBNA cells and analyzed by
indirect immunofluorescence. Cells were transfected with pFlag-FAZF or
pFlag parental vector (as a negative control for specificity of the
Flag antisera) and processed 20 hours later for confocal microscopy.
Figure 5 (see page 3740) shows
representative views of FAZF expression patterns. Consistent with
observations reported for PLZF and other BTB/POZ-containing proteins,
FAZF is almost entirely nuclear, with most cells containing foci
superimposed on a microspeckled nuclear
pattern.26,40,47,60,67 Like PLZF foci, FAZF nuclear foci
varied in size and number, but were present in nearly all cells
observed (Table 2). Staining was absent in cells transfected with pFlag.
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| Fig 5.
FANCC and FAZF colocalize in nuclear bodies. 293 EBNA
cells were transfected with pFlag-FAZF, pHA-FANCC, or pHA-L554P, or the
pFlag parental vector (control panel), as indicated. The fixed and
permeabilized cells were stained with antibodies specific for the
epitope tags, followed by staining with fluorescent-conjugated
secondary antibodies (Red = FAZF and Green = FANCC or L554P).
Immunofluoresence was examined by confocal laser microscopy. For double
labeling, the green and red channels were recorded independently and
then overlaid. (A) Subcellular localization of FAZF. Original
magnification ×63. (B) Colocalization of FANCC and FAZF in nuclear
foci. Original magnification ×60. (C) The mutant L554P protein does
not colocalize with FAZF.
|
|
Wild-type but not mutant FANCC colocalizes with FAZF in nuclear
bodies.
To determine if FAZF and FANCC proteins colocalize, we performed
experiments using indirect immunofluorescence and confocal microscopy
to examine cells that were doubly transfected with constructs encoding
tagged FAZF and FANCC. A survey of cells expressing FANCC (either
singly or coexpressed with FAZF) showed that FANCC is distributed in
both cytoplasmic and nuclear compartments, as reported
previously.16-19 In addition, we observed that the
distribution of FANCC between cytoplasm and nucleus varied, and that
among cells expressing FANCC in an asynchronous population, 24% of
cells contained two or more nuclear foci (Table 2 and Fig 5B, FANCC panel). The amount of FANCC appearing in the nucleus, or in foci, did
not seem to be correlated with higher expression levels. Indeed, in
many cells where FANCC expression was highest, no FANCC was observed in
the nuclear compartment at all (data not shown). In cells that
contained foci, the typical number ranged from approximately 5 to 20. The foci were typically superimposed on a diffuse nuclear stain, which
was excluded from the nucleoli. Table 2 summarizes the percentage of
cells containing FANCC foci observed in this experiment. Similar
results were obtained from a 293 cell line stably expressing FANCC
without an epitope tag using a FANCC-specific MoAb (data not shown).
Interestingly, a similarly complex and variable pattern was recently
described for another FA protein, FANCA, by D'Andrea et
al,68 suggesting that foci formation may be an important
and newly appreciated feature of FANCC and FANCA subnuclear
localization. Not all cells expressing FANCC contain foci, as described
above, but when FAZF and FANCC foci did appear in the same cell, we
observed extensive overlap. Figure 5C shows a representative cell in
which there is overlap of nuclear foci. No colocalization was observed
between FANCC and PLZF, or between promyelocytic leukemia gene product
(PML) and FANCC in colocalization experiments performed similarly (data
not shown).
Although the FAZF interaction site on FANCC appears to be between aa
#106 and 168, well away from the C-terminus of the FANCC, we wondered
whether the L554P mutant protein could colocalize with the FAZF nuclear
foci, because other features of the defective protein besides this
interaction might influence colocalization. To answer this question, we
expressed an HA-epitope-tagged L554P protein in 293 EBNA cells and
observed the staining pattern of L554P with and without FAZF
coexpression. In contrast with the pattern observed for wild-type
FANCC, L554P staining shows intense, diffuse cytoplasmic staining with
minimal nuclear staining (Fig 5C, L554P panel). In addition, although
the expression of L554P was comparable with that of wild-type FANCC,
nuclear foci were very rare; of 100 cells expressing L554P, only one
foci in each of two cells was observed (Table 2), suggesting that the
ability of L554P to enter the nucleus is compromised under these
conditions. A recent report by Savoia and others described similar
observations for L554P overexpressed in Hela cells.69 When
L554P and FAZF are expressed in the same cells, the proteins are
localized to different parts of the cells (Fig 5C [overlap]). In the
cell shown, the FAZF staining shows a micropunctate nuclear staining
pattern, and the L554P staining is mainly cytoplasmic. When these two
photos were overlaid, no colocalization was observed. No FAZF/L554P
colocalization was observed in other cells examined, although other
cells coexpressing L554P and FAZF contained larger FAZF foci (data not
shown) consistent with FAZF's overall variable nuclear staining pattern.
FAZF binds to PLZF target sequences.
The striking homology between PLZF and FAZF suggested that the two
proteins may have similar functions. PLZF has been shown to bind to
specific DNA target sequences.61 To determine if FAZF could
bind to the same sequences as PLZF, we performed an EMSA with nuclear
extracts of 293T cell transfected with the PLZF or FAZF expression
constructs, using a radiolabeled high-affinity PLZF binding site (Table
1, Site B) derived from a CpG island library.51a
Figure 6A shows that both PLZF and FAZF
formed DNA-protein complexes with this site, indicated by the
appearance of shifted bands in lanes containing FAZF or PLZF extracts.
No shifted bands appear in the control lanes where no extracts, or mock
transfected extracts, were added. The complexes caused by the
epitope-tagged FAZF protein could be supershifted by the addition of an
anti-Flag antibody (Fig 6A, right panel), confirming that the shifted
band contains tagged FAZF. In a competition assay, the PLZF- and
FAZF-DNA complexes were abrogated by addition of a molar excess of
unlabeled site B, a high affinity site from the IL-3 receptor promoter
(Fig 6B) as well as the Lex operator (data not shown), which is also a
target of PLZF.62 A missense mutant within the IL3R site
abrogated binding to both PLZF and FAZF. Together, these data suggest
that PLZF and FAZF may bind the same or a closely overlapping set of target genes.
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| Fig 6.
FAZF and PLZF bind to the same DNA sequences. (A) Nuclear
extracts from untransfected cells (control) or from 293T cells
transfected with a PLZF expression vector or a Flag-FAZF expression
vector were allowed to interact with a high affinity PLZF binding site
(Site B, Table 1), followed by nondenaturing gel electrophoresis. A
supershift assay of the Flag-FAZF complexes was performed by the
addition of Flag M2 MoAb to the reaction. (B) Competition analysis of
FAZF and PLZF DNA binding. Extracts containing PLZF or FAZF were
allowed to bind to a high affinity PLZF site (Site B, Table 1) in the
absence of competitor or in the presence of a 10-fold, 100-fold, or
1,000-fold molar excess of the indicated unlabeled competitor
oligonucleotide. The DNA protein complexes were resolved as described
above.
|
|
FAZF interacts with PLZF.
Because BTB/POZ domain-containing proteins have been known to form
functional dimers,26,59 we next determined whether PLZF and
FAZF might physically interact. PLZF and FAZF combinations were
coexpressed as AD/BD fusion proteins in yeast, and assayed for reporter
activity. We found that PLZF protein self-associated in this assay, as
expected, and the POZ domain of PLZF (POZ-PLZF) was sufficient for this
interaction (Fig 7A). FAZF (AD) could similarly interact with a BD (full-length) PLZF or BD-POZ-PLZF. We
conclude that the PLZF POZ domain is sufficient for interaction of PLZF
and FAZF. To confirm the interaction observed in yeast, we tested for
PLZF-FAZF interaction by coimmunoprecipitation in mammalian cells. In
293T cells cotransfected with expression vectors encoding PLZF and a
Flag-tagged FAZF expression vector, PLZF could be detected after
immunoprecipitation of the epitope-tagged FAZF (Fig 7B).
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| Fig 7.
PLZF and FAZF interact in vivo. (A) Yeast two-hybrid
assay. PJ69-4A yeast were transformed with a bait plasmid encoding
full-length PLZF or the POZ domain of PLZF linked to the GAL4 DNA
binding domain and the indicated prey plasmids containing full-length
FAZF or PLZF or the POZ domain of PLZF linked to an acidic activation
domain. Yeast colonies grown on leucine and tryptophan deficient media
were expanded, and a liquid -galactosidase assay was performed.
Levels of -galactosidase were normalized, setting the level of
-galactosidase obtained in the presence of a PLZF bait and PLZF prey
plasmid to 1.0. The GAL4 protein acted as positive control for
transcription. (B) In vivo-coimmunoprecipitation. 293T cells were
transfected with PLZF or Flag-FAZF plasmids as indicated. Lysates from
the cells were subjected to immunoprecipitation with the Flag M2 MoAb
followed by immunoblotting with a PLZF polyclonal
antibody.47
|
|
FAZF is a transcriptional repressor.
To determine if FAZF is able to act as a transcriptional repressor, we
tested FAZF by coexpressing the protein along with a reporter gene
linked to a promoter containing multimerized IL3R-PLZF target sites. We
found that both PLZF and FAZF specifically repressed expression of this
reporter gene, but not the parental tk-luciferase reporter
(Fig 8A). The transcriptonal repression was
dependent on the amount of plasmid transfected, and when both PLZF and
FAZF were expressed, there was an additive effect on repression (Fig 8B). Taken together, the binding and repression data suggest that not
only do PLZF and FAZF proteins bind to the same DNA sequence, but they
repress transcription by a similar mechanism.
View larger version (19K):
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| Fig 8.
FAZF is a transcriptional repressor. (A) 293T cells were
cotransfected in quadruplicate with a luciferase reporter gene
containing four copies of a high-affinity PLZF binding site (0.15 µg)
from the IL-3-receptor promoter linked 5' to the HSV-tk promoter
or the parental tk-luciferase reporter (0.15 µg) along with 0.85 µg
of expression vectors for PLZF or FAZF as indicated or the pSG5
expression vector. At 48 hours after transfection, the cells were
assayed for luciferase activity. (B) Increasing amounts of the PLZF or
FAZF expression vector or a combination of both vectors was
cotransfected in quadruplicate along with the
IL3-tk-luciferase promoter (0.1 µg). Luciferase
activity was assayed as above.
|
|
| |
DISCUSSION |
We report here that FANCC interacts with a novel protein, FAZF, which
we show functions as a transcriptional repressor. The FAZF/FANCC
interaction maps to a central region in FANCC, which is deleted in
patients with a severe FA phenotype.14,53 Moreover, we
found that wild-type FANCC, but not the L554P mutant protein, can
colocalize with FAZF in nuclear foci. Thus, disturbing the FANCC/FAZF
interaction may play a role in FA.
The FAZF protein is similar to transcriptional repressors of the
BTB/POZ subfamily of Krüppel-like zinc finger proteins (reviewed in Albagli et al25 and Bardwell et al26), which
have been shown to be important for development, tissue-specific
proliferation and differentiation, and neoplasia. In particular, FAZF
is similar to the PLZF gene, which is translocated and fused to the
RAR locus t(11;17)(q23;q21) in a subset of patients with
APL.27,29,35 Recently, the mechanism of transcriptional
repression of PLZF has been shown to occur through binding of specific
promoter sequences61,62 followed by recruitment of nuclear
receptor Sin-3/histone deactylase corepressor
complexes.30-32,70-72 Like PLZF, we found that FAZF is able
to repress transcription, suggesting that the function of these two
proteins is, at least in part, overlapping.
The BTB/POZ protein-protein interaction motif is a highly conserved
domain found in a number of eukaryotic proteins involved in cancer
including the PLZF protein,29 BCL-6,37 and HIC
(hypermethylated in cancer).73 The BTB/POZ domain mediates
self-association26,59,60 and specific heterodimeric
association with other BTB/POZ proteins (eg, PLZF,74 LRF,
and LACZ-3/BCL-641). Recent evidence suggests that even
though BTB/POZ domains may have significant homology and are able to
interact with a related set of corepressor components, they may differ
in their interactions with the corepressor complex.58,75 Thus, crosstalk through heterodimer formation combined with the subtle
differences in corepressor interactions suggests that transcriptional regulation by the proteins in this family is highly permuted. In this
regard, we found that the FANCC/FAZF interaction is apparently not
dependent on the BTB/POZ domain of FAZF, potentially allowing this
region to form homodimeric and heterodimeric complexes with other
proteins. Indeed, we report here that FAZF forms homodimers and
heterodimerizes with PLZF. It will be interesting to determine if
unique binding partners exist for the FAZF POZ domain.
The FANCC/FAZF interaction suggests that FANCC may play a role in the
repression of gene expression, potentially in response to DNA damage.
The nature of the target genes is unknown. The only PLZF target gene
reported so far is cyclin A2. Enforced expression of PLZF delayed the
transit of cells though the G1 and S phases of the cell cycle, an
effect that was reversed by ectopic expression of cyclin A in these
cells.76,77 The cyclin A2 promoter contains two PLZF
binding sites that are required for the protein to repress this
promoter. It is plausible that FAZF could have a similar effect on
cyclin A expression and the cell cycle, perhaps reflecting a role in
inhibiting cell growth in response to DNA damage. Although the FAZF
protein can bind to the same binding site as PLZF, the fact that the
FAZF protein only contains three zinc fingers whereas PLZF contains
nine motifs suggests that the range of target genes bound by these two
proteins may only partially overlap. Future studies will determine the
effects of FAZF in cell growth control and DNA damage response.
Immunofluoresence analysis of FANCC in this study indicated that FANCC
is expressed in both cytoplasmic and nuclear compartments, as reported
previously.16-19 However, L554P was not found in the nuclear compartment at wild-type levels. Failure, or reduced ability, of L554P to enter the nucleus could account for its defect.
Confirmation awaits comparison of endogenous wild-type and L554P
localization that is currently not possible because of low endogenous
expression levels. We also observed that a subset of FANCC-expressing
cells contained nuclear foci, and suggest that this is a feature of wild-type FANCC localization. The following evidence suggests that
these foci might be functionally significant. First, although expressed
at comparable levels, the signal observed for the L554P mutant protein
was greatly reduced in the nucleus as suggested by earlier
studies,19 and was consequently unable to form nuclear foci. That L554P could be highly expressed in the cytoplasm and not
"forced" into the nucleus also argues against the notion that nuclear localization and nuclear foci are simply an artifact of overexpression. Second, FANCA has also been observed in nuclear foci.68 Third, D'Andrea et al18,78 have
reported that the nuclear fractions of FANCA and FANCC, but not L554P,
form a complex and that this nuclear complex is functional. Taken
together, these observations support the notion that foci formation may
be biologically significant. Finally, we determined that FAZF, like
other members of its family, is located primarily in nuclear
foci.26,40,47,60,67 Our coimmunoprecipitation and
colocalization experiments independently support the FAZF/FANCC
interaction detected by two-hybrid analysis and in vitro binding. We
found that in some cells, colocalization of FANCC and FAZF foci was
extensive, whereas only partial overlap was observed in other cells,
suggesting that the FAZF/FANCC interaction may be transient or
conditional. This pattern of partially overlapping foci has been
described for a series of colocalized proteins including BRCA1/hRad51,79 Arenavirus proteins/PML,80
retinoblastoma protein/PML,81 and PML/PLZF.74
Given these complexities, and the fact that the FA pathway is novel,
further studies will be required to understand the consequences of the
FAZF/FANCC interaction and the potential role of transcriptional
repression in the genomic instability observed in FA.
| |
ACKNOWLEDGMENT |
The authors are grateful to their colleagues Mike Forte and Beth
Blanchly-Dyson for initial assistance with the two-hybrid studies. The
authors thank the members of the Fanconi anemia research community for
stimulating discussions, in particular Hans Joenje, Mike Heinrich,
Johan de Winter, Quinten Waisfisz, and Henri van de Vrugt for their
interest and advice.
| |
FOOTNOTES |
Submitted May 21, 1999; accepted July 22, 1999.
Supported by NIH grants HL56045 (M.E.H.), CA59938 (J.D.L., A.Z.), and
K08 CA73762 (A.M.); the Medical Research Foundation of Oregon and the
Fanconi Anemia Research Fund (M.E.H.); and the American Cancer Society
DHP160 (J.D.L.). J.D.L. is a Scholar of the Leukemia Society of America.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Maureen E. Hoatlin, PhD,
Division of Hematology and Medical Oncology, Oregon Health Sciences
University, 3181 SW Sam Jackson Park Rd, Portland, OR 97201; e-mail:
hoatlinm{at}OHSU.edu.
| |
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ABSTRACT
INTRODUCTION