From Le Centre de Recherche en Cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Département de Biologie Médicale, Faculté de Médecine, Québec G1R 2J6, Canada
Received for publication, November 28, 2000
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ABSTRACT |
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Fetoprotein transcription factor
(FTF) is an orphan nuclear receptor that activates the
At 8-8.5 days of mouse embryogenesis, endodermal cells of the
ventral foregut interact with the cardiac mesoderm and become committed
to the hepatic differentiation program; these newly specified cells
then migrate and proliferate in the mesenchyme of the septum
transversum where liver morphogenesis becomes apparent at One of the earliest events marking endodermal specification to liver
function is the activation of the Early developmental patterns of FTF expression in several species (14,
17, 19) and its activation of the AFP locus in hepatocyte progenitors
suggested an important role for FTF in endodermal differentiation
pathways. To gain better insight into FTF functions and its position in
liver induction pathways, we isolated and functionally characterized
the mouse FTF gene promoter in search for upstream FTF gene regulators,
and we also searched for new FTF downstream gene targets. The combined
results make a compelling case for FTF as a key intermediary between
initial signals of liver specification and cascade activations of other transcription factors that enhance hepatic differentiation.
cis-activating elements pin-pointed in the mFTF promoter may lead to
new important effectors of endodermal differentiation pathways.
Isolation and Mapping of the Mouse FTF Gene--
A mouse 129 SV
genomic DNA library in bacteriophage
The mFTF gene transcription start site was mapped by primer extension
of mouse liver RNA using a 25-mer oligonucleotide (FTF-PE1) spanning
mouse FTF gene sequence +37 to +61 (underlined in Fig. 1B). FTF-PE1 was radiolabeled (106 cpm/ng) with
[
Chromosomal localization of the mouse FTF gene was carried out by
fluorescence in situ hybridization (Genome Systems). Mouse FTF genomic DNA (P1 clone) was labeled with digoxigenin dUTP by nick
translation, mixed with sheared mouse DNA, and hybridized to metaphase
chromosomes from normal mouse embryo fibroblasts in 50% formamide,
10% dextran sulfate, and 2× SSC. Hybridization signals were
revealed with fluoresceinated anti-digoxigenin antibodies followed by
counterstaining with 4,6-diamidino-2-phenylindole. Of 80 metaphases
analyzed, 72 exhibited specific labeling; FTF gene assignment was
further confirmed by cohybridization with a telomeric probe specific to
chromosome 1.
FTF Gene Constructs--
FTF gene promoter activity was analyzed
with FTF/CAT reporter constructs spanning 4 kb or 280 bp of contiguous
DNA 5'-adjacent to the mouse FTF transcription start site. A
HindIII-BamHI fragment from pSV0-CAT (9) was
cloned into HindIII-BamHI-digested pBluescript SK+ to generate vector SK-CAT; the EcoRI FTF
gene fragment Hnf1 Electrophoretic Mobility Shift Assays and Transient Cell
Transfections--
Bandshift assays were conducted as described before
(10, 11) with total nuclear proteins from adult rat liver and
oligonucleotides listed in Table II,
using 0.2 ng of 32P-labeled probe, 3 µg of nuclear
proteins, and 5-500-fold molar excess of cold competitor
oligonucleotides. For FTF binding assays, probe and competitors were
coincubated with the nuclear extract for 30 min at 4 °C; for HNF4
Transfection assays were carried out by the calcium phosphate procedure
detailed previously (9, 10), using HepG2, Hep3B, HeLa, and F9 cells
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) containing 10% fetal bovine serum (Wisent) and 1%
penicillin/streptomycin (F9 cultures used gelatin-coated dishes). Cells
were plated at 1.5 × 106 cells in 6-cm Petri dishes
or 75-cm2 flasks and transfected 24 h later with 2-5
µg of CAT reporter plasmid, 2-10 µg of transcription factor
expression vector, and 1.5-2.5 µg of RSV-lacZ to
standardize for transfection efficiency. Cells were washed after
16 h and harvested 48 h after transfection. CAT activity was
measured by thin layer chromatography and phosphorimaging. Luciferase
activity was measured with a EG & G Berthold Lumat LB9507 luminometer.
Expression vectors for transcription factors used viral
enhancer-promoter elements from cytomegalovirus (FTF, HNF4 Genetic Mapping of the Mouse FTF Locus--
We retrieved three
independent clones (ZF2, ZF6, and ZF25) from the mouse genomic library
screened with a rat FTF cDNA probe; clones ZF2 and ZF25 gave
stronger signals on Southern blots and were further characterized.
Clone ZF2 contained a contiguous sequence closely matching the mouse
FTF/LRH-1 cDNA sequence (11) but with many base changes, deletions,
insertions, and no introns, all indicative of a retro-pseudogene. Clone
ZF25 contained FTF coding and interspersed DNA sequences corresponding
to the exon 3-6 domain in Fig.
1A. Intronic oligonucleotides
were used for further screening of a mouse P1 library, which yielded an
insert extending 35 kb upstream from ZF25. The P1 insert revealed
additional exons and a cluster of transcription factor recognition
sites 5'-flanking exon 1 and suggestive of a promoter domain (Fig.
1B). Intron/exon boundaries mapped from
The mFTF gene transcription initiation site was identified by primer
extension of adult mouse liver RNA using antisense primer +61/+37
(underlined in Fig. 1B); a predominant
transcription start site was mapped (Fig. 1B) at a G residue
79 nt upstream from the previously reported 5'-end of mouse FTF
cDNA and 238 nt upstream from the first translation initiation
codon (11) (other extension primers downstream of +79 bp yielded smears
of reverse transcription products terminating around +79; some mRNA
structure perhaps blocked polymerase extension but alternative start
sites around +79 cannot be excluded).
Chromosomal assignment of the mFTF gene was carried out by
fluorescence in situ hybridization with a P1-FTF genomic probe minimizing cross-hybridization with the mouse FTF pseudogene. Out of 80 metaphases analyzed, 72 showed single specific labeling of the middle
portion of chromosome 1 (identified by 4,6-diamidino-2-phenylindole staining and cohybridization with a specific telomeric probe); measurements of chromosomal distances on 10 fluorescence in situ hybridization metaphases located the mouse FTF gene at chromosome 1 band E4 (Fig. 1C).
Functional Analysis of the mFTF Gene Promoter--
Functional
analyses were initiated with mFTF gene construct 4F-CAT carrying FTF
gene segment
mFTF gene activation by HNF4
To prove their role in mFTF promoter activity, point mutations were
introduced in the HNF4, GATA, and Nkx elements in the natural 4-kb
context of construct 4F-CAT; mutations were also introduced in tandem E
box motifs at FTF Binds and Activates the Hnf3 FTF Gene Structure and Splice Products--
This is the first
molecular cloning of a mammalian FTF genomic locus. The partial mFTF
gene structure established here indicates an intron/exon organization
similar to that of the Sf1 gene, which was duplicated from
the same ancestral gene (14). Like in man, the mouse Sf1 and
FTF genes reside on different chromosomes (16, 29, 30). The mouse FTF
gene structure is largely conserved in zebrafish (17) but with an
apparent evolutionary remodeling in the exon 2-3 domain, as discussed below.
The mouse exon mapping indicates 5'-exon heterogeneity between FTF
cDNAs cloned from different species. Clones of human FTF cDNAs
uniformly lack a sequence corresponding to mouse exon 2 (the hFTF
cDNA clone we initially reported in Ref. 16 corresponds to human
FTF cDNA variant hB1F2 in Ref. 29 and CPF variant 1 in Ref. 31; we
reviewed our original sequence data and found one compressed C missing
at position 344 in Ref. 16, disrupting the hFTF N-terminal reading
frame). One human FTF variant (hB1F) (29) (CPF) (31) lacks both exons 2 and 3. Rat FTF (rFTZ-F1
FTF also exists as C-terminal splice variants; human and
Xenopus "short" variants (hFTFs and xFF1rAs) diverge to
noncoding sequences at the 3'-splice junction of exon 6 (16, 33). Other FTF cloning products include human FTF clone CPF-2 (31) truncated in
exon 6 to restart with exon 7, and zebrafish ff1 clones
"B" which also interrupt a C-terminal exon (17). Such transcripts devoid of the AF2 activatory domain could in principle exert dominant negative functions (11, 21, 33); however, no significant amounts of
such products have been detected in vivo (17, 33). The FTF
exons 2-3 domain is further considered below.
Regulation of the mFTF Gene Promoter--
This report is also the
first functional characterization of an FTF gene promoter. Many
cis-acting elements pinpointed here are stringently conserved in
zebrafish (17); such evolutionary conservation suggests that these
elements fulfill important regulatory functions. Given the broad
phylogenetic role of GATA factors in endodermal specification (4), a
triple GATA-binding domain also suggests tight connections of the
GATA-FTF pathway with endodermal differentiation, which conceivably
could extend to invertebrate FTF/Ftz-F1 homologues (to the best of our
knowledge, FTF is the first liver-enriched transcription factor gene
directly activated by GATA).
In cotransfection assays, GATA and HNF4
Point mutations selecting the downstream GATA site or its adjacent E
box element resulted in lower levels of basal FTF promoter activity,
indicating that in hepatoma cells the proximal FTF regulatory domain
The more distal GATA/Nkx mFTF promoter domain between An Alternative mFTF Promoter?--
FTF translational analyses
in vitro and in vivo (11) have clearly
established that rodent FTF exon 1 is transcribed in prenatal liver,
and therefore that the promoter characterized here is used at fetal
stages of development (hFTF exon1 transcripts are also present in fetal
human liver) (29). But data in lower vertebrates point to a second FTF
promoter downstream from exon 1 and used preferentially at early stages
of development. FTF cloning from embryonic cDNA libraries has
yielded transcripts with a translation start site corresponding either
to the Met62 codon of mouse exon 3 (amphibian xFF1rA and
rrFTZ-F1 Novel FTF-activated Gene Targets--
The
Hnf3
The proximal FTF site in the HNF1
In conclusion, this combined analysis of FTF promoter regulatory
elements and gene targets suggests that FTF is an early intermediary in
the endodermal chain of events leading to hepatic differentiation. Our
results are consistent with a simplified scheme (Fig.
6) where GATA cooperates with bHLH or
homeodomain factors to activate the FTF gene in hepatocyte precursors,
and the FTF gene product in turn activates early liver functions (AFP)
and other transcription factors enhancing cross-regulatory networks to
set the hepatic phenotype. FTF gene disruption in the mouse is
embryonic lethal at 1-fetoprotein gene during early liver developmental growth. Here we sought to define better the position of
FTF in transcriptional cascades leading to hepatic differentiation. The
mouse FTF gene was isolated and assigned to chromosome 1 band E4 (one
mFTF pseudogene was also found). Exon/intron mapping shows an mFTF gene
structure similar to that of its close homologue SF1, with two more
N-terminal exons in the mFTF gene; exon mapping also delimits several
FTF mRNA 5'- and 3'-splice variants. The mFTF transcription
initiation site was located in adult liver at 238 nucleotides from the
first translation initiator codon, with six canonical GATA, E box, and
Nkx motifs clustered between
50/
140 base pairs (bp) from the cap
site; DNA/protein binding assays also pinpointed an HNF4-binding
element at +36 bp and an FTF-binding element at
257 bp. Transfection
assays and point mutations showed that the mFTF promoter is activated
by GATA, HNF4
, FTF, Nkx, and basic helix-loop-helix factors, with
marked cooperativity between GATA and HNF4
. A tandem GATA/E box
activatory motif in the proximal mFTF promoter is strikingly similar to
a composite motif coactivated by differentiation inducers in the hematopoietic lineage; a tandem GATA-Nkx motif in the distal mFTF promoter is also similar to a composite motif transducing
differentiation signals from transforming growth factor-
-like
receptors in the cardiogenic lineage. Three genes encoding
transcription factors critical to early hepatic differentiation,
Hnf3
, Hnf4
, and
Hnf1
, each contain dual FTF-binding elements
in their proximal promoters, and all three promoters are activated by
FTF in transfection assays. Direct DNA binding action and cooperativity
was demonstrated between FTF and HNF3
on the Hnf3
promoter and between FTF and HNF4
on the Hnf1
promoter. These combined results suggest that FTF is an early
intermediary between endodermal specification signals and downstream
genes that establish and amplify the hepatic phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E10.5 (1,
2). Initial induction of hepatic functions is driven in part by growth
factors of the FGF1 family,
secreted by cardiac mesodermal cells and acting via transmembrane receptor kinases at the endodermal cell surface (3). The process also
involves potentiating transcription factors among which GATA factors
appear essential to endodermal determination across vertebrates as well
as invertebrates (Ref. 4 and references therein). Following liver
specification, transcriptional activation cascades further develop
among early hepatic transcription factors creating interactive regulatory networks that amplify the induction signals and imprint the
liver phenotype; the HNF4
gene product, for example, activates the
Hnf1
gene whose product further enhances the
Hnf4
gene promoter, and both HNF1
and HNF4
activate
a broad spectrum of liver functions (5, 6).
1-fetoprotein (AFP) locus, one of four albumin-related genes tandemly organized in the
genome but differentially expressed during development (7). The AFP
gene is the first to be activated in the foregut endoderm (8), and
therefore transcription factors that transduce early cell specification
signals to unfold AFP chromatin are likely to exert high ranking liver
differentiation functions. One prime candidate in that regard is an
orphan nuclear receptor originally pinpointed as a potent, highly
specific and mandatory activator of the proximal AFP gene promoter
(9-11). The fetoprotein transcription factor (FTF) (NR5A2) (FTF
designation as per Genome Data Base Nomenclature Committee (accession
number 9837397); NR5A2 as per Nuclear Receptor Nomenclature Committee
(12)) belongs to a subgroup of nuclear receptors related to the
archetypal Drosophila segmentation gene Ftz-F1
(13) and that bind as monomers to the DNA motif PyCAAGGPyCPu
(where Py is C or T and Pu is A or G)(11). FTF is widespread and
tightly conserved among vertebrates (14-17); its closest mammalian
relative is SF1 (18) that is mainly expressed in steroidogenic cell
lineages, whereas FTF is mainly expressed in gut derivatives.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DASH II (Stratagene) was
screened by plaque hybridization using rat FTF cDNA (11) as a
probe. The PCR method was used to screen a mouse 129 SV P1
bacteriophage library (Genome Systems) with the following primers from
mouse FTF gene introns: 5'-AGTTGAATCTCTGCTGCCCGTGTCC-3' (intron 2) and
5'- TAGCCCGAGAGTGTAAAACCAGGAA-3' (intron 3).
DASH II DNA from the
FTF-positive clones was prepared by the plate lysate method, and the P1
clone DNA was prepared by the alkaline lysis procedure. DNA was
digested with various restriction enzymes, and DNA fragments were
ligated into pBluescript SK+ (Stratagene) and used to
transform Escherichia coli DH5
; plasmid DNA was isolated,
and positive subclones were identified by Southern analysis using mouse
FTF/LRH-1 (11) cDNA fragments. DNA was sequenced on both strands by
the dideoxynucleotide method, and exon/intron boundaries were mapped
against the mFTF/LRH1 cDNA sequence (11); mFTF genomic sequences
were deposited in GenBankTM under accession number AF239709
(BankIt 322861).
-32P]ATP and T4 polynucleotide kinase (Life
Technologies, Inc.) and purified by gel electrophoresis and
electroelution; 5 × 105 cpm of the FTF-PE1 primer was
annealed to 200 µg of total RNA from adult mouse liver overnight at
50 °C in 32 µl of Superscript II reverse transcriptase buffer
(Life Technologies, Inc.); the reaction was cooled to 42 °C and
pursued for 2 h in the presence of 10 mM
dithiothreitol, 1 mM dNTPs, and 100 units of Superscript II
reverse transcriptase. Reverse transcription products were subjected to
RNase A digestion, phenol/chloroform extraction, and ethanol/sodium
acetate precipitation and analyzed by electrophoresis on denaturing 6%
polyacrylamide gels; reference DNA sequences used the FTF-PE1 primer
with DNA from FTF gene plasmid 4F-CAT (below).
3.9 kb to +79 bp (leftmost fragment in Fig.
1A) was isolated, blunted, and inserted in
HindIII-blunted SK-CAT to generate construct 4F-CAT. To
obtain pF-CAT, the 4F-CAT vector was digested with PstI to leave only FTF gene segment
184/+79 upstream from CAT, and a PstI
280/
185 FTF segment was inserted at
184 in the
correct orientation. Mutations in the FTF gene promoter were derived by PCRs using plasmid 4F-CAT and oligonucleotides listed in Table I; two
complementary oligonucleotides (125 ng of each) overlapping the
targeted region were mixed with 50 ng of 4F-CAT DNA and amplified with
Pfu DNA polymerase (Stratagene) for 30 s at 95 °C, 1 min at 55 °C, and 2 min/kb of DNA template at 68 °C (18 cycles),
followed by 1 h at 37 °C with 10 units of DpnI
(Stratagene). The PCR products were transformed into E. coli
DH5
, and mutations were confirmed by sequencing. Mutant 4FmG123 was
derived from 4FmG3.
, Hnf3
, and Hnf4
Promoter Vectors--
Reporter
vector pH1-CAT driven by the Hnf1
promoter was obtained
by PCR amplification of mouse Hnf1
gene region
174/+10 using mouse genomic DNA and the following primers:
5'-GGCTCGAGTGCTCACTCCCAATTGCAGGCCATGACTCC-3' and
5'-CCAAGCTTGGCCAGTGAATCAGGGCCCCTGCCTGCTC-3'. The PCR product was
digested with XhoI-HindIII and cloned in SK-CAT
to generate pH1-CAT. Mutations were introduced in pH1-CAT by PCRs as
described above for the FTF constructs, using oligonucleotides H1mF1
and H1mF2 (Table I) with pH1-CAT plasmid
DNA or its derivative pH1mF1-CAT. HNF3
-CAT reporters pH3-CAT and
H3mF1 are constructs HNF3
184/+69 and its UF2-H3
mutant,
described elsewhere (20); vector H3mF12 was derived from H3mF1 using
PCR with a pTZ18U internal primer and oligonucleotide H3mF2 (Table I).
The PCR product was digested with ScaI and SacI
and used to replace the ScaI/SacI segment of H3mF1. HNF4
reporter vector pH4-luc is a luciferase expression vector carrying a
363/+182-bp mouse Hnf4
gene segment
(6).
Oligonucleotides used for mutagenesis
supershift assays, 1 µl of anti-HNF4
serum (Santa Cruz
Biotechnology) was preincubated with the nuclear extract 1 h at
room temperature, and then the probe and competitors were added for an
additional 30 min.
Oligonucleotides used in electrophoretic mobility shift assay
,
C/EBP
, HNF1
, HNF3
, HNF3
, HNF6, SP1, and Nkx2.5), Rous sarcoma virus (GATA1, GATA2, GATA4, GATA5, and GATA6), and simian virus
40 (COUP-TFII); the FTF vector carried full-length human FTF cDNA
(16, 21).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or P1 clones all
conformed to the GT/AG splicing rule. Mouse FTF exons 3-6 correspond
to SF1 exons 1-4 (18), and two additional 5' FTF exons encode the longer FTF N-terminal (A/B) domain (11); the conserved exon/intron junctions of SF1 and FTF predict that the FTF 3'-gene domain contains three more exons (7-9 in the mouse) encoding C terminus amino acids
390-560 (11). mFTF exons 1 and 4-6 are similar in size to zebrafish
ff1 exons 1 and 3-5 (17).
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Fig. 1.
Genetic mapping of the mouse FTF locus.
A, restriction sites (E, EcoRI and
S, StuI) and mapping of mFTF gene exons I-VI
(superscript numbers, encoded amino acids; ZF1
and ZF2, DNA-binding zinc finger 1 and 2 domains;
arrow, translation initiation). The dashed line
is a gene segment measured by PCR and unmapped with restriction
enzymes. B, transcription initiation site and promoter
sequence. Left, the mFTF mRNA start site was located by
primer extension of mouse liver RNA with FTF antisense primer +37/+61
(sequencing ladders used the same primer with 4F-CAT plasmid DNA); the
major reverse transcription product is marked by the arrow.
Right, mFTF promoter sequence compared with the
corresponding zebrafish FTF gene sequence (zff1) (17);
dots indicate conserved nucleotides; transcription factor
recognition elements are marked in bold; arrows
+1, mRNA initiation sites; underlined
sequence: oligonucleotide used for primer extension. C,
chromosomal mapping of the mFTF gene by fluorescence in situ
hybridization using a mouse P1 FTF probe (middle panel,
cohybridization with a chromosome 1 telomeric probe); arrows
point specific signals that assign the mFTF gene to chromosome 1 band
E4.
4 kb/+79 bp. Transfection assays showed strong reporter
gene activity in HepG2 and Hep3B hepatoma cells (which express
endogenous FTF) (21) and no detectable activity in (FTF-negative) HeLa
or F9 cells (inset, Fig.
2B). Reporter CAT activity was
also higher in Hep3B than HepG2 cells, correlating with higher
expression of endogenous FTF in Hep3B cells (21). These results thus
indicated that mFTF gene segment
4 kb/+79 bp correctly reproduced
hepatic specificity and relative activities of endogenous FTF gene
promoter functions. Electromobility shift assays were then conducted
with presumptive HNF4 and FTF recognition sequences located at +36 and
257 (Fig. 1B), slightly divergent from consensus binding
sites. Radiolabeled DNA probe +36/+50 (oligonucleotide FH4, Table II)
yielded a single retarded liver complex that was competed by cold oligo
FH4 and more efficiently by an optimized HNF4-binding site (oligo DR1)
(Fig. 2A, lanes 1-7) but not at all by 100-fold excess of a
mutated FH4 sequence (oligo mFH4 in Table II) (Fig. 2A, lane
10). Furthermore, the retarded complex was completely supershifted
by anti-HNF4
antibodies (Fig. 2A, lane 11). These results
showed that FTF promoter segment +36/+50 is a highly specific HNF4
recognition site, of lower affinity than a canonical HNF4 DR1 element.
Similar assays using as a probe the strong FTF-binding element of the
AFP gene promoter (10, 11) (oligonucleotide FTF
in Table II)
indicated that mFTF gene segment
254/
268 (oligo FF, Table II) can
also efficiently bind FTF, with lower affinity than the FTF
site
(Fig. 2A, lanes 12-17).
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Fig. 2.
A, bandshift reactions showing the
interaction of liver transcription factor HNF4 with mFTF gene
sequence +36/+50 (oligo FH4, Table II) (mFH4, mutated FH4 sequence;
DR1, optimized HNF4-binding site), and the binding of FTF to promoter
sequence
254/
268 (oligo FF, Table II) (FTF
and mFTF
are the
wild-type and mutated FTF-binding sequence from the AFP promoter).
Reactions used 3 µg of rat liver nuclear proteins (arrows
point the four rat FTF isoforms) (10, 11) and 5-100-fold excess
(numbers above lanes) of cold competitors (C, no
competitor; lane 11 shows a supershift reaction by
preincubation of the extract with anti-HNF4
antiserum; all bandshift
is removed indicating that the FH4 element is highly restricted to
HNF4
). B, transient transfection assays conducted in
HepG2 and Hep3B hepatoma cells with mFTF gene reporter constructs
4F-CAT and pF-CAT cotransfected with transcription factor expression
vectors (C, control cotransfections with void vector pCI).
Results are averages ± 1 S.D. from three sets of duplicate or
triplicate transfections using two plasmid preparations and referred to
pCI controls given an independent value of 1 for HepG2 or Hep3B
transfections. Inset, autoradiograms are CAT assays using
FTF gene construct 4F-CAT transiently transfected in the four cell
lines indicated (arrows, chloramphenicol-acetylated
products; <0.05%, undetectable activity).
or FTF was tested by transient
transfection in Hep3B and HepG2 cells. Cotransfection of construct 4F-CAT with an HNF4
expression vector resulted in 5-6-fold
enhancement of reporter gene activity in both cell lines (Fig.
2B, lane 3); cotransfection with an FTF expression vector
also resulted in significant increase of CAT activity in Hep3B cells
(Fig. 2B, lane 4) but not in HepG2 cells (perhaps because
HepG2 cells express abundant SF1, which binds and activates the same
DNA motif as FTF) (11, 21). Three consensus GATA-binding sites (WGATAR) were conspicuous in the proximal mFTF gene domain
80/
138 (Fig. 1B), and we tested activatory effects of GATA expression
vectors including the GATA6 and GATA4 variants expressed in the foregut endoderm and liver primordium (22, 23). Cotransfection of GATA6 or
GATA4 (and GATA1, GATA2, or GATA5 as well) raised 4F-CAT activity
2-3-fold in HepG2 or Hep3B cells (Fig. 2B, lane 2);
coexpression of GATA6 or GATA4 with HNF4
showed additive or
cooperative effects enhancing FTF promoter activity as much as 12-fold
in Hep3B cells (Fig. 2B, lane 6). We also found significant
activation of the 4F-CAT construct with the Nkx2.5 expression vector
(Fig. 2B, lane 5), which is consistent with the recognition
of a cognate high affinity (24) Nkx2.5-binding element at
99/
105 bp
(Fig. 1B); coexpression of GATA4 and Nkx2.5 enhanced 4F-CAT
activity only slightly more than each factor alone (not shown). No
stimulation of mFTF promoter activity was detected with expression
vectors encoding SP1, HNF1
, HNF3
, HNF3
, HNF6, or C/EBP
(Fig. 2B, lanes 7-12) (no binding sequences for these
factors were apparent in the mFTF promoter sequence), nor with c-Myc,
USF1, or USF2 expression vectors (not shown). Generally similar results
were obtained in cotransfections using the shorter FTF gene construct
pF-CAT carrying only 280 bp of 5'-flanking DNA (Fig. 2B, lanes
13-17); this suggested a direct action of the activating factors
on their cognate DNA motifs clustered around the cap site. Basal
activity of pF-CAT was similar to that of 4F-CAT and provided no
indication for regulatory components operating between
280 bp and
4
kb.
66 and
58 bp. Mutation of the HNF4 sequence (FmH4 in
Table I) reduced basal mFTF promoter activity in HepG2 or Hep3B cells
(Fig. 3B, lane 2), and in
cotransfection assays it abolished mFTF promoter activation by HNF4
(Fig. 3C, lanes 1 versus 4). Mutations
targeting the three GATA elements (mutant mG123) or only the proximal
GATA site (mG3) also reduced basal FTF promoter activity (Fig.
3B, lanes 3 and 5), and mG3 attenuated the
response to cotransfected GATA4 (Fig. 3C, lanes 2 versus 7), whereas mutation of the distal GATA
sites (mG12) abolished all induction by GATA4 (Fig. 3C,
lane 8). Mutation of the Nkx element also eliminated
promoter activation by Nkx2.5 (Fig. 3C, lanes 3 versus 11). These combined data showed that mFTF
promoter activation in cotransfection assays using HNF4
, GATA, and
Nkx2.5 vectors resulted from a direct action of these factors on their
cognate DNA-binding elements surrounding the FTF cap site. Notably, in
contrast to the proximal GATA and HNF4 mutants (mG3, mH4), the distal
GATA and Nkx mutants (mG12, mNk) showed significant increase of basal
promoter activity in both HepG2 and Hep3B cells (Fig. 3B, lanes
2 and 3 versus 4 and
6). Also, when the two E box motifs were mutated (mE12),
strong reduction of basal FTF promoter activity was observed (Fig.
3B, lane 9), whereas a single mutation of either the distal
or the proximal E box had opposite effects on basal promoter activity
(mE1 versus mE2 in Fig. 3B, lanes 7 and
8). Double mutant mE12 maintained similar relative responses
to exogenous GATA4 or HNF4
(Fig. 3C, lanes 1 versus
9 and 2 versus 10), like
GATA mutants to HNF4
and vice versa (Fig. 3C, lanes 1 versus 6 and 2 versus
5, and data not shown), suggesting that each factor can
activate the mFTF gene promoter independently.
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Fig. 3.
Mutational analysis of the mFTF gene
promoter. A, diagram of 4F-CAT mutations (using PCR
primers shown in Table I) introduced in the GATA (G1, G2,
and G3), E box (E1 and E2), Nkx, and
HNF4 promoter sites (Fig. 1B). B, mutation
effects on basal mFTF promoter activity in transient transfection
assays; results are averages ± 1 S.D. from three sets of
transfections with three plasmid preparations, referred to wild-type
4F-CAT activity given an independent value of 1 for HepG2 or Hep3B
cells. C, mutation effects on the 4F-CAT response to
cotransfected expression vectors; results are averages ± 1 S.D.
from three transfections with two preparations of plasmids, referred to
void vector pCi transfected in parallel and given an independent value
of 1 for HepG2 or Hep3B cells.
, Hnf4
, and Hnf1
Promoters--
Among liver-enriched transcription factors, HNF3
,
HNF4
, and HNF1
exert preponderant functions in the establishment
and maintenance of the hepatic phenotype (4, 5, 25). We observed that
the promoter sequence of the Hnf3
, Hnf4
,
and Hnf1
genes all contained putative FTF-binding sites;
the proximal Hnf1
promoter had potential FTF recognition
elements at
47 and
99 bp, on each side of the HNF4-binding element
(Fig. 4A and Ref. 5); the Hnf3
promoter also had two proximal FTF-like sequences
organized in tandem at
35 and
58 downstream from the HNF3
autoregulatory element (Fig.
5A and Ref. 20); and the
Hnf4
promoter had two FTF-like sequences at
224 and
290, upstream from the HNF1 recognition element (Fig. 5B
and Ref. 6). Electromobility shift assays confirmed that all FTF motifs
identified in the Hnf1
, Hnf3
, and
Hnf4
promoters were efficient competitors in bandshift
reactions using the high affinity AFP/FTF-binding sequence as a probe
(Figs. 4A and 5, A and B).
Furthermore, in transfection assays, Hnf1
, Hnf3
, and Hnf4
promoter constructs were all
activated by cotransfection of the FTF expression vector (Fig.
4B, lane 2, Fig. 5A, lane 2, and Fig. 5B,
lane 3) to a similar extent as by other known direct activators,
i.e. the Hnf1
promoter by HNF4
(Fig.
4B, lane 3), the Hnf4
promoter by HNF1
(Fig. 5B, lane 2), or the Hnf3
promoter by
HNF3
(Fig. 5A, lane 3). In addition, mutation of the two
FTF-binding sites in the Hnf1
or Hnf3
promoters eliminated all promoter response to exogenous FTF (Fig.
4B, lane 11, and Fig. 5A, lane 8) and not to
HNF4
or HNF3
(Fig. 4B, lane 12, and Fig. 5A,
lane 9). Transfection assays confirmed coactivatory effects of
HNF4
and COUP on the Hnf1
promoter (27) (Fig.
4B, lanes 3 and 5) and showed cooperativity
between FTF and HNF3
in activating the Hnf3
promoter
(Fig. 5A, lane 4) and between FTF, HNF4
, and COUP in
activating the Hnf1
promoter (Fig. 4A,
lanes 4 and 6). Notably, the Hnf4
promoter responded strongly to GATA6 in HepG2 cells (Fig.
5B), and it contains no apparent GATA-binding sites; this may indicate activatory protein-protein interaction between GATA6 and
SF1 (28). SF1 is highly expressed in HepG2 cells (21) and presumably
occupies FTF-binding sites on the Hnf4
promoter.
View larger version (40K):
[in a new window]
Fig. 4.
Activation of the Hnf1
promoter by FTF. A, left,
bandshift reactions conducted (like in Fig. 2) with rat liver nuclear
extract and oligonucleotides shown in Table II (H1FTF-1 and H1FTF-2
correspond to FTF sites 1 and 2 in the Hnf1
promoter).
Right, diagram of FTF site mutations introduced in
Hnf1
promoter construct pH1-CAT (mutated sequences H1mF1
and/or H1mF2 are shown in Table I). B, transfection assays
in Hep3B or HepG2 cells showing the response of pH1-CAT or its FTF site
mutants to coexpressed transcription factors; results are averages ± 1 S.D. from three transfections using two plasmid preparations,
referred to parallel transfection with void vector pCI (column
C) given an independent value of 1 for HepG2 or Hep3B cells (basal
activities of pH1-CAT, H1mF1, H1mF2, or H1mF12 did not vary
significantly).
View larger version (37K):
[in a new window]
Fig. 5.
Activation of the Hnf3
and Hnf4
promoters by FTF. A,
diagram, regulatory elements of the Hnf3
promoter (26) and FTF site mutations (cf. sequences in Table
I) introduced in reporter construct pH3-CAT. Autoradiogram,
FTF binding to Hnf3
promoter sites H3FTF-1 and H3FTF-2,
shown by bandshift reactions using a radiolabeled AFP/FTF probe
(FTF
) incubated with 3 µg of rat liver nuclear proteins and
20-fold molar excess of unlabeled oligonucleotides (sequences are given
in Table II) (C, no competitor). Histograms,
transfection assays in HepG2 cells showing the effect of FTF site
mutations on basal pH3-CAT activity (lanes C) and on HNF3
promoter inducibility by cotransfected FTF and/or HNF3
; results are
averages ± 1 S.D. from four sets of transfections using two
plasmid preparations referred to pH3-CAT/pCI cotransfection (lane
1) given a value of 1. B, autoradiogram,
bandshift reactions showing FTF binding to Hnf4
promoter
element FTF-2 (oligo H4FTF-2 in Table II) (results were similar with
oligo H4FTF-1); reactions were conducted as in A with
20-500-fold molar excess of cold competitors. Histogram,
transfection assays in HepG2 cells showing enhancement of pH4-luc
activity by coexpressed FTF, HNF1
, or GATA6 (no additive effects
were found between these three factors); results are averages ± 1 S.D. from three transfections using two plasmid preparations, referred
to void vector pCI (C) given a value of 1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (32) also exists as two 5'-transcripts, one
21 amino acids shorter than the other and probably skipping exon 2. In
zebrafish, one FTF transcript (ff1-I) (17) contains a
17-amino acid exon 1 corresponding to mouse exon 1 and spliced to an
exon corresponding to mouse exon 4, i.e. also skipping the
mouse homologue 2-3 domain. Thus, some FTF transcripts are 5'-splicing
variants that lack most of the N-terminal protein (A/B) domain flanking
the FTF DNA-binding domain. FTF splice variant 1:4 (hB1F, CPF) is more
abundant than variant 1:3 (hFTF, hB1F2, CPF-1) in human liver (29), and
the shorter rat FTF variant (presumably 1:3) (rFTZ-F1
2) is also more abundant than the longer transcript (rFTZ-F1
1) (32). These observations may indicate that the FTF N-terminal (A/B) domain fulfills
no particular activatory functions, which would fit transfection data
showing comparable transactivations by full-length FTF cDNA versus FTF devoid of the first 3 exons (pf1
versus pf4 in Ref. 11).
were highly efficient
partners in coactivating the mFTF promoter, consistent with abundant
coexpression of GATA4, HNF4
, and FTF in mammalian liver. However,
FTF is also abundant in other tissues such as the adult exocrine
pancreas in which HNF4
is not significantly expressed. Furthermore,
during development FTF gene expression precedes that of HNF4
(FTF
can be unambiguously detected by in situ hybridization at
E7.0 in the foregut
endoderm,2 which is
consistent with AFP gene transcription beginning at least from E8.0
(8)), and HNF4
knockout experiments also indicate that the FTF (and
AFP) genes are efficiently expressed without HNF4
at early stages of
liver differentiation (25). Therefore the FTF promoter segment analyzed
here would bypass HNF4
at prehepatic endodermal stages.
85/
61 is subject to up-regulation by GATA and basic
helix-loop-helix (bHLH) factors. bHLH proteins play important
differentiation functions in diverse cell lineages and are plausible
activators of the FTF gene at an early stage of development. The tandem
GATA/E box motif is also intriguingly similar to a composite element of
the hematopoietic lineage, with the same orientation and spacing, and
coactivated by a protein complex made of GATA1, bHLH factor Tal1, and
several bridging molecules that include the LIM domain protein Lmo2
(34). GATA1, Tal1, and Lmo2 are each required for proper development of
the erythropoietic lineage, and genes activated by the composite GATA/E
box motif are predicted to play critical differentiation functions in
the blood cell lineage. FTF could thus plausibly qualify as a first
target gene for such a GATA/E box multiprotein activatory complex in
the endodermal lineage. Furthermore, a second FTF promoter E box
element is present two nucleotides downstream from the first one and
appears to down-regulate FTF promoter activity (Fig. 3B, lane
8); this strikingly reproduces a variant hematopoietic GATA/E box
motif in which the second E box serves to attenuate the adjacent
GATA1-Tal1 multiprotein activatory complex (35). Differential mFTF
promoter activity between tissues or developmental stages might then
finely respond to changing combinations of activatory or repressor
components forming the GATA/E boxes complex. We note here that the
second E box is not conserved in the zebrafish promoter (see Fig.
1B), the usage of which is much less restricted among adult
tissues (abundant FTF transcripts ff1-I are detected in ovary, testis, and muscle) (17).
99 and
138 bp
conveys clear inductive responses to exogenous GATA and Nkx2.5 in
cotransfection assays, but mutations in these elements do not reduce
but rather enhance basal FTF promoter activity in hepatoma cells. This
suggests that the distal GATA/Nkx segment may be less efficiently used
in hepatocytes than in other cell types or liver differentiation
states. Like bHLH factors, Nk2-related homeoproteins such as Nkx2.5
broadly serve mammalian cells to execute differentiation programs; Nkx
factors are also represented in hepatic cells (36). Notably, the
combination of GATA4/Nkx2.5 factors that enhanced the mFTF promoter are
well known to induce pleiotropic differentiation program in the
cardiogenic lineage, and they cooperatively activate heart-specific
gene promoters via tandem motifs similar to that in the distal FTF
promoter (37). Interestingly, cardiogenic pathways use GATA4/Nkx2.5 as
terminal transducers of differentiation cascades triggered by
cell-surface TGF-
-related receptors (38); similar TGF-
signals
operate in endodermal specification (39), and TGF-
or FGF relays (3) could plausibly reach the distal FTF promoter domain and activate its
preferential use in specific cells and times.
) (14, 40) (zebrafish ff1 variant II (17) also
initiates translation at Met62) or to the Met94
codon of mouse exon 4 (frog rrFTZ-F1
, chicken OR2.0) (15, 40).
Furthermore, the N-terminal exon of xFF1rA, ff1-II, and rrFTZ-F1
contains a 3'-end sequence matching mouse exon 3 segment Met62-Val89 (see Fig. 1A) (17, 40),
but its 5'-side sequence becomes unrelated to mouse exon 3 (17, 33,
40), and it spans
500 nucleotides in xFF1rAs (14, 33). This readily
suggests that FTF gene transcription starts in an intronic domain in
which the exon 2-3 structure has been evolutionarily rearranged. In
addition, fish/amphibian transcripts containing exon 1 are undetectable at embryonic stages, whereas transcripts excluding exon 1 increase abundantly in early embryos and then decline (14, 17). Thus, all
indicate that at least in lower vertebrates FTF gene transcription proceeds from both the upstream promoter characterized here and from an
internal "early" promoter yet to be mapped and independently regulated. The early promoter also seems broadly activated in fish,
amphibian, or avian embryonic cells (especially brain and steroidogenic
tissues), whereas the upstream promoter is less promiscuous and more
active in gut derivatives (14, 15, 17, 40). We note that no SF1
transcripts have been found in fish (41); an alternate or
ancestral FTF gene promoter possibly evolved in early vertebrates to
better fulfill both FTF and SF1 functions in time and space. Whether a
second mFTF gene promoter may actively operate between exons 1 and 3 remains conjectural. But FTF isoforms referred to translational
variants, and fluctuating between adult liver and fetal liver or
embryonic stem cells (11) might also indicate modulations in both
promoter and splicing products; for instance, the four rat FTF isoforms
(10) (see Fig. 2A) interpreted as translational variants
Met1, Met62, Met83, and
Met87 (11) could also be accounted for by exon variants
1:2, 1:3, 1:4, and 3:4. An alternative mFTF promoter might take charge
of FTF transcription detected as early as the 2-cell stage embryo and
in all cells of the E3.5 blastocyst (our unpublished results with a
lacZ-marked mFTF genomic locus).2
, Hnf4
, and
Hnf1
genes encode transcription factors critically
involved in early liver differentiation, and the promoters of all three
genes were found here to be activated by FTF. Combined with temporal
patterns of expression of these four factors (19, 25, 42, 43) and the
fact that the FTF promoter, but not the Hnf3
,
Hnf4
, and Hnf1
promoters, contain
bona fide GATA-inducible sites, these results clearly
suggest that FTF lies between GATA and HNF3
, HNF4
, and HNF1
in
transcriptional cascades of liver cell differentiation. Thus, as FTF
initiates hepatic functions at the AFP promoter, it would also enhance
other transcription factors engaged into liver-induction networks. We
recall here that AFP gene activity is restricted to early proliferative
stages of liver development and that the FTF regulation domain in the AFP promoter is deactivated by hormonal signals that interrupt liver
cell growth (9, 11, 44); this implies that the FTF function may be
dependent upon or enhanced by cell growth stimuli. Although other
potential FTF liver gene targets have recently been proposed (21, 29,
31, 45, 46), it is uncertain whether all FTF effects can actually
proceed in nongrowing hepatocytes since most results were obtained by
cotransfection in growing cultured cells. For one, the hepatitis B
viral core promoter, strongly activated by three FTF-binding elements
(21, 29), could have evolved FTF activation sites to adapt sustained
hepatitis B virus replication with proliferative stages of
hepatitis. FTF action on the Hnf3
,
Hnf4
, and Hnf1
promoters may then be
exerted only transiently at early developmental growth stages and be
substituted by alternative factors in mature nondividing hepatocytes;
the Hnf3
, Hnf4
, and
Hnf1
promoters all contain substitute proximal elements
expected to operate efficiently under adult stage conditions. But FTF
clearly plays a role in hepatocyte bile acid synthesis pathways; FTF
activates the rate-limiting Cyp7A gene promoter in adult
hepatocytes (47), and FTF is a direct target for CYP7A feedback
repression by FTF heterodimeric partner SHP induced by bile acid
receptor FXR (48-50). FTF could thus use alternative regulatory means
to modulate differentially early developmental and late metabolic liver
program. It is interesting that the Hnf1
, Hnf3
, Hnf4
, hepatitis B virus, and AFP
promoters, but not Cyp7A, all contain a close doublet of
FTF-binding sites (Fig. 4 and 5, Ref. 21).2 This may point
to novel regulatory means for DNA-bound FTF monomers operating during
cell growth; transient FTF-FTF interactions via a cell
cycle-dependent FTF modification, ligand, or coregulator is
an intriguing possibility.
promoter is particularly
interesting with regard to developmental growth signaling. That site is
stringently conserved in the Xenopus Hnf1
promoter (51), which undergoes developmental enhancement (52) closely
parallel to that of the Xenopus FTF gene (xFF1rA)
(14). Furthermore, the FTF site in the xHnf1
promoter
lies in a 30-bp domain essential to promoter activity (deletant 238/207
in Ref. 51), and it is contained in a short 69-bp promoter segment
inducible by activin A (53), also a cell surface receptor ligand of the
TGF-
superfamily. The 69-bp domain carries the conserved
HNF4-binding site (5, 51) which, however, is dispensable for activation
of the 69-bp domain in endodermal cells (54). It is thus very tempting
to infer that the endodermal activin A response element in the
xHnf1
promoter is indeed FTF (xFF1rA). Given
the fast kinetics of xHnf1
promoter induction by activin
A (<6 h) (53), this may then imply that TGF-
and/or FGF signaling
could reach both the FTF gene promoter (as discussed previously) and
the FTF protein itself.
E7.5,2 and it will be important to
see if FTF-mutant ES cells contribute to the hepatocyte lineage of
chimeric mice.
View larger version (12K):
[in a new window]
Fig. 6.
FTF relationships with other genes involved
in liver differentiation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Robert Costa for collaboration and the gift of plasmids; Dr. Mona Nemer and Dr. Stephen Duncan for useful information and materials; Dr. Frances Sladek for the pH4-luc vector; Julie Vézina and Daniel Malenfant for experimental assistance; and Denise Rioux and Marie-France Voyer for excellent secretarial support.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant MT-6478 from the Canadian Institutes for Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a doctoral studentship from Fonds de la Recherche en
Santé du Québec and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.
§ To whom correspondence should be addressed: Cancer Research Centre, L'Hôtel-Dieu de Québec, Québec G1R 2J6, Canada. Tel.: 418-691-5543; Fax: 418-691-5489; E-mail: luc.belanger@crhdq.ulaval.ca.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M010737200
2 J.-F. Paré, A. Lamontagne, and L. Bélanger, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FGF, fibroblast
growth factor;
AFP, 1-fetoprotein;
bHLH, basic
helix-loop-helix;
FTF, fetoprotein transcription factor;
mFTF, mouse
FTF;
HNF, hepatocyte nuclear factor;
SF1, steroidogenic factor 1;
CAT, chloramphenicol acetyltransferase;
PCR, polymerase chain reaction;
bp, base pair;
kb, kilobase pairs;
nt, nucleotide;
oligo, oligonucleotide;
TGF-
, transforming growth factor-
.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Le Douarin, N. (1975) Med. Biol. 53, 427-455[Medline] [Order article via Infotrieve] |
2. | Zaret, K. (1998) Curr. Opin. Genet. & Dev. 8, 526-531[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Jung, J.,
Zheng, M.,
Goldfarb, M.,
and Zaret, K. S.
(1999)
Science
284,
1998-2003 |
4. | Zaret, K. (1999) Dev. Biol. 209, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
5. | Kuo, C. J., Conley, P. B., Chen, L., Sladek, F. M., Darnell, J. E., Jr., and Crabtree, G. R. (1992) Nature 355, 457-461[CrossRef][Medline] [Order article via Infotrieve] |
6. | Zhong, W., Mirkovitch, J., and Darnell, J. E., Jr. (1994) Mol. Cell. Biol. 14, 7276-7284[Abstract] |
7. |
Bélanger, L.,
Roy, S.,
and Allard, D.
(1994)
J. Biol. Chem.
269,
5481-5484 |
8. | Gualdi, R., Bossard, P., Zheng, M., Hamada, Y., Coleman, J. R., and Zaret, K. S. (1996) Genes Dev. 10, 1670-1682[Abstract] |
9. | Guertin, M., LaRue, H., Bernier, D., Wrange, O., Chevrette, M., Gingras, M.-C., and Bélanger, L. (1988) Mol. Cell. Biol. 8, 1398-1407[Medline] [Order article via Infotrieve] |
10. | Bernier, D., Thomassin, H., Allard, D., Guertin, M., Hamel, D., Blaquière, M., Beauchemin, M., LaRue, H., Estable-Puig, M., and Bélanger, L. (1993) Mol. Cell. Biol. 13, 1619-1633[Abstract] |
11. | Galarneau, L., Paré, J.-F., Allard, D., Hamel, D., Lévesque, L., Tugwood, J. D., Green, S., and Bélanger, L. (1996) Mol. Cell. Biol. 16, 3853-3865[Abstract] |
12. | Nuclear Receptors Nomenclature Committee. (1999) Cell 97, 161-163[Medline] [Order article via Infotrieve] |
13. | Lavorgna, G., Ueda, H., Clos, J., and Wu, C. (1991) Science 252, 848-851[Medline] [Order article via Infotrieve] |
14. | Ellinger-Ziegelbauer, H., Hihi, A. K., Laudet, V., Keller, H., Wahli, W., and Dreyer, C. (1994) Mol. Cell. Biol. 14, 2786-2797[Abstract] |
15. | Kudo, T., and Sutou, S. (1997) Gene (Amst.) 197, 261-268[CrossRef][Medline] [Order article via Infotrieve] |
16. | Galarneau, L., Drouin, R., and Bélanger, L. (1998) Cytogenet. Cell Genet. 82, 269-270[Medline] [Order article via Infotrieve] |
17. | Lin, W.-W., Wang, H.-W., Sum, C., Liu, D., Hew, C. L., and Chung, B.-C. (2000) Biochem. J. 348, 439-446[CrossRef][Medline] [Order article via Infotrieve] |
18. | Ikeda, Y., Lala, D. S., Luo, X., Kim, E., Moisan, M.-P., and Parker, K. L. (1993) Mol. Endocrinol. 7, 852-860[Abstract] |
19. | Rausa, F. M., Galarneau, L., Bélanger, L., and Costa, R. H. (1999) Mech. Dev. 89, 185-188[CrossRef][Medline] [Order article via Infotrieve] |
20. | Pani, L., Qian, X., Clevidence, D., and Costa, R. H. (1992) Mol. Cell. Biol. 12, 552-562[Abstract] |
21. |
Gilbert, S.,
Galarneau, L.,
Lamontagne, A.,
Roy, S.,
and Bélanger, L.
(2000)
J. Virol.
74,
5032-5039 |
22. |
Bossard, P.,
and Zaret, K. S.
(1998)
Development
125,
4909-4917 |
23. | Koutsourakis, M., Langeveld, A., Patient, R., Beddington, R., and Grosveld, F. (1999) Development 126, 723-732[Abstract] |
24. |
Chen, C. Y.,
and Schwartz, R. J.
(1995)
J. Biol. Chem.
270,
15628-15633 |
25. |
Li, J.,
Ning, G.,
and Duncan, S. A.
(2000)
Genes Dev.
14,
464-474 |
26. | Samadani, U., and Costa, R. H. (1996) Mol. Cell. Biol. 16, 6273-6284[Abstract] |
27. | Ktistaki, E., and Talianidis, I. (1997) Mol. Cell. Biol. 17, 2790-2797[Abstract] |
28. |
Tremblay, J. J.,
and Viger, R. S.
(1999)
Mol. Endocrinol.
13,
1388-1401 |
29. |
Li, M.,
Xie, Y.-H.,
Kong, Y.-Y.,
Wu, X.,
Zhu, L.,
and Wang, Y.
(1998)
J. Biol. Chem.
273,
29022-29031 |
30. | Taketo, M., Parker, K. L., Howard, T. A., Tsukiyama, T., Wong, M., Niwa, O., Morton, C. C., Miron, P. M., and Seldin, M. F. (1995) Genomics 25, 565-567[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Nitta, M.,
Ku, S.,
Brown, C.,
Okamoto, A. Y.,
and Shan, B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6660-6665 |
32. | Nawata, H., Yanase, T., Oba, K., Ichino, I., Saito, M., Goto, K., Ikuyama, S., Sakai, H., and Takayanagi, R. (1999) J. Steroid Biochem. Mol. Biol. 69, 323-328[CrossRef][Medline] [Order article via Infotrieve] |
33. | Ellinger-Ziegelbauer, H., Gläser, B., and Dreyer, C. (1995) Mol. Endocrinol. 9, 872-886[Abstract] |
34. |
Wadman, I. A.,
Osada, H.,
Grütz, G. G.,
Agulnick, A. D.,
Westphal, H.,
Forster, A.,
and Rabbitts, T. H.
(1997)
EMBO J.
16,
3145-3157 |
35. |
Cohen-Kaminsky, S.,
Maouche-Chrétien, L.,
Vitelli, L.,
Vinit, M.-A.,
Blanchard, I.,
Yamamoto, M.,
Peschle, C.,
and Roméo, P.-H.
(1998)
EMBO J.
17,
5151-5160 |
36. |
Apergis, G. A.,
Crawford, N.,
Ghosh, D.,
Steppan, C. M.,
Vorachek, W. R.,
Wen, P.,
and Locker, J.
(1998)
J. Biol. Chem.
273,
2917-2925 |
37. |
Durocher, D.,
Charron, F.,
Warren, R.,
Schwartz, R. J.,
and Nemer, M.
(1997)
EMBO J.
16,
5687-5696 |
38. |
Monzen, K.,
Shiojima, I.,
Hiroi, Y.,
Kudoh, S.,
Oka, T.,
Takimoto, E.,
Hayashi, D.,
Hosoda, T.,
Habara-Ohkubo, A.,
Nakaoka, T.,
Fujita, T.,
Yazaki, Y.,
and Komuro, I.
(1999)
Mol. Cell. Biol.
19,
7096-7105 |
39. |
Tremblay, K. D.,
Hoodless, P. A.,
Bikoff, E. K.,
and Robertson, E. J.
(2000)
Development
127,
3079-3090 |
40. | Nakajima, T., Takase, M., Miura, I., and Nakamura, M. (2000) Gene (Amst.) 248, 203-212[CrossRef][Medline] [Order article via Infotrieve] |
41. | Chai, C., and Chan, W.-K. (2000) Mech. Dev. 91, 421-426[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Ang, S.-L.,
Wierda, A.,
Wong, D.,
Stevens, K. A.,
Cascio, S.,
Rossant, J.,
and Zaret, K. S.
(1993)
Development
119,
1301-1315 |
43. | Blumenfeld, M., Maury, M., Chouard, T., Yaniv, M., and Condamine, H. (1991) Development 113, 589-599[Abstract] |
44. | Bélanger, L., Hamel, D., Lachance, L., Dufour, D., Tremblay, M., and Gagnon, P. M. (1975) Nature 256, 657-659[Medline] [Order article via Infotrieve] |
45. |
Lee, Y.-K.,
Parker, K. L.,
Choi, H.-S.,
and Moore, D. D.
(1999)
J. Biol. Chem.
274,
20869-20873 |
46. |
del Castillo-Olivares, A.,
and Gil, G.
(2000)
J. Biol. Chem.
275,
17793-17799 |
47. |
del Castillo-Olivares, A.,
and Gil, G.
(2000)
Nucleic Acids Res.
28,
3587-3593 |
48. | Lu, T. T., Makishima, M., Repa, J. J., Schoonjans, K., Kerr, T. A., Auwerx, J., and Mangelsdorf, D. J. (2000) Mol. Cell 6, 507-515[Medline] [Order article via Infotrieve] |
49. | Goodwin, B., Jones, S. A., Price, R. R., Watson, M. A., McKee, D. D., Moore, L. B., Galardi, C., Willson, J. G., Lewis, M. C., Roth, M. E., Maloney, P. R., Wilson, T. M., and Kliewer, S. A. (2000) Mol. Cell 6, 517-526[Medline] [Order article via Infotrieve] |
50. | Sinal, C. J., Tohkin, M., Miyata, M., Ward, J. M., Lambert, G., and Gonzalez, F. J. (2000) Cell 102, 731-744[Medline] [Order article via Infotrieve] |
51. | Zapp, D., Bartkowski, S., Holewa, B., Zoidl, C., Klein-Hitpass, L., and Ryffel, G. U. (1993) Mol. Cell. Biol. 13, 6416-6426[Abstract] |
52. | Bartkowski, S., Zapp, D., Weber, H., Eberle, G., Zoidl, C., Senkel, S., Klein-Hitpass, L., and Ryffel, G. U. (1993) Mol. Cell. Biol. 13, 421-431[Abstract] |
53. |
Weber, H.,
Holewa, B.,
Jones, E. A.,
and Ryffel, G. U.
(1996)
Development
122,
1975-1984 |
54. | Ryffel, G. U., and Lingott, A. (2000) Mech. Dev. 90, 65-75[CrossRef][Medline] [Order article via Infotrieve] |