From the Hormone and Metabolic Research Unit, Louvain University Medical School and the Christian de Duve Institute of Cellular Pathology, Avenue Hippocrate 75, B-1200 Brussels, Belgium
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ABSTRACT |
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Transcription factors of the ONECUT class, whose
prototype is hepatocyte nuclear factor (HNF)-6, are characterized by
the presence of a single cut domain and by a peculiar homeodomain (Lannoy, V. J., Bürglin, T. R., Rousseau, G. G.,
and Lemaigre, F. P. (1998) J. Biol. Chem. 273, 13552-13562). We report here the identification and characterization
of human OC-2, the second mammalian member of this class. The
OC-2 gene is located on human chromosome 18. The
distribution of OC-2 mRNA in humans is
tissue-restricted, the strongest expression being detected in the liver
and skin. The amino acid sequence of OC-2 contains several regions of
high similarity to HNF-6. The recognition properties of OC-2 for
binding sites present in regulatory regions of
liver-expressed genes differ from, but overlap with, those of HNF-6.
Like HNF-6, OC-2 stimulates transcription of the hnf-3 The identification of transcription factors has provided insight
into the mechanisms of the control of gene expression. Some of these
factors contain a DNA-binding region called the homeodomain. The
homeodomain proteins are evolutionarily conserved and play an important
role in cell differentiation and in morphogenesis (1-4). Several
homeodomain proteins contain a second type of DNA-binding domain. This
is the case for the proteins of the CUT superclass, in which the second
DNA-binding domain is called cut because it was initially described in
the Drosophila CUT protein (5-7). The CUT superclass
comprises three classes (8). The CUX class, whose members have three
cut domains, includes the Drosophila CUT protein and its
mammalian homologs, namely human CDP, rat CDP-2, dog CLOX, and mouse
CUX and CUX-2. The SATB class, whose members have two cut domains,
includes the human homeodomain proteins called matrix attachment
region-binding proteins or SATB (special
AT-rich binding) proteins. A third class,
called ONECUT because its members have a single cut domain, was
identified (8) thanks to the cloning of rat hepatocyte nuclear factor-6
(HNF-6)1 (9). The ONECUT
class includes mammalian HNF-6 and four Caenorhabditis elegans cDNAs or open reading frames (ORFs) (8).
The proteins of the ONECUT class are characterized not only by their
single cut domain, but also by a homeodomain with a peculiar amino acid
composition. Homeodomains, which are 60 residues long, are organized in
three Our previous experiments showed that HNF-6 can bind to a number of DNA
sites, which differ slightly in terms of sequence, and that the cut
domain is required for binding of HNF-6 to all the sites tested (8).
These experiments ascribed a dual role to the peculiar homeodomain of
HNF-6. They showed that this domain is involved in DNA binding, but
only for a subset of the sites recognized by HNF-6. They also showed
that the homeodomain is involved in transcriptional activation, but
only of those genes for which the binding of HNF-6 does not require its
homeodomain. By mutational analysis, we demonstrated that phenylalanine
48 and methionine 50 play a role in this transcriptional activating function of the HNF-6 homeodomain (8). This work indicated that the
linker region between the cut domain and homeodomain of HNF-6 is
important for DNA binding (8). We identified two rat isoforms (HNF-6 As mentioned above, the ONECUT class contains several C. elegans members, but only one mammalian member, namely HNF-6. The DNA-binding domains of these C. elegans proteins display DNA
binding properties similar to those of HNF-6 (8). This indicates that these properties have been evolutionarily conserved and are therefore important for basic regulatory processes that are common to nematodes and mammals. The existence of several members of the ONECUT class in
C. elegans prompted us to search for mammalian members of
this class that are distinct from HNF-6. We describe here a new member, which we call OC-2, of the ONECUT class.
Oligonucleotides--
The polymerase chain reaction (PCR)
primers were as follows: bACT5, 5'-GGCATCGTGATGGACTCCG-3'; bACT3,
5'-GCTGGAAGGTGGACAGCGA-3'; FM2.1, 5'-ATTTCCCAGCAGCTGGG-3'; FM2.2,
5'-TATCTGTTGTCCTCATTTGG-3'; FM2.2A, 5'-CAGCTAGGAATCCGGTGTC-3'; FM2.9,
5'-TGGGGCTGGCCAGCAGCT-3'; FM2.10, 5'-AAAGTCTGGGCACTTTGC-3'; HNF6.6,
5'-GCTAGACGAGCAAAATCACACTCC-3'; HNF6.5,
5'-AAACCAAAGACTTAGCTCACCTGCA-3'; VLIIA,
5'-CCCAAGCTTACCATGGCAATCAATACCAAAGAGGTGGCTC-3'; and VLIIB,
5'-CGGGATCCTCACAGCCACTTCCACATCCTCCG-3'. The primers used as probes in
Southern blotting experiments were as follows: HOM2,
5'-CCYMGRYTSGYSTTYAC-3'; HOM1, 5'-CKNSYRTTCATRAARAA-3'; HNF6.3,
5'-AGGAAAGAGCAAGAACATGGGAAGG-3'; and HNF6.4,
5'-CTGCTCATCATTTGTCTTGCCAAG-3'. The double-stranded oligonucleotide
probes used in electrophoretic mobility shift assays were as follows:
HNF-4, 5'-AGGATAGAAGTCAATGATCTGGGA-3' ( Cloning of OC-2 cDNA--
A 193-base pair (bp)-long OC-2
probe was synthesized using the PCR primers FM2.1 and FM2.2 and the
IMAGE clone 566080 as template. This clone contains an expressed
sequence tag (GenBankTM accession number AA121823) derived
from a human fetal retina cDNA library. The OC-2 probe was used to
screen a HeLa cell cDNA library (kindly provided by J.-M. Garnier)
prepared in Radiation Hybrid Mapping--
The chromosomal location of the
human OC-2 gene was characterized by radiation hybrid
mapping using the GeneBridge 4 Radiation Hybrid panel (Research
Genetics). This panel of 93 human × hamster somatic cell hybrids
was screened by PCR (35 cycles at 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 1 min) with primers FM2.1 and FM2.2A in 50-µl
incubations and visualized on an ethidium bromide-stained agarose gel.
The data were analyzed using the Whitehead Institute/Massachusetts
Institute of Technology Center for Genome Research radiation hybrid
map.3
RT-PCR--
To determine the tissue distribution of
OC-2 mRNA, RT-PCR was performed (GeneAmp kit,
Perkin-Elmer) with 500 ng of total RNA isolated from human tissues. The
integrity of the RNA preparations was tested by amplification of a
610-bp-long Expression Vectors and Reporter Constructs--
The
expression vector pCMV-HNF6 Transfections and Cell Extracts--
Rat hepatoma FTO-2B
cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12
medium supplemented with 10% fetal calf serum. Cells (1 × 105 cells/well on 24-well plates) were transfected in
medium without fetal calf serum by lipofection using LipofectAMINE PLUS
(Life Technologies, Inc.), 400 ng of reporter construct
(pHNF6/HNF3
COS-7 cells (1.5 × 105 cells/6-cm dish) were
transfected in Dulbecco's modified Eagle's medium without fetal calf
serum by lipofection using
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-triethylammonium methyl sulfate (Boehringer Mannheim) and 5 µg of expression vector (pECE-HNF6 Electrophoretic Mobility Shift Assays--
COS-7 cell lysates (5 µl) were incubated on ice for 20 min in a final volume of 20 µl
containing 10 mM HEPES (pH 7.6), 1 mM dithiothreitol, 1 mM MgCl2, 0.5 mM
EGTA, 50 mM KCl, 10% (v/v) glycerol, 4 µg of
poly(dI-dC), and the 32P-labeled probe (30,000 cpm). The
samples were loaded on a 7% acrylamide gel (29:1
acrylamide/bisacrylamide ratio) in 0.25× Tris borate/EDTA buffer and
electrophoresed at 200 V.
Identification of OC-2--
During a search for HNF-6-related
proteins in GenBankTM using the Basic Local Alignment
Search Tool (BLAST) (24), we found an expressed sequence tag
(GenBankTM accession number AA121823) from a fetal human
retina cDNA library that contains a partial cDNA showing
significant similarity to the HNF-6 homeobox. Two primers (FM2.1 and
FM2.2; see "Experimental Procedures") whose sequences were based on
the expressed sequence tag were used to screen, by PCR, two cDNA
libraries, one from adult human retina and one from HeLa cells.
Identical 193-bp-long bands were seen with the two libraries, yielding
a probe that was used to screen the HeLa cell cDNA library by phage
plaque hybridization. Six independent clones were obtained. All these clones lacked the 5'-end of the coding sequence, but contained nearly
identical 5'-extremities. This probably reflects the presence of
secondary structures in the mRNA that blocked reverse transcription during the preparation of the library. Rapid amplification of cDNA
ends/PCR failed to amplify the missing portion, which turned out to be
GC-rich (see below and Fig.
1A). To clone the 5'-end, we
therefore screened a human genomic DNA library with a probe encompassing nucleotides 1027-1214 (Fig. 1A). An additional
5'-sequence was obtained (nucleotides 1-585 in Fig. 1A),
and RT-PCR with high processivity polymerase on total RNA from human
liver and skin confirmed that this sequence is expressed as mRNA.
The fully coding cDNA and the derived 485-amino acid-long sequence
are shown in Fig. 1A. The ATG codon at nucleotides 88-90
lies within a perfect Kozak consensus sequence, strongly suggesting
that it codes for the initiator methionine. Sequence comparisons showed
the presence of a single cut domain (residues 314-379) and a
homeodomain (residues 407-466). The homeodomain was typical of the
ONECUT class, with a phenylalanine at position 48 and a methionine at
position 50. We therefore named the novel protein OC-2 (for
ONECUT-2).
Fig. 1B shows an alignment of the amino acid sequences of
OC-2 and HNF-6. This alignment shows that the sequences of the cut domain and homeodomain of OC-2 are 97 and 87% identical to those of
HNF-6, respectively, and that HNF-6 and OC-2 display 92% amino acid
sequence identity over the 157 residues (boxed in Fig.
1B) that encompass the entire DNA-binding region. There are
additional regions of similarity between OC-2 and HNF-6 (Fig.
1B), such as a serine-rich tail of 19 residues in OC-2 and
of 21 residues in HNF-6. The linker between the cut domain and
homeodomain has the same length (27 residues) in OC-2 and HNF-6
Recent data bank searches showed that the human genome contains
sequences that code for additional putative ONECUT proteins very
similar to HNF-6 and OC-2. The amino acid sequences of two ORFs, one
present in fosmid F37502 (GenBankTM accession number
AC004755) and one present in cosmid F21967 (GenBankTM
accession number AC005256), are shown in Fig. 1B. Fosmid
F37502 and cosmid F21967 contain sequences present on the same
chromosome (human chromosome 19) and separated by a 1-kilobase pair
gap. The distance between the two ORFs shown in Fig. 1B is
19 kilobase pairs. The ORF of fosmid F37502 contains a typical ONECUT
homeodomain with a phenylalanine at position 48 and a methionine at
position 50. The ORF of cosmid F21967 contains a cut domain that is
quasi-identical to those of the bona fide ONECUT proteins. It also
contains an STP box and a polyglycine stretch. It could be that cosmid
F21967 and fosmid F37502 each contain one-half of a single
ONECUT gene whose organization resembles that of the
hnf-6 gene, namely with a very long intronic region between
the cut domain and homeodomain (17). In this case, the two
corresponding ORFs shown in Fig. 1B would belong to two
parts of the same OC-3 protein. If these ORFs do not correspond to
pseudogenes and if these ORFs are expressed as peptides, these peptides
very likely correspond to ONECUT protein(s) different from HNF-6 and
OC-2.
Chromosomal Localization of OC-2--
The chromosomal localization
of OC-2 in humans was determined by radiation hybrid
mapping. PCR amplifications were performed, and the data were analyzed
as indicated under "Experimental Procedures." This showed that the
OC-2 gene is located on chromosome 18, 10.31 cR3000 telomeric from marker WI-8740 and 9.2 cR3000 centromeric from marker CHLC.GATA30B03 (lod
score > 3.0). Human HNF-6 is located on chromosome
15q21.1-21.2 (27),5 and, as
said above, the putative human OC-3 gene is located on chromosome 19. These data show that in humans, the genes coding for the
homeodomain proteins of the ONECUT class are not located in a cluster.
Like OC-2, marker D18S69 is located between markers WI-8740
and CHLC.GATA30B03. Since D18S69 is cytogenetically located at 18q21.1-18q21.2 (28), we conclude that OC-2 is most probably also located cytogenetically at 18q21.1-18q21.2. Deletions encompassing 18q21.1-21.3 are associated with retinal cone-rod dystrophy (CORD)-1 (29). Considering that OC-2 is expressed in the retina (see "Cloning of OC-2 cDNA," above) and that the loss of the
homeodomain-containing transcription factor CRX causes the related
syndrome CORD2 (30, 31), further work should identify the retinal cell
types expressing OC-2 and the possible association between
CORD1 and OC-2 dysfunctions.
OC-2 Gene Expression Is Tissue-restricted--
To determine the
tissue distribution of OC-2, we screened, by RT-PCR, 12 human tissues
for expression of its mRNA. The tissue distribution of
HNF-6 mRNA, not yet studied in humans, was analyzed in
parallel. The PCR products were subjected to Southern blot analysis
using overlapping radioactive PCR probes. As a control, OC-2 and HNF-6 Have Distinct but Overlapping DNA Binding and
Transcriptional Activation Properties--
The remarkable amino acid
sequence similarity of the two DNA-binding domains of OC-2 and HNF-6
HNF-6 regulates the transcription of genes involved in glucose
metabolism (9, 32).6 As shown
in Fig. 3 (lanes 14-19), we found that OC-2 and
HNF-6
Experiments on dedifferentiated hepatoma lines and on embryoid bodies
provided evidence for a network of liver-enriched transcription factors
that is required for hepatocyte differentiation and morphogenesis (33-38). HNF-3
To study if OC-2 may be involved in the network of liver transcription
factors by controlling transcription of the hnf-3 Conclusions--
Human OC-2 is a mammalian member of the ONECUT
class of homeodomain transcription factors whose sequence is very
similar to that of HNF-6. On the basis of their respective tissue
distribution, DNA binding specificities, and transcriptional activation
properties, HNF-6 and OC-2 have different but overlapping functions.
The expression of OC-2 in the retina and its chromosomal
localization raise the question of the involvement of OC-2 in CORD1. In
liver, OC-2 is expected to participate in the network of transcription
factors that regulates differentiation and morphogenesis.
gene in transient transfection experiments, suggesting that OC-2
participates in the network of transcription factors required for liver
differentiation and metabolism.
INTRODUCTION
Top
Abstract
Introduction
References
-helices (for a review on homeodomain-DNA interactions, see
Refs. 10-12). The third helix, called the recognition helix, contacts
the DNA and is crucial for sequence-specific binding. Within this
helix, residue 48 is part of a hydrophobic core. Whereas residue 48 is
a tryptophan in all known homeodomains, it is a phenylalanine in the
ONECUT proteins. Residue 50 is also located in the recognition helix.
Mutations at this position often lead to changes in the sequence
specificity of DNA binding (13-15). This is consistent with the
crystallization data that demonstrate that the amino acid at position
50 is in contact with bases (for a review, see Ref. 16). In the ONECUT
proteins, a methionine is found at position 50. This amino acid is
never found at this position in other homeodomains.
and HNF-6
) that originate from the same gene by alternative splicing
(17). HNF-6
(491 residues) is identical to HNF-6
(465 residues),
except that it contains an insert of 26 amino acids in the linker
region. These two isoforms differ in DNA binding specificity and
kinetics (8).
EXPERIMENTAL PROCEDURES
394 to
371 of the mouse
hnf-4 promoter); HNF-3
,
5'-AGCTTAAGGCCCGATATTGATTTTTTTTTCTCC-3' (
150 to
118 of the rat
hnf-3
promoter); TTR, 5'-GTCTGCTAAGTCAATAATCAGAAT-3' (
110 to
87 of the mouse transthyretin promoter); PEPCK,
5'-CAAAGTTTAGTCAATCAAACGTTG-3' (
263 to
240 of the rat
phosphoenolpyruvate carboxykinase (pepck) gene
promoter); and GRU, 5'-AAAAAAATCCATAACTTTCA-3' (sequence in
the GRU of the first intron of rat 6-phosphofructo-2-kinase (pfk-2) gene A).
Zap II (Stratagene) by hybridization at 42 °C in 6×
SSC and 50% formamide. Filters were washed at 55 °C in 3× SSC, and
positive clones were isolated according to the supplier's
instructions. Clones lacking the 5'-end of the cDNA were obtained.
To find this 5'-end, a human genomic
EMBL3 library was screened with
a probe containing the OC-2 cut domain. This probe was
obtained by PCR using primers VLIIA and VLIIB and a HeLa cell
OC-2 cDNA clone as template. A 4.5-kilobase pair-long
EcoRI-EcoRI fragment was obtained from a purified
genomic clone and subcloned in pBluescript KS+
(Stratagene). A cDNA containing a full-length coding sequence was
constructed by fusing 585 bp of the genomic subclone (nucleotides 1-585 in Fig. 1A) to 1069 bp of a HeLa cell OC-2
cDNA clone (nucleotides 586-1654 in Fig. 1A). The
genomic portion used to construct the fully coding cDNA is
expressed as mRNA as confirmed by RT-PCR with liver or skin RNA.
These RT-PCR experiments were performed with the Titan RT-PCR system
(Boehringer Mannheim) in the presence of 2 M betaine using
primers FM2.9 and FM2.10. The sequence of the two DNA strands was
determined by automated DNA sequencing using the dideoxy chain
termination method. DNA and protein sequence analysis was performed
using the GCG software package (Genetics Computer Group, Inc.,
University of Wisconsin, Madison, WI). To identify predicted
structurally conserved regions, multiple sequence alignments were
performed using the Match-Box algorithm with matrix blosum62 (18,
19).2
-actin cDNA fragment with primers bACT5 and bACT3.
OC-2 cDNA (193-bp-long fragment) was amplified with
primers FM2.1 and FM2.2. HNF-6 cDNA (387-bp-long fragment) was amplified with primers HNF6.5 and HNF6.6. Primers FM2.2
and HNF6.6 were used in the reverse transcription step. The specificity
of the amplified products was verified in Southern blotting experiments
as described (20) using, as 32P-labeled probes, PCR
fragments obtained with primers HOM1 and HOM2 for the identification of
OC-2 products and with primers HNF6.3 and HNF6.4 for the
identification of HNF-6 products.
contains an
AccI-EcoRI fragment of the HNF-6
cDNA, derived from pSP-HNF6
(9), cloned downstream of a
cytomegalovirus promoter. pECE-HNF6
has been described (9).
pXJ42-OC2 contains a fully coding human OC-2 cDNA cloned
in the XhoI and EcoRI sites of pXJ42 (21). The
pHNF6/HNF3
(6×)-TATA-luc, pHNF3
-luc, pHNF3
mut-luc, and
pHNF4-0.7-luc reporter vectors have been described (8, 22, 23). The
internal control vector pRL138 contains the pfk-2 promoter
(
138 to +86) cloned in pRLnull (Promega).
(6×)-TATA-luc, pHNF3
-luc, pHNF3
mut-luc, or
pHNF4-0.7-luc), 15 ng of pCMV-HNF6
or 15 ng of pXJ42-OC2; and 15 ng
of pRL138 as internal control. After 6 h, the cells were washed
with phosphate-buffered saline and further incubated for 45 h in
Dulbecco's modified Eagle's medium/Ham's F-12 medium plus 10% fetal
calf serum before measuring luciferase activities with the
Dual-Luciferase kit (Promega). Luciferase activities were measured with
a Lumac luminometer and expressed as the ratio of reporter activity
(firefly luciferase) to internal control activity (Renilla luciferase).
or pXJ42-OC2). Forty-eight h after transfection, the cells were washed with phosphate-buffered saline and harvested in 1 ml
of 40 mM Tris-Cl (pH 7.5), 1 mM EDTA, and 150 mM NaCl. The cells were pelleted and resuspended in 60 µl
of 50 mM Tris-Cl (pH 7.9), 500 mM KCl, 0.5 mM EDTA, 2.5 µg/ml leupeptin, 1 mM
dithiothreitol, 0.1% (v/v) Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, and 20% (v/v) glycerol. After three
freeze-thaw cycles, the lysates were centrifuged, and the supernatants
were collected.
RESULTS AND DISCUSSION
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Fig. 1.
OC-2 is a novel mammalian member of the
ONECUT class of homeodomain proteins. A, nucleotide
sequence of OC-2 cDNA and deduced amino acid sequence.
The cut domain and homeodomain are boxed. B,
alignment of the amino acid sequences of human (h) OC-2,
human HNF-6, C. elegans (ce) ORF R07D10.x, and
human ORFs found in cosmid F21967 and fosmid F37502. Residues identical
to those in OC-2 are on a black background; similar residues
are indicated by gray shading. Boxed sequences
correspond to regions whose structures predicted by the Match-Box
algorithm are similar among all the sequences listed.
Asterisks point to Phe-48 and Met-50 of the
homeodomain.
, and
the amino acid sequence of this linker is quasi-identical (83%) in
both proteins. A search by RT-PCR with RNA from tissues expressing
OC-2 (see below) and primers located in the cut domain and
homeodomain failed to provide evidence for an OC-2 isoform containing
an alternatively spliced insert between these domains, as is the case
for HNF-6
(data not shown). As in HNF-6, a polyhistidine tract
occurs in OC-2 at about the same distance upstream of the cut domain. A sequence of 14 consecutive glycines (residues 22-35) is present in
OC-2. Such stretches of identical amino acids have been found in many
homeodomain proteins (25) and may be functionally important. Indeed,
alterations of a polyalanine stretch in the HOXD13 protein causes
polydactyly (26). Yet another conserved region (92% identity between
OC-2 and HNF-6) is a serine/threonine- and proline-rich sequence (STP
box) of 24 residues located just upstream of the polyhistidine tract.
Our experiments on HNF-6
suggest that this STP box plays a role in
transcriptional activation.4
Fig. 1B also shows an alignment of the amino acid sequences
of OC-2 and HNF-6
with that of the C. elegans
ONECUT gene product closest to these mammalian proteins,
namely R07D10.x (an ORF found on cosmid C17H12). One sees that not only
the homeodomain and the cut domain, but also the STP box, have been
conserved in the ONECUT class since the nematodes.
-actin
mRNA was amplified from the same RNA preparations. The data (Fig.
2) show that OC-2 mRNA is
abundant in the liver and skin. Lower amounts were found in the testis,
brain (occipital cortex), and urinary bladder. The kidney yielded a
very weak signal. The expression of HNF-6 mRNA was
strongest in the liver and clearly detectable in the testis and skin.
We conclude that OC-2 has a tissue-restricted pattern of
expression that differs from, but overlaps with, that of HNF-6.
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Fig. 2.
Tissue distribution of human OC-2 and human
HNF-6. The tissue distribution of OC-2 and HNF-6 was determined by
RT-PCR, and amplified products were identified by Southern blotting. As
a control for RNA integrity, -actin mRNA was amplified by
RT-PCR, and the products were visualized on a nondenaturing acrylamide
gel stained with ethidium bromide. Ki, kidney;
Lu, lung; Mu, skeletal muscle; Br,
brain; Sp, spleen; Te, testis; He,
heart; My, myometrium; Li, Liver; Bl,
urinary bladder; Sk, skin; PBL, peripheral blood
lymphocytes.
and of the linker region between these domains suggested that the two
proteins display similar DNA binding specificities. HNF-6 binds to two
types of sequences. Binding to the first type, exemplified by a probe
derived from the transthyretin gene (TTR probe), requires both the cut
domain and homeodomain of HNF-6. Binding to the second type of
sequence, exemplified by a probe derived from the hnf-3
gene (HNF-3
probe), requires only the cut domain of HNF-6 (8). We
therefore tested, by electrophoretic mobility shift assay, whether OC-2
binds to these two probes. This was the case (Fig.
3, lanes 3 and
8 versus lanes 1 and 6).
Binding of OC-2 was specific as shown in incubations containing an
excess of unlabeled probe (Fig. 3, lanes 4 and
9). Cross-competitions suggested that OC-2 binds with higher
affinity to the HNF-3
probe than to the TTR probe (Fig. 3,
lanes 5 and 10). In contrast, HNF-6
bound equally well to the two probes, as shown in Fig. 3
(lanes 2 and 7) and by our earlier
results (8).
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Fig. 3.
DNA binding specificity of OC-2 and HNF-6 as
determined by electrophoretic mobility shift assay. Extracts from
nontransfected (nt.) COS-7 cells or from COS-7 cells
transfected with HNF-6 or OC-2 expression vectors were used as a source
of proteins, as indicated above the lanes. The radioactive probes used
are indicated below. The unlabeled competitor oligonucleotides used in
the binding reaction are indicated above.
bind to the same cis-acting sequences in the
pepck (PEPCK probe) and pfk-2 (GRU probe) genes.
Like HNF-6, OC-2 is therefore expected to play a role in the regulation
of liver gluconeogenesis and glycolysis.
is an essential component of this network. It
stimulates expression of hnf-4. On the other hand, HNF-4
activates transcription of the hnf-1 gene. Our earlier
experiments indicated that HNF-6 binds to, and controls the expression
of, the hnf-3
and hnf-4 genes (22). We
therefore determined whether OC-2 binds to the same sites as HNF-6 in
these two genes. While OC-2 bound to the same HNF-3
probe as HNF-6
(see above), OC-2 did not bind to the HNF-6-binding site derived from
the hnf-4 gene promoter (HNF-4 probe) (Fig. 3,
lanes 11-13). The discrimination of OC-2 between the HNF-3
and HNF-4 probes is similar to that described earlier for
HNF-6
(8). However, OC-2 binds to a probe (PEPCK) to which HNF-6
binds very poorly. Therefore, OC-2 has a DNA binding specificity that
differs from, but overlaps with, that of HNF-6
and HNF-6
(Table
I).
DNA binding specificity of OC-2, HNF-6, and HNF-6
gene,
rat hepatoma FTO-2B cells were cotransfected with an OC-2 expression
vector and a construct containing the luciferase reporter under the
control of six copies of the HNF-6/OC-2 site found in the
hnf-3
gene. Cells similarly transfected, but with
HNF-6
instead of OC-2, were examined in parallel. As shown in Fig.
4A, OC-2 stimulated the
reporter construct 50-fold and was as effective as HNF-6
. To compare
the activities of HNF-6
and OC-2 on the wild-type
hnf-3
promoter, we cotransfected, in FTO-2B cells, their
expression vectors with a reporter construct containing the
hnf-3
gene promoter (pHNF3
-luc) or the same promoter
in which the HNF-6/OC-2 site has been destroyed by mutation
(pHNF3
mut-luc). Fig. 4B shows that overexpression of OC-2
or HNF-6
stimulated transcription from the wild-type promoter
2-3-fold, but was without effect on the mutated promoter. Consistent
with earlier results (22), the mutated promoter displayed a 50%
reduction in basal activity compared with the wild-type promoter,
probably as a result of the loss of action of endogenous ONECUT
transcription factors. Finally, as expected from the lack of binding of
OC-2 to the probe derived from the hnf-4 gene (Fig. 3),
overexpression of OC-2 did not activate the hnf-4 promoter
(pHNF4-0.7-luc) (Fig. 4B) in transfection experiments,
whereas HNF-6
did. We conclude from these transfection experiments
that OC-2 stimulates transcription of the hnf-3
gene. Both HNF-6 and OC-2 might therefore be key players in the network of
liver transcription factors by controlling a different set of
genes.
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Fig. 4.
OC-2 is a transcription factor that
stimulates transcription of the hnf-3
gene. FTO-2B cells were transiently transfected with
the empty expression vector pXJ42 (black bars) as a control
or with an OC-2 (shaded bars) or HNF-6 (white
bars) expression vector. The cotransfected firefly luciferase
reporter constructs pHNF3
(6×)-luc, pHNF3
-luc,
pHNF3
mut-luc, and pHNF4-0.7-luc were used as indicated.
Firefly luciferase values were normalized for Renilla
luciferase values from the internal control plasmid pRL138. Data are
expressed as -fold stimulation of reporter gene activity by OC-2 or
HNF-6 (means ± S.E., n = 3) and are normalized
for the reporter activity in the presence of empty expression vector.
With the empty expression vector, the absolute activity of
pHNF3
mut-luc was 2-fold lower than that of pHNF3
-luc, as shown by
Landry et al. (22).
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ACKNOWLEDGEMENTS |
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We thank J.-M. Garnier for the HeLa cell
cDNA library, J. Nathans for the retina cDNA library, I. Davidson for pXJ42, R. Costa for pHNF6/HNF3(6×)-TATA-luc, F. Brasseur for human RNA samples, the United Kingdom Human Genome Mapping
Project Resource Center for the IMAGE clone 566080, and S. Durviaux and
S. Neou for expert technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Belgian State Program on Interuniversity Poles of Attraction, Prime Minister's Office, Federal Office for Scientific, Technical, and Cultural Affairs; from the Délégation Générale Higher Education and Scientific Research of the French Community of Belgium; from the Fund for Scientific Medical Research (Belgium); and from the National Fund for Scientific Research (Belgium).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y18198.
These two authors equally contributed to this work.
§ Recipient of a fellowship from the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture (Belgium).
¶ Senior Research Associate from the National Fund for Scientific Research (Belgium). To whom correspondence should be addressed. Tel.: 32-2-764-7583; Fax: 32-2-762-7455; E-mail: lemaigre{at}horm.ucl.ac.be.
The abbreviations used are: HNF, hepatocyte nuclear factor; ORF, open reading frame; RT-PCR, reverse transcription-polymerase chain reaction; TTR, transthyretin; PEPCK, phosphoenolpyruvate carboxykinase; GRU, glucocorticoid-responsive unit; bp, base pair(s); CORD, cone-rod dystrophy.
2 http://www.fundp.ac.be/sciences/biologie/bms/matchbox_submit.html.
3 http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl.
4 V. J. Lannoy, G. G. Rousseau, and F. P. Lemaigre, manuscript in preparation.
5 A. M. Møller, J. Ek, S. M. Durviaux, S. A. Urhammer, J. O. Clausen, H. Eiberg, T. Hansen, G. G. Rousseau, F. P. Lemaigre, and O. Pedersen, submitted for publication.
6 C. Pierreux, J. Stafford, D. Demonte, D. K. Scott, J. Vandenhaute, R .M. O'Brien, D. K. Granner, G. G. Rousseau, and F. P. Lemaigre, submitted for publication.
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