From the Hormone and Metabolic Research Unit, Louvain
University Medical School and Christian de Duve Institute of Cellular
Pathology (ICP), B-1200 Brussels, Belgium, and the ¶ Department of
Cell Biology, Biozentrum, University of Basel, CH-4056 Basel,
Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hepatocyte nuclear factor-6 (HNF-6) contains a
single cut domain and a homeodomain characterized by a phenylalanine at
position 48 and a methionine at position 50. We describe here two
isoforms of HNF-6 which differ by the linker that separates these
domains. Both isoforms stimulated transcription. The affinity of
HNF-6 and HNF-6
for DNA differed, depending on the target
sequence. Binding of HNF-6 to DNA involved the cut domain and the
homeodomain, but the latter was not required for binding to a subset of
sites. Mutations of the F48M50 dyad that did not affect DNA binding
reduced the transcriptional stimulation of constructs that do not
require the homeodomain for DNA binding, but did not affect the
stimulation of constructs that do require the homeodomain. Comparative
trees of mammalian, Drosophila, and Caenorhabditis
elegans proteins showed that HNF-6 defines a new class, which we
call ONECUT, of homeodomain proteins. C. elegans proteins
of this class bound to HNF-6 DNA targets. Thus, depending on their
sequence, these targets determine for HNF-6 at least two modes of DNA
binding, which hinge on the homeodomain and on the linker that
separates it from the cut domain, and two modes of transcriptional
stimulation, which hinge on the homeodomain.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The phenotype of multicellular organisms is determined in part by
the cell type-specific expression of genes. Since the initial observation that expression of liver-specific genes is controlled at
the level of transcription (1), several liver-enriched transcription factors have been identified and extensively studied. These factors contain DNA-binding domains that have been conserved throughout evolution. Based on the structure of their functional domains, the
liver-enriched transcription factors were classified into five families
(2, 3). These include the CCAAT/enhancer binding proteins and the
proline acid-rich factors, which both contain a leucine zipper, the
homeodomain proteins of the hepatocyte nuclear factor
(HNF)1-1 family, the winged
helix proteins HNF-3, -
, and -
, and the zinc finger orphan
receptor HNF-4 family. These factors are expressed not only in liver
but also in a restricted number of other tissues, where they control
gene transcription both in the adult and during development (2).
We have recently cloned (4) a new type of liver-enriched transcription
factor that we called HNF-6. This factor, which was originally
characterized as a transcriptional activator of the liver promoter of
the 6-phosphofructo-2-kinase (pfk-2) gene, is expressed in
liver, brain, spleen, pancreas, and testis. Moreover, the developmental
pattern of expression of HNF-6 in the mouse and the demonstration that
HNF-6 can control transcription of the hnf-3 and
hnf-4 genes (5) suggest a role of HNF-6 in several developmental programs. In adult rat liver, HNF-6 mediates
sex-dependent effects of growth hormone (6). HNF-6 contains
a cut domain and a homeodomain. Cut homeodomain proteins were
originally described as the products of the Drosophila cut
gene (7) and of its mammalian homologs, the mclox genes
(8-12). These proteins contain three cut domains upstream of a
homeodomain. Such cut repeats, which are well conserved sequences of
66-88 amino acids, function as DNA-binding domains, alone or in
combination with the homeodomain (13-17).
In contrast, HNF-6 contains a single cut domain. Moreover, its homeodomain differs by the nature of its residues 48 and 50 from the 400-odd homeodomains described so far (4). Residue 48 of homeodomains, which is part of their hydrophobic core, is a phenylalanine in HNF-6, whereas it is invariably a tryptophan in the other homeodomain proteins, including the cut homeodomain proteins. Residue 50, which is located in the DNA recognition helix and is a key determinant of sequence-specific DNA binding (18, 19; for a review on homeodomain-DNA interactions, see Ref. 20), is a methionine in HNF-6, an amino acid never found at this position in other homeodomain proteins. These characteristics of the cut domain and of the homeodomain of HNF-6 have been evolutionarily conserved since we found them in the ceh-21 and F22D3.1/ceh-38 genes of Caenorhabditis elegans (4).2 In proteins that contain two DNA-binding domains, the distance between these domains may be crucial for their proper function (21). Our preliminary data suggested the existence of an isoform of HNF-6 characterized by an additional sequence of 26 amino acids in the linker region between the cut domain and the homeodomain (4). All these features of HNF-6 raised important questions that we have addressed in the present work, namely (i) does the putative isoform of HNF-6 differ from the bona fide HNF-6 only by the linker region? (ii) how does the linker sequence affect the DNA-binding properties of HNF-6? (iii) are the single cut domain and the peculiar homeodomain of HNF-6 involved in DNA binding and transcriptional stimulation? (iv) could HNF-6 dimerize on DNA? and (v) are the DNA-binding properties conserved among the cut homeodomain proteins related to HNF-6?
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reverse Transcription (RT)-PCR--
To obtain the isoform of
HNF-6 we performed RT-PCR (Gene Amp kit from Perkin-Elmer) on rat liver
RNA (200 ng) with primers whose 5' ends correspond to positions 210 and
1374 of rat HNF-6 cDNA (4), namely the BGL primer
(5'-gaagatctATGTTGCAGCCCGCTCACCC-3') and the RKE primer
(5'-CTTCCCGTGTTCTTGCTCTTTCC-3'), followed by Southern blotting of
the PCR product with a primer (78S) specific for the
isoform
(5'-ATGGGTGGCAGTGTGCCTTC-3').
Plasmid Constructions--
pSPHNF6 and pSPHNF6
contain the rat HNF-6
and -
cDNA cloned in the
BamHI and SacI sites of pSP72 (Promega).
pSPcut-hd and pSPcut-78-hd code for amino acids 292-444 and 292-470
of HNF-6
and -
, respectively. The corresponding cDNA
fragments were obtained by RT-PCR on rat liver RNA with the cut 5'
(5'-CCCAAGCTTACCATGGCAATCAATACCAAAGAGGTGCTC-3') and hd 3'
(5'-CGGGATCCTCAGTCCAGACTCCTCCTTCTCGC-3') primers and ligated in the
HindIII and BamHI sites of pSP72. pSPHNF6
hd
and pSPHNF6
hd code for amino acids 1-384 and 1-410 of HNF-6
and -
, respectively, and were obtained by an
EcoNI-EcoRV deletion. pSPHNF-6
cut codes for
amino acids 1-291 and 371-465 of HNF-6
and was generated by an
XmnI-EcoNI deletion. In pSPHNF6
cut, which
codes for amino acids 1-291 and 366-491 of HNF-6
, the cut domain
was deleted by a BlpI-XmnI digestion.
pSPHNF6
(cut + hd) codes for amino acids 1-291 and was made by
deleting the portion 3' of the XmnI site. The site-directed
mutants pSPHNF6F48W, pSPHNF6M50H, and pSPHNF6F48W,M50H were made using
a PCR-based strategy with the following primers:
5'-CTGTCAGCAACTGGTTCATGAATGCG-3' (F48W), 5'-CAGCAACTTCTTCCACAATGCGAGAAG-3' (M50H), and
5'-CAGCAACTGGTTCCACAATGCGAGAAGG-3' (F48W,M50H);
mutated codons are underlined. pHNF-6/HNF-3
(6×)-TATA-luc and
pHNF-6/TTR (6×)-TATA-luc contain six HNF-6-binding sites from the
hnf-3
promoter (
141 to
127) or from the transthyretin
(TTR) promoter (
11 to
86) upstream of a TATA box and a luciferase gene. pECE-HNF6
, which is identical to pECE-HNF6 described in Lemaigre et al. (4), and pECE-HNF6
contain the cDNA
of HNF-6
and -
downstream of the SV40 early promoter in pECE72.
pECE-HNF6
F48W, pECE-HNF6
M50H, and pECE-HNF6
F48W,M50H were
generated by deleting a BstEII/XbaI fragment from
pECE-HNF6
and replacing it by the corresponding
BstEII/EcoRV fragment derived from
pSPHNF6
F48W, pSPHNF6
M50H, and pSPHNF6
F48W,M50H. pRL-138
contains the rat pfk-2 liver promoter (
138 to +87,
i.e. devoid of HNF-6-binding site) cloned upstream of the
Renilla luciferase coding sequence. pGEM-3 was from Promega. The
cDNA of F22D3.1/ceh-38 (yk34h2) and ceh-21 (yk90c4) was excised from
ZAPII (Stratagene)
libraries by standard procedures. The fragments containing the cut
domain plus the homeodomain of F22D3.1/ceh-38 and of
ceh-21 were isolated by PCR from the corresponding cDNA
with the F22D3.1/ceh-38 cut 5'
(5'-ACCATGGCAATAGACACAAAAGATCTTTGC-3') and
F22D3.1/ceh-38 hd 3' (5'-TCAACCGAGTCGACTTCTACGACG-3')
primers and with the ceh-21 hd cut 5'
(5'-ACCATGGCACTCGACACTGTCGACATTGCTC-3') and ceh-21 hd 3'
(5'-TCAGTCAATGCGAAGACGACGCGC-3') primers, respectively. The F22D3.1/ceh-38 and ceh-21 fragments were
ligated in the HindIII and BamHI and in the
XbaI and XhoI sites of pSP72 to yield
pSPF22D3.1/ceh-38 and pSPceh-21,
respectively. pGEXHNF6
, coding for a GST-HNF6
fusion protein, was
obtained by subcloning a ClaI/HindIII fragment from pSPHNF6
into the EcoRI and HindIII sites
of pGEX-KG.
In Vitro Synthesis of Recombinant Proteins and Electrophoretic
Mobility Shift Assay (EMSA)--
To produce wild-type or mutant
recombinant HNF-6 and -
proteins, 1 µg of the corresponding
HNF-6 cDNA under the control of the SP6 promoter was combined with
25 µl of TNT-coupled wheat germ extract system (Promega) in a 50-µl
incubation mixture according to the supplier's instructions. To
produce the DNA-binding domains of Ceh-38 and of Ceh-21,
pSPF22D3.1/ceh-38 and pSPceh-21 were incubated with the wheat germ extract to yield translated products of
169 (Ceh-38) and of 157 (Ceh-21) amino acids. For EMSAs, 5 µl of the
reaction mixture 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 (20,000 cpm, except
for Scatchard analysis, see below). These solutions were loaded on an
8% acrylamide gel (acrylamide/bisacrylamide ratio was 29:1) in 0.25×
TBE buffer and electrophoresed at 200 V. The sequence of the
double-stranded oligonucleotides used as probes was as follows: oligo
HNF-4, 5'-AGGATAGAAGTCAATGATCTGGGA-3' (
394 to
371 of the mouse
hnf-4 promoter); oligo HNF-3
,
5'-AGCTTAAGGCCCGATATTGATTTTTTTTTCTCC-3' (
150 to
118 of the rat
hnf-3
promoter); oligo phosphoenolpyruvate carboxykinase,
5'-CAAAGTTTAGTCAATCAAACGTTG-3' (
263 to
240 of the rat
phosphoenolpyruvate carboxykinase (pepck) gene promoter); oligo TTR, 5'-GTCTGCTAAGTCAATAATCAGAAT-3' (
110 to
87 of the mouse
transthyretin promoter); oligo GRUc,
5'-AGCTTCAAACAAACAAAAAAAATCCATAACTTTCA-3' (in the glucocorticoid
response unit of the first intron of the rat pfk-2 gene A);
oligo PFK-2, 5'-gatcGCTTTGAAATTGATTTCAAAGC-3' (
195 to
216 of
the pfk-2 L promoter); oligo CYP2C12,
5'-GCAAAATATTGATTTTTATGGTG-3'(
52 to
30 of the rat
cyp2c12 gene promoter. For affinity constant (KD) determination, EMSAs were performed with a
constant amount of recombinant protein and various concentrations of
probe (1.3-20.8 nM) of decreasing specific radioactivity
to avoid overlabeling of the gels. Incubations lasted 1 h on ice
to reach equilibrium. Bound and free probe were quantified with an
Instant Imager (Packard). The bound/free ratio was calculated, and the
values were plotted against the concentration of bound probe
(nM) to obtain Scatchard plots in which the
KD =
1/slope.
Cell Culture and Transfection--
Human hepatoma HepG2 cells
were grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum (FCS). Cells (8 × 105 cells per
60-mm dish) were transfected overnight in medium without FCS using
DOTAP, 3 µg of pHNF-6/HNF3(6×)-TATA-luc or
pHNF-6/TTR(6×)-TATA-luc reporter construct, 400 ng of pECE-HNF6
,
pECE-HNF6
, pECE-HNF6
F48W, pECE-HNF6
M50H, or
pECE-HNF6
F48W,M50H, 500 ng of pRL-138 as internal control, and
pGEM-3 to adjust plasmid amounts to 5 µg. The cells were washed with
phosphate-buffered saline and further incubated for 24 h in
Dulbecco's modified Eagle's medium plus 10% FCS 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).
Sequence Analysis--
Data base searches were performed using
BLAST at the National Center for Biotechnology Information (22).
Sequences were extracted using FETCH from the GCG package (23). Further
packages used from GCG were GAP for pairwise comparisons and PILEUP for multiple sequence comparisons and for classification of sequences. Multiple sequence alignments were performed using MSE (generously provided by W. Gilbert). C. elegans sequence searches were
performed using the blast
server.3 Open reading frames
of unfinished C. elegans cosmid sequences were analyzed
using GENEFINDER within ACeDB (24) on a SUN SPARCStation5. Cosmids were
examined in detail using a special dot matrix
program4 to ascertain that no
additional cut domains were present in the C. elegans ONECUT
genes. Accession numbers are as follows: T26C11 (containing
ceh-21 also known as T26C11.6, ceh-39
also known as T26C11.7, and T26C11.5), U41017;
F22D3 (containing ceh-38 also known as F22D3.1),
U28993; F17A9.6, AF016417; dgclox, X69017; hCDP, M74099, P39880; rcdp-2, U09229;
rhnf-6, X96553; rhnf-6
, Y14933;
mhnf-6, U95945; hHNF-6, U77975; mcux,
X75013; mcux-2 (also known as cutl2), U45665;
msatb1, U05252; hSATB1, Q01826, M97287; cm18e7, AF023470. cDNAs for ceh-21,
cm18e7 and yk90c4; cDNA for F22D3.1/ceh-38, yk34h2. R07D10.x is an open
reading frame found on the overlapping cosmids RO7D10 and C17H12.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There Are Two Isoforms of HNF-6--
Besides the fully coding
cDNA originally described for the 465-residue rat HNF-6 (HNF-6),
we had found a partial cDNA that was identical to the 3'-half of
HNF-6
cDNA, except for a 78-bp insert within the spacer that
separates the cut domain from the homeodomain (4). To extend this
partial sequence, we performed an RT-PCR reaction on rat liver RNA with
a PCR primer (oligo RKE) located 3' of the 78-bp insert and a PCR
primer (oligo BGL) located 5' of the HNF-6
ATG initiator codon.
Southern blotting of the PCR products with an oligonucleotide (oligo
78S) specific for the 78-bp insert showed a 1130-bp band that
corresponded to the missing portion of the cDNA (data not shown).
Data base scanning with the sequence of this fragment revealed that it
was identical to the corresponding HNF-6
cDNA fragment, with the
exception of the 78-bp insert. This defined an isoform of HNF-6
(HNF-6
, 491 residues) that differs from HNF-6
(465 residues) only
by the presence of an additional sequence of 26 amino acids in the linker between the cut domain and the homeodomain. Cloning of the rat
HNF-6 gene indicated that the two isoforms originate from the same gene
by alternative splicing.5
HNF-6 and HNF-6
Differ in DNA-binding
Properties--
HNF-6
differs from HNF-6
by the length of the
linker region between the cut domain and the homeodomain. This
suggested that the two HNF-6 isoforms may have distinct DNA-binding
properties. We therefore compared the affinity of HNF-6
and -
for
the hnf-3
promoter site (HNF3
probe). To do so, the
corresponding HNF-6 cDNAs were transcribed and translated in a
wheat germ extract. The integrity of the recombinant proteins was
controlled by SDS-polyacrylamide gel electrophoresis after
incorporation of [14C]leucine (data not shown). This
showed a major band of the expected molecular weight and a band of
lower molecular weight that was interpreted as a product of degradation
or of initiation from an internal initiation codon. Accordingly, EMSAs
with the synthesized proteins showed an upper major band and a lower
minor band (Fig. 1A). These
bands were competed for by the cold probe and were not seen with
unprogrammed extracts (Fig. 1J). We excluded that the upper
band was the result of dimerization of HNF-6 with the probe (see
below). Scatchard plots were performed to determine the equilibrium
dissociation constants (KD) of HNF-6
and -
. In
these experiments, various amounts of probe were used with a fixed
amount of protein. The KD determined in this manner
is independent of protein concentration and of the proportion of active
protein in the preparation. HNF-6
bound to the HNF-3
probe with a
12-fold higher affinity than HNF-6
(Fig. 1I).
Representative experiments are shown in Fig. 1, C and D. To determine whether this was due to a faster on-rate or
to a slower off-rate of HNF-6
, we measured off-rate constants
(koff) experimentally and deduced the on-rate
constants (kon) from the relationship
kon = koff/KD (Fig. 1I).
As shown in Fig. 1E, the koff of
HNF-6
was actually 3-fold higher than that of HNF-6
, whereas its
kon (values calculated from the
KD in Fig. 1, C and D) was
60-fold higher than that of HNF-6
. Thus, if HNF-6
and HNF-6
were to compete at identical concentrations for the HNF-3
probe,
HNF-6
would leave little chance to HNF-6
for occupying this
binding site.
|
|
The Role of the Cut Domain and of the Homeodomain in DNA Binding
Depends on the cis-Acting Sequence--
To delineate the DNA-binding
region of HNF-6, and to gain insight into the respective roles of the
cut domain and of the homeodomain, we constructed deletion mutants and
synthesized them in vitro (Fig.
3A). The integrity of the
recombinant proteins was controlled by SDS-polyacrylamide gel
electrophoresis after incorporation of [14C]leucine (data
not shown). The ability of the HNF-6 mutants to bind specifically to
DNA was tested by EMSA with the HNF-3 probe (Fig. 3C).
Consistent with the presence of the 26-residue insert, the HNF-6
proteins migrated slower than the HNF-6
proteins. Deletion of the
amino-terminal half (amino acids 1-291) of HNF-6
and -
(HNF-6
cut + hd) did not prevent binding (Fig. 3C, lanes 9 and
10 versus lanes 2 and 3). Deletion of the
homeodomain (HNF-6
hd) was still compatible with binding (Fig.
3C, lanes 4 and 5). Deletion of the cut domain
(HNF-6
cut) led to the loss of DNA binding (Fig. 3C, lanes
6 and 7), indicating that the homeodomain alone was
unable to bind to the HNF-3
probe. Deletion of both the cut domain
and the homeodomain (HNF-6
(cut + hd)) also abolished DNA binding
(Fig. 3C, lane 8). An isolated cut domain or homeodomain could not be tested as they were very unstable in the wheat germ extracts (data not shown).
|
HNF-6 Binds as a Monomer to DNA--
The homeodomain of the Paired
class of homeodomain proteins serves as a dimerization interface (27),
and the Clox cut domain and the Clox homeodomain are known to interact
with each other (13). We therefore investigated the possibility that
HNF-6 and -
bind to DNA as dimers and perhaps form heterodimers.
To address this point, we used the assay described by Hope and Struhl (28), in which a short and a long version of the protein studied are
mixed and tested by EMSA. If dimerization occurs, one observes a
complex corresponding to the long homodimer, a faster migrating complex
corresponding to the short homodimer, and a complex of intermediate
mobility corresponding to the heterodimer. Full-length and deleted
forms of HNF-6 were synthesized in vitro and tested in this
way with the TTR probe. When incubated alone with DNA, the full-length
HNF-6
(Fig. 4A, lane 2) and
the carboxyl-terminal portion of HNF-6 that contains the cut domain and
the homeodomain of the
isoform (HNF-6
cut + hd) (Fig. 4A,
lane 3) or of the
isoform (HNF-6
cut + hd) (Fig. 4A,
lane 7) produced the expected complexes. When full-length HNF-6
was combined with these deletants, no complex indicative of
dimerization was seen (Fig. 4A, lanes 4 and 8),
excluding the occurrence of a dimerization interface in the DNA-binding
region. There was no dimerization domain in the remaining portion of
the protein either, as no complex indicative of dimerization was seen
when combining full-length HNF-6
with the amino-terminal portion of
HNF-6 (HNF-6
(cut + hd), which does not bind alone, see lane
5) (Fig. 4A, lane 6). Identical results were obtained
by using extracts containing cotranslated proteins of different length
rather than by mixing the extracts primed for synthesizing individual
proteins (data not shown). In addition, when a fusion protein
containing full-length HNF-6
fused to glutathione
sulfhydryltransferase (GST) was incubated with full-length HNF-6
, no
intermediate complex was detected (data not shown). Results identical
to those observed with the TTR probe were obtained with the HNF-3
probe (data not shown). These results suggest that the two isoforms of
HNF-6 bind to DNA as monomers and that they do not form
heterodimers.
|
HNF-6 and C. elegans Genes Define a New Class of Cut Homeobox Genes-- The sequence of the DNA-binding domains of HNF-6 is quasi-identical in rat, mouse, and man (Fig. 5). As basic developmental processes of mammals are very similar to those in primitive organisms, it is not surprising that DNA-binding domains of developmentally crucial transcription factors have been conserved between distantly related species. Data base searches with HNF-6 revealed that several C. elegans open reading frames are most similar to HNF-6. Of these, ceh-21 (cm18e7) was first found as an expressed sequence tag by the genome project (29), which upon sequencing was found to have a highly divergent homeodomain.6 Currently, the genome sequencing project (30) has finished more than 75% of the C. elegans genome, and we have undertaken a systematic search of these sequences for cut-like genes. Fig. 5 shows the sequence alignments of the cut domains and homeodomains of all cut-related genes found to date. This list includes six C. elegans genes, as well as a MAR/SAR DNA-binding protein, SATB1, which was initially not recognized as containing cut domains and a homeodomain (31).
|
|
|
Mutations of Phe-48 and Met-50 Affect the Transcriptional
Activation Properties of HNF-6, Depending on the Target
Sequence--
The conserved F48M50 dyad in the homeodomain of the
ONECUT class suggested that these amino acids confer specific
properties to the proteins of this class. We therefore mutated Phe-48
of HNF-6 into a tryptophan (HNF-6
F48W) or Met-50 into a
histidine (HNF-6
M50H) and also constructed the double mutant (HNF-6
F48W,M50H) to obtain the dyad that is typical of the CUX-type
homeodomain (see Fig. 5). These mutants were tested with probes that
require (TTR probe) or not (HNF-3
probe) the presence of the HNF-6
homeodomain for binding. As shown in Fig.
8A, these mutations still
allowed binding of HNF-6 to the two probes. Similar data were obtained with the GRUc probe (Fig. 8A). Scatchard plots were
constructed with the TTR probe. These demonstrated that, compared with
wild-type HNF-6
, the affinity of HNF-6
F48W was unchanged, the
affinity of HNF-6
M50H was 3-fold lower, and the affinity of
HNF-6
F48W,M50H was 2-fold lower (Fig. 8B).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have characterized here an isoform of HNF-6 (HNF-6) which
differs from HNF-6
by a 26-amino acid insert in the linker region
between the cut domain and the homeodomain, and we have shown that this
isoform can activate transcription. Several homeodomain proteins
contain a second type of DNA-binding domain. The role of the linker
between these two domains has been extensively studied in the POU
factors, in which a POU homeodomain is associated with a POU-specific
domain. In this family of proteins, the use of mutant (21) and chimeric
(41) proteins showed that both linker length and linker composition
affect DNA binding affinity and specificity. For OCT-1, the POU
homeodomain and the POU-specific domain bind to DNA cooperatively, even
when expressed as separate peptides not joined by the linker (42).
Therefore, the linker of the POU factors facilitates binding by
maintaining a high local concentration of the two domains. To our
knowledge, HNF-6 provides the first example of naturally occurring
variants of the linker length. In HNF-6, this linker influenced the
affinity in a different way for different targets. Thus, some genes
will bind HNF-6
much better than HNF-6
, whereas others that bind
HNF-6
might not bind HNF-6
. A kinetic analysis of HNF-6 binding
to the HNF-3
site showed that this difference results from a strong
effect of linker length on the docking (kon of
HNF-6
60-fold higher than that of HNF-6
) without much influence
on the stability of the complex (koff of
HNF-6
3-fold higher than that of HNF-6
). Since these two isoforms
of HNF-6 originate from the same gene,5 alternative
splicing leads to an increase in the DNA binding repertoire of HNF-6.
This mechanism has not been described so far for cut homeodomain
proteins.
As HNF-6 and HNF-6
can be expressed in the same tissues (4), we
have explored the possibility that these isoforms influence each
other's activity when binding to the same target. This was ruled out.
First, there was no evidence for formation of heterodimers. Second,
when HNF-6
stimulated the hnf-3
promoter in
transfection experiments, addition of increasing amounts of HNF-6
failed to amplify or inhibit this effect (data not shown).
Another question that we addressed in this work is the role of the cut
domain and of the homeodomain of HNF-6. Others have studied the role of
the three cut domains of human CDP and of its canine homolog Clox (13,
14, 16, 17). They showed that GST fusion proteins containing either one
of these cut repeats can bind DNA, with a broad but overlapping
sequence-specificity characterized by the presence of a 5'-ATCGAT-3'
motif. This motif resembles the 5'-ATCAAT-3' sequence found in the
HNF-6 binding consensus. The CDP/Clox homeodomain alone also binds DNA
when fused to GST, and it does so with a sequence specificity that overlaps that of the cut domains (13). Furthermore, the CDP/Clox cut
repeats and homeodomain synergize in DNA binding (16), probably as a
consequence of interactions between the cut domain and the homeodomain
(13). Interestingly, in target selection experiments by PCR a cut
domain alone and a cut domain associated with the homeodomain yield the
same consensus, which contains the cut core 5-ATCGAT-3' (17). The
DNA-binding properties of HNF-6 bear similarities with those of
CDP/Clox but also display major differences. In HNF-6, the cut domain
was essential for binding. Contrary to what is seen with CDP/Clox, the
HNF-6 homeodomain could not bind DNA on its own. Still, the presence of
a HNF-6 homeodomain was an absolute requirement for binding to a subset
of targets. Thus, there are at least two ways in which HNF-6 binds to
DNA, depending on the target sequence. The requirement of a
homeodomain, which in itself is unable to bind DNA, for high affinity
DNA binding with another type of domain was recently observed with the
SATB-1 protein, a member of the CUT superclass of homeodomain proteins. The atypical homeodomain of SATB-1 promotes DNA recognition by its
matrix attachment region binding domain (43). Coincubation of HNF-6
hd and HNF-6
cut in EMSA and double hybrid experiments failed to
show protein-protein contacts between the cut domain and the
homeodomain of HNF-6.8 In any case, our data suggest that
the linker, the cut domain, and the homeodomain are all used
combinatorially to determine the binding affinity, the binding
kinetics, and the sequence specificity of HNF-6.
Sequence comparisons between the cut domains and the homeodomains of cut homeodomain proteins led us to update the classification of these proteins and to define the ONECUT class and the CUT superclass. The ONECUT class is characterized by the presence of a single cut domain and by a homeodomain that contains the F48M50 dyad. The corresponding amino acids in the CUX and SATB classes are W48H50 and F48Q50, respectively. Amino acid 48 in homeodomains is part of the hydrophobic core. Given the chemical similarity between phenylalanine and tryptophan, it is not surprising that the F48W mutation in HNF-6 was devoid of effect on DNA binding. As to amino acid 50, it plays a crucial role in the DNA recognition properties of homeodomains by interacting with nucleotides 3' of the canonical TAAT consensus (Ref. 44; for a review on homeodomain-DNA interactions, see Ref. 20). Mutation of this amino acid can modify the sequence specificity as in, for example, the case in Bicoid (19) or alter the discrimination among probes, for example, as in POU factors (45) or else leave the DNA-binding properties unaffected as the Q50A mutation in the engrailed homeodomain (18). We found here that the homeodomain of HNF-6 is required for binding to probes such as the TTR probe. As binding of the HNF-6 homeodomain alone was undetectable and crystallographic data are lacking, we cannot conclude whether the homeodomain itself contacts the TTR probe or whether it influences the binding of the cut domain. Our unpublished data8 indicate that the TAAT sequence in the TTR probe is not crucial for HNF-6 binding. Therefore, the HNF-6 homeodomain is unlikely to interact with DNA in a way similar to that of homeoproteins known to bind optimally to TAAT-containing sequences. Wilson et al. (44) have drawn an amino acid consensus for homeodomains that interact with TAAT-containing sequences. Interestingly, our alignment (not shown) of this consensus with the entire homeodomain of ONECUT proteins showed that the latter diverges significantly from the consensus only in the third helix, which is the DNA-recognition helix in homeodomain crystals. Our experiments indicated that mutation of the HNF-6 F48M50 dyad into W48H50, the dyad found in CUX proteins, is conservative in terms of DNA binding. However, the same mutation did decrease transcriptional stimulation of a target to which HNF-6 bound without need for the homeodomain. In contrast, this mutation did not affect activation of a construct containing a target strictly requiring the homeodomain for HNF-6 binding. Thus, when the homeodomain is not involved in DNA binding, it might be available for protein-protein contacts involved in transcriptional activation. Some homeodomains of other homeoproteins are involved in transcriptional repression. For instance, the interaction between the amino-terminal arm of the Msx-1 homeodomain and the general transcription factor TBP leads to transcriptional repression (46). Also, transcriptional repression by the HOXD8 or HOXA7 homeodomain requires its first or third helix, respectively (38, 39). Our study of HNF-6 now provides evidence that the homeodomain can stimulate transcription via amino acids that are specific to a class of homeodomain proteins. Indeed, C. elegans members of the ONECUT class are able to bind to HNF-6 sites and, like HNF-6, they possess an F48M50 dyad. It is therefore tempting to speculate that the C. elegans proteins will regulate transcription in a way similar to HNF-6. Also, it is likely that the evolutionary conservation of the dyad in the ONECUT class relies on similarities in the mode of transcriptional activation and DNA binding.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank L. Hillier, Genome Sequencing
Center, Washington University, St. Louis, for communication of DNA
sequence data prior to publication. We thank G. Ruvkun in whose lab
part of the C. elegans project was initiated; Y. Kohara
(National Institute of Genetics, Japan) and A. Coulson (Sanger Center,
UK) for providing C. elegans clones; R. Costa for the
HNF-6/HNF-3(6×)-TATA and HNF-6/TTR(6×)-TATA reporter constructs;
L. Bertrand and J. P. Herveg for assistance in computer work;
J.-L. Danan and H. Nacer-Cherif for communication of data; and S. Neou
for technical help.
![]() |
FOOTNOTES |
---|
* This work was supported in part 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); from the National Fund for Scientific Research (Belgium); and from the Fonds de Développement Scientifique (Louvain University).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) Y14933.
§ Holds a fellowship from the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture (Belgium).
Supported by Grants NF 3130-038786.93 and NF 3100-040843.94 from the Swiss National Science Foundation.
** Research Associate of the National Fund for Scientific Research (Belgium). To whom correspondence should be addressed: Hormone and Metabolic Research Unit, Louvain University Medical School and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium. Tel.: 32 2 764 7583; Fax: 32 2 762 74 55; E-mail: lemaigre{at}horm.ucl.ac.be.
1 The abbreviations used are: HNF-6, hepatocyte nuclear factor-6; EMSA, electrophoretic mobility shift assay; PFK-2, 6-phosphofructo-2-kinase; RT-PCR, reverse transcription-polymerase chain reaction; FCS, fetal calf serum; bp, base pair; oligo, oligonucleotide(s); TTR, transthyretin
2 T. R. Bürglin and G. Ruvkun, unpublished data.
3 Blast server available at http://www.sanger.ac.uk/DataSearch/.
4 T. R. Bürglin, manuscript in preparation.
5 M. Rastegar, G. G. Rousseau, and F. P. Lemaigre, unpublished observations.
6 T. R. Bürglin and G. Ruvkun, unpublished observations.
7 L. Hillier, personal communication.
8 V. J. Lannoy, G. G. Rousseau, and F. P. Lemaigre, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|