From the Department of Medical Biochemistry and Genetics,
Biochemical Laboratory C, The Panum Institute, University of
Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
The lactase-phlorizin hydrolase (LPH) gene is
expressed specifically in the enterocytes of the small intestine. LPH
levels are high in newborn mammals, but decrease after weaning. We have previously suggested that the promoter element CE-LPH1, located at
40
to
54, plays an important role in this down-regulation, because the
DNA binding activity of a nuclear factor that binds to this site is
present specifically in small intestinal extracts and is down-regulated
after weaning. In an effort to clone CE-LPH1-binding factors, a yeast
one-hybrid genetic selection was used, resulting in the isolation of a
partial cDNA encoding the human homeodomain protein HOXC11. The
full-length HOXC11 sequence was obtained by rapid amplification of
cDNA ends. It was shown in a yeast assay and by electrophoretic
mobility shift assay that HOXC11 binds to the CE-LPH1 element with
similar specificity to the endogenous intestinal factor. Two HOXC11
transcript sizes were identified by Northern blot analysis. The larger
transcript (2.1 kilobase pairs) is likely to contain a translational
start site in good context and is present in HeLa cells. The shorter
1.7-kilobase pair transcript, present in HeLa and Caco-2 cells,
probably encodes a protein lacking 114 amino acids at the N-terminal
end. Both forms of HOXC11 potentiate transcriptional activation of the
LPH promoter by HNF1
. The expression of HOXC11 mRNA in human
fetal intestine suggests a role in early intestinal development.
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INTRODUCTION |
Lactase-phlorizin hydrolase
(LPH)1 is a membrane-bound
small intestinal enzyme that hydrolyzes the lactose present in milk. LPH is highly expressed in the newborn mammal, but is down-regulated after weaning. However, in some humans, mainly of Northern European descent, LPH activity remains high throughout adult life. In other humans, down-regulation of LPH leads to milk intolerance in adolescence and adult life, a clinical condition referred to as adult-type hypolactasia. Several studies have indicated that the down-regulation of LPH activity in mammals is mainly at a transcriptional level, although post-transcriptional mechanisms play a modifying role (1-4).
A 1-kb promoter region of the pig LPH gene is able to drive appropriate
tissue-specific and developmental regulation of a linked reporter gene,
suggesting that the cis elements required for
transcriptional regulation are located within this region (3).
Previous work in our group has sought to identify the cis
elements of the lactase gene promoter, and the corresponding
DNA-binding factors. A factor present in nuclear extracts prepared from
pig small intestine was shown to bind to the CE-LPH1 site at
40 to
54 in the promoter, adjacent to the putative TATA-box. This DNA binding activity, termed NF-LPH1, was found to be intestine-specific and was present at higher levels in newborn pigs than in adult animals
(5). NF-LPH1 is also present in Caco-2 cells (6), a cell line derived
from a human colon carcinoma, which express LPH when allowed to
differentiate (7). By using competition electrophoretic mobility shift
assays (EMSAs) and photoaffinity-labeling, it was suggested that
NF-LPH1 in Caco-2 nuclear extracts is functionally related to a nuclear
factor binding to the SIF1 element of the sucrase-isomaltase gene. Both
factors recognize a TTTA(T/C) core sequence (6).
The SIF1-binding protein was recently cloned from mouse and identified
as a homeodomain protein, Cdx-2 (8). The hamster homologue, termed
Cdx-3 or shCdx-2, was previously isolated as a factor binding to an
insulin gene promoter element (9). We have shown that Cdx-2 also binds
to CE-LPH1 and activates LPH gene expression, dependent on a functional
CE-LPH1 element, in co-transfection studies (10). The ability of Cdx-2
to bind to both the CE-LPH1 and SIF1 elements may be important in
specifying intestinal expression of the corresponding genes. However,
transcription factors in addition to Cdx-2 are probably required for
the final developmental expression pattern of LPH and
sucrase-isomaltase. Whereas LPH activity is high at birth and is
down-regulated in most mammals after weaning, sucrase-isomaltase levels
are low at birth but increase significantly after birth.
Previous EMSA analysis suggests that Cdx-2 is an abundant
CE-LPH1-binding protein in differentiated Caco-2 cells. Formation of
the main CE-LPH1-protein complex with Caco-2 nuclear extract was
weakened by antibodies to Cdx-2, although no supershifted complex was
generated (10, 11). However, this does not preclude that other
regulatory factors bind to CE-LPH1 under alternative binding
conditions, or if a fractionated nuclear extract were used. The CE-LPH1
element contains two subregions which are both protected in DNase
I-footprinting (5). Cdx-2 appears to bind to only the upstream TTTAC
subregion. We have suggested that a repressor binds to the other
half-site because a mutation here, which does not affect Cdx-2 binding,
leads to higher transcriptional activity of a linked reporter gene
(10).
A one-hybrid genetic screen in yeast (12) was carried out to identify
cDNAs from Caco-2 cells, which encode factors able to bind
specifically to the CE-LPH1 element. This approach allows for the
detection of DNA binding activity in vivo under
physiological conditions, in contrast to EMSA analysis. DNA-binding
factors that have previously been isolated using a one-hybrid screen
include Olf1, an olfactory-specific transcription factor (12), ORC6, a
protein that recognizes the yeast origin of DNA replication (13), and a
metal response element-binding protein (14). Using this screen, a
1.65-kb cDNA was isolated, which was shown to contain the homeobox
of the human HOXC11 protein, previously called HOX3H (15). We describe
here the isolation of the full-length human HOXC11 sequence from HeLa
cDNA using RACE (rapid amplification of cDNA ends). The
relative affinity of HOXC11 protein for various DNA elements, the
expression pattern of HOXC11 mRNA, and the ability of HOXC11 to
activate transcription were analyzed.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
Standard molecular biology techniques
were used (16), and sequencing of double-stranded DNA was carried out
with ThermoSequenase (Amersham Pharmacia Biotech). All inserts
generated by PCR (Advantage KlenTaq polymerase,
CLONTECH) were sequenced completely to check polymerase fidelity. Oligonucleotides were obtained from Amersham Pharmacia Biotech.
In order to construct a reporter plasmid driven by four CE-LPH1
elements, two copies of the annealed oligonucleotides
5'-GATCTTTTACAACCTCAGTTGTTTTACAACCTCAGTTG-3' and
5'-GATCCAACTGAGTTGTAAACAACTGAGGTTGTAAAA-3' were cloned head-to-tail into pRS315HIS (kindly provided by R. Reed; Ref. 12) to prepare the
reporter plasmid pRS-LPH. To construct the other reporter plasmids,
annealed oligonucleotides containing BamHI and
BglII overhangs were multimerized and cloned into
BamHI/BglII-digested pBluescript, in which the
SpeI site had first been replaced by a BglII
site. The appropriate DNA fragments cut out with BamHI and
SacI were cloned into pRS315HIS. The plasmids pRS-LPHmut1, pRS-LPHmut2, pRS-LPHmut3, and pRS-LPHmut4 contained four head-to-tail copies of the mut1 LPH1, mut2 LPH1, mut3 LPH1, or mut4 LPH1
oligonucleotides, respectively (Fig. 1).
pRS-SIF contained two copies of the annealed oligonucleotides
5'-GATCTGTGCAATAAAACTTTATGAGTAGTGCAATAAAACTTTATGAGTAG-3' and
5'-GATCCTACTCATAAAGTTTTATTGCACTACTCATAAAGTTTTATTGCACA-3', resulting in
four SIF1 binding sites from the sucrase-isomaltase gene promoter
(17).

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Fig. 1.
The double-stranded oligonucleotides used in
this study are shown. The TTTA(T/C) core sequence is
underlined. The bases that differ from the CE-LPH1-17
element are written in lowercase. BSK-17 represents a
sequence present in the pRS315HIS vector upstream of the polylinker
site that has extended homology to CE-LPH1; ABD-B-17 contains the
optimal binding site for Abdominal-B, which was obtained by binding
site selection (34).
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The shCdx-2 insert of pBAT7Cdx-3 (kindly provided by M. German; Ref. 9)
was subcloned into the SacI/EcoRI sites of
pBluescript and used as template for PCR with the universal M13
primer and a primer annealing to the translational start site
(5'-GGAAGCTTGTCGACCATGTACGTGAGCTACC-3'). The 1-kb PCR product obtained
was cloned into the SalI/EcoRI site of pPC86
(12), resulting in p86Cdx. The SalI/ApaI insert
of pF4 was subcloned into pBluescript and used as template for PCR with
the universal M13 primer and a primer annealing to the first in-frame
methionine codon 5'-TAGCGGCCGCCACCATGAAAAACG-3'. The 600-bp PCR product
was cloned into the NotI/ApaI site of pRC-CMV (Invitrogen), resulting in pRC-HOX
N. In order to construct pRC-HOX, the 250-bp BglII/HincII fragment of the 92B1
plasmid (I.M.A.G.E. Consortium; Ref. 18) was first ligated in front of
the HincII/ApaI insert of pF4. The resulting
750-bp BglII/ApaI fragment was ligated into
pRC-CMV, together with a 200-bp
NotI/BglII-digested PCR fragment amplified from a
RACE-generated plasmid with the primers
5'-TTAGCGGCCGCAACGATGTTTAACTCGGTC-3' and
5'-GGGTCGACGCTGTTCTTGTTGACTG-3'.
In order to construct the HOXC11 template for riboprobe synthesis, the
410-bp HincII fragment of the plasmid 92B1 was cloned into
pBluescript. The resulting plasmid p92B410 was linearized with
XhoI. For the LPH riboprobe template, the 380-bp
PstI fragment of the plasmid HULAC (19) was cloned into
pBluescript. The resulting plasmid pLPH380 was linearized with
BamHI which removed a 100-bp fragment from the LPH
insert.
The pGL3-LPH227WT plasmid was constructed by cloning the
SstI/XhoI fragment of
pGL2-LPH227,2 which contains
the pig LPH promoter region from
227 to
17, upstream of the
luciferase reporter gene in pGL3-basic (Promega). The mutations that
were generated by PCR overlap extension changed the TTTA(T/C) core
sequence of CE-LPH1a and -1c to TTGC(T/C) in the plasmid
pGL3-LPH227mut1ac, and the additional mutation of CE-LPH2c from GTTAC
to GTCCC in the plasmid pGL3-LPH227mut1ac,2c.
Preparation of the cDNA Library--
Total RNA was prepared
from differentiated Caco-2 cells (1 week after confluence) using the
guanidinium thiocyanate/acid phenol method (20). Poly(A)+
mRNA was isolated from total RNA using oligo(dT)25
Dynabeads (Dynal). The Superscript cDNA synthesis system (Life
Technologies, Inc.) was used for the directional synthesis of cDNA
containing SalI and NotI overhangs. The
size-fractionated cDNA was ligated into pPC86 (12) and
electroporated into the Escherichia coli strain DH10B (Life
Technologies, Inc.). The bacterial transformants (6 × 105 with an average insert size of 1 kb) were scraped from
the plates into LB medium, and plasmid DNA was prepared (Qiagen).
Yeast Methods--
The yeast strain used throughout was yWAM2
(MAT
gal4 URA3::GAL1-lacZ lys2801amber
his3-
200 trp1-
63 leu2ade2-101ochreCYH2; kindly provided by
R. Reed; Ref. 12). Yeast YPD and synthetic minimal media (SD
His) containing adenine sulfate, uracil, and lysine were prepared as
described (21). Where appropriate, the minimal SD medium was
supplemented with histidine, leucine, and/or tryptophan (Sigma) (21).
Yeast transformed with the reporter plasmids were stably maintained in
SD medium supplemented with tryptophan and histidine. Yeast were
transformed by the polyethylene glycol/LiAc method (22), and plasmid
DNA was recovered from yeast (23) by electroporation of E. coli XL1-B cells (Stratagene).
Screening of the cDNA Library--
Yeast containing the
pRS-LPH plasmid were transformed with 6 µg of library cDNA and
plated onto 6 SD
His 14-cm plates at an average density of
1.7 × 105 transformants/plate. After 4 days of
incubation at 30 °C, 24 colonies were able to grow. These were
colony-purified, and plasmid DNA was recovered and individually
re-transformed into reporter plasmid-containing yeast. Transformant
colonies (at least three independent ones for each) were resuspended in
TE buffer at approximately 1000 viable cells/µl. 1 µl of each
was plated onto selective media plates (SD + His or SD
His) and
grown 2-4 days at 30 °C.
Cloning the 5' End of HOXC11--
In order to clone the 5' end
of HOXC11 cDNA, a modified RACE protocol was carried out using
Marathon-Ready HeLa cDNA (CLONTECH) according
to the manufacturer's protocol except that Advantage Tth
DNA polymerase (CLONTECH) was used. The
gene-specific RACE primer used was F4PR1L (sequence
5'-GAAGAAACGGTCGAAGGCTTGAGGCAG-3', see Fig. 3). The reaction mix was
overlaid with oil and cycled in a Hybaid thermal cycler where the tube
temperature was programmed for 1 cycle of 94 °C for 1 min; 5 cycles
of 94 °C for 30 s and 72 °C for 4 min; 35 cycles of 94 °C
for 30 s and 70 °C for 4 min; and 1 cycle of 70 °C for 5 min. A portion of the reaction was loaded onto an agarose gel and
analyzed by Southern blotting with a 410-bp HincII-digested
fragment of 92B1, which was radioactively labeled using the Rediprime
kit (Amersham Pharmacia Biotech). The 550-bp RACE product thus
identified was cut out and cloned (TA cloning kit, Invitrogen).
Cell Culture, Transfections, Preparation of Whole Cell Extracts,
and EMSAs--
Caco-2 and HeLa cells were grown in minimum essential
medium (Life Technologies, Inc.) containing 10% calf serum. Caco-2
cells were seeded at 2 × 106 cells/75-cm2
flask, and were harvested after 4 days (80% confluent) and 14 days (1 week post-confluence). HeLa cells were transfected by calcium phosphate
precipitation with either pRC-HOX
N or water (untransfected control)
as described previously (10). Whole cell extracts were prepared 48 h later using the protocol of Schöler et al. (24).
Protein concentration was determined using Bradford reagents (Sigma
Diagnostics). EMSAs were carried out as described previously (10).
Transient transfections of Caco-2 cells by the calcium phosphate method
were carried out as described previously (10). All transfections
contained 1 µg of a
-galactosidase expression plasmid as an
internal standard for transfection efficiency (PCH110; Amersham Pharmacia Biotech) and 2.5 µg of pGL3-LPH227 wild-type or mutated constructs. In addition, 1.25 µg of pRC-CMV, pRC-HOX
N, pRC-HOX, and/or 0.63 µg of RSV-HNF, made up to 2.5 µg with pRC-CMV, were co-transfected.
Analysis of RNA Expression--
Northern analysis was carried
out according to standard techniques (25) using 2 µg each of
poly(A)+ RNA from HeLa cells and small intestine
(CLONTECH) and undifferentiated (80% confluent)
and differentiated (1 week post-confluent) Caco-2 cells.
Poly(A)+ RNA was prepared from Caco-2 cells using the
Fast-Track kit from Invitrogen. A 400-bp RsaI fragment from
the untranslated 3' region of the HOXC11 cDNA was purified (Qiaex,
Qiagen) and labeled to high specific activity by random priming with
the Rediprime labeling kit (Amersham Pharmacia Biotech). Hybridization
with 0.5 × 106 cpm/ml Express Hyb solution
(CLONTECH) was carried out according to the
manufacturer's protocol.
For ribonuclease protection assay, the Promega T3/T7 polymerase kit was
used to synthesize antisense riboprobes from linearized p92B410 and
pLPH380 plasmid templates to high specific activity (1 × 109 cpm/µg). Both probes were gel-purified according to
the protocol provided with the Ambion RNAPII kit. The cyclophilin
control antisense riboprobe (Ambion) was labeled to low specific
activity (7 × 105 cpm/µg) and purified by RNAID
(Bio101). 1 µg of yeast tRNA or poly(A)+ RNA, each
supplemented with 10 µg of yeast tRNA, was hybridized overnight at
50 °C with 105 cpm HOXC11 riboprobe or with a mixture of
3 × 105 cpm LPH riboprobe and 3000 cpm cyclophilin
control riboprobe and analyzed after RNase digestion on a
non-denaturing polyacrylamide gel, according to the Ambion protocol,
together with radioactively labeled double-stranded DNA markers.
PCR with double-stranded cDNA templates was carried out with 10 pmol each of the following primer pairs: glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (5'-GACCACAGTCCATGCCATCACT-3' and
5'-TCCACCACCCTGTTGCTGTAG-3'), LPH (5'-AGGATACACAGTTTGGAGTGCGATG-3' and
5'-GTTGGCTTCGTTGTGTTTTCCCTTG-3'), or HOXC11
(5'-ACAAATCCCAGCTCGTCCGGTTCAG-3' and 5'-GCAGCAAGACATTGTCGCCGAGGTG-3') and 0.5 ng of cDNA in each 50 µl of reaction mix. The HeLa, fetal intestine (from 18-28-week Caucasian fetuses), and small intestine (from a 15-year-old Caucasian) cDNAs were Marathon-Ready cDNAs from CLONTECH, whereas the differentiated Caco-2
cDNA was prepared from mRNA from Caco-2 cells 1 week after
confluence using the two-hybrid cDNA synthesis kit from
CLONTECH. A portion of each PCR reaction was
analyzed on an agarose gel. PCR was also carried out under the above
conditions on CLONTECH's human fetal multiple tissue cDNA panel. The HOXC11 PCR reactions were analyzed by
Southern blotting with the end-labeled oligonucleotide
5'-GCGAGAGTCAGATAGAGGGTCCAG-3'.
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RESULTS |
Cloning of a CE-LPH1-binding Protein--
In order to clone a
factor capable of binding to the CE-LPH1 element of the LPH gene
promoter, the CE-LPH1 element was multimerized and placed in front of a
HIS3 reporter gene in the construct pRS-LPH. Yeast
containing the pRS-LPH reporter plasmid were transformed with a
cDNA library prepared from differentiated Caco-2 cells, in which
cDNA-encoded proteins are expressed as a fusion (hybrid) with the
activation domain of the yeast transcriptional activator GAL4 (Fig.
2A). All yeast
co-transformants grow on minimal medium supplemented with histidine (SD + His); the ability to grow on medium lacking histidine (SD
His) correlates with the extent of HIS3 reporter gene
activation by the GAL4 fusion protein. The plasmid pPC86, which lacks a
cDNA insert, is unable to cause activation of the HIS3
gene, so there is no growth on SD
His (Fig. 2B).

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Fig. 2.
A, outline of the one-hybrid genetic
screen in yeast. The Caco-2 cDNA library is expressed as a fusion
protein with the yeast GAL4 activation domain. If the cDNA-encoded
peptide binds to the CE-LPH1 elements in the pRS-LPH reporter plasmid,
transcription of the HIS3 reporter gene is activated and the
yeast can grow on minimal medium, which lacks histidine. B,
yWAM2 yeast strains containing the indicated reporter plasmid were
transformed with either pPC86, p86Cdx, or pF4 plasmid DNA.
Approximately 1000 cells from each co-transformant were plated onto SD + His or SD His medium and grown for 2-4 days at
30 °C.
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Out of 1 × 106 library transformants, 24 were able to
grow in the absence of histidine. Two of these grew slowly on selective media, contained insert-less plasmid DNA, and were not studied further.
An additional 20 clones encoded proteins that activated the reporter
gene equally well in the presence (pRS-LPH) or absence of CE-LPH1 sites
(pRS315HIS and p59.7). The p59.7 plasmid contains three copies of an
unrelated sequence element, which binds an olfactory transcription
factor, Olf-1 (12). The significant number of false positives are
presumed to encode proteins that bind DNA nonspecifically.
The remaining two yeast positives, pF4 and pF6, did not activate from a
reporter gene driven by heterologous Olf-1 binding sites (p59.7). In
addition, only the two true positives (pF4 and pF6) were able to grow
under increased stringency conditions, i.e. in the presence
of 5 mM 3-aminotriazole (Sigma; data not shown). Both
plasmids contained identical 1.65-kb cDNA inserts, as judged by
restriction digest. The insert of pF4 was sequenced completely on both
strands, and shown to contain an open reading frame of 194 amino acids
including the homeodomain of human HOXC11, in-frame with the
vector-encoded GAL4 activation domain (Fig. 3). The isolated pF4 plasmid DNA was
re-transformed into yeast containing either the pRS-LPH reporter
plasmid used in the library screen or other constructs designed to test
the specificity of binding (Fig. 2B). For comparison, yeast
were also transformed with the plasmid p86Cdx, which encodes a fusion
protein of the activation domain of GAL4 with full-length shCdx-2 (9).
Cdx-2 has previously been shown to bind to the CE-LPH1 element
(10).

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Fig. 3.
The nucleotide sequence of the full-length
human HOXC11 cDNA, and its conceptual translation using the
one-letter amino acid code. The 60-amino acid
homeodomain is underlined. The start of the pF4 cDNA
insert and the 92B1 genomic DNA insert are indicated below the
sequence. The position at which the two sequences start to diverge,
presumably due to the location of an intron in the genomic 92B1 insert,
is shown with a filled triangle. The annealing site for the
5' RACE primer F4PR1L is indicated. The 5' ends of five different
cloned RACE products are shown by filled circles. A signal
for poly(A) tail addition (double underlined) precedes the
poly(A) tail.
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As shown in Fig. 2B, both the p86Cdx and pF4-encoded fusion
proteins activate HIS3 reporter gene expression when driven
by SIF1, CE-LPH1, or mut2 LPH1 elements, resulting in growth on SD
His plates. These elements also bind NF-LPH1 from Caco-2 cells (6).
On the other hand, there is no activation, or reduced activation, from
mut1 LPH1, mut3 LPH1, mut4 LPH1, and p59.7-driven reporter genes. The
mut1 and mut4 LPH1 oligonucleotides do not bind NF-LPH1 from Caco-2
cells, whereas mut3 LPH1 binds weakly, as shown by competition EMSA
analysis (6). Although the parental pRS315HIS reporter plasmid lacks
CE-LPH1 elements, there is some activation of the HIS3 gene
by p86Cdx and pF4, resulting in weak growth on minimal medium lacking
histidine (Fig. 2B). This is probably due to the presence of
a CE-LPH1-like sequence just upstream of the polylinker. A
double-stranded oligonucleotide covering this vector sequence, referred
to as BSK-17 in Fig. 1, was able to compete for HOXC11 binding to the
CE-LPH1-24 probe in EMSA analysis (Fig. 5, lane 9).
Isolation of Full-length HOXC11 DNA--
Hox genes in
man and mouse are organized in four chromosomal clusters, which have
probably arisen during evolution by gene duplications (reviewed in Ref.
26). It was expected that HOXC11 would show greatest sequence homology
with the human HOXA11 and HOXD11 paralogues, which are located at a
similar position on the A and D chromosomal clusters; there is no
corresponding transcript on the B cluster (27). A putative sequence
entry for the N terminus of human HOXC11 was identified by a data base
search with the N-terminal sequence of mouse Hoxa11. The corresponding
plasmid 92B1 (accession number Z63886) was acquired from the I.M.A.G.E. Consortium (18) and sequenced. The 92B1 genomic insert contains the
first 351 nucleotides of the pF4 cDNA insert flanked on the 5' side
by 325 additional nucleotides and on the 3' side by divergent, presumably intronic, sequence (Fig. 3). The presumed intron precedes the homeobox, and corresponds in its location to the intron present in
the mouse Hoxa11 and Hoxd11 genes (Fig.
4).

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Fig. 4.
A, comparison of the amino acid sequence
of human HOXC11 with mouse Hoxa11 and Hoxd11 (GenBank accession numbers
AJOO0041, U20370, and X60395, respectively). The Macaw program was used
to prepare the alignment. Regions of extended homology are shown with
uppercase letters, and include the N terminus, the
homeodomain (underlined), and the C-terminal extension.
Residues identical in all three proteins are indicated by an
asterisk (*), and in two of the proteins by a + above the
particular residue. The doubly underlined amino acid
residues are interrupted in the genomic sequence by an intron.
B, the homeodomains and C-terminal residues of
Drosophila Abdominal-B (ABD-B); human HOXC11;
mouse Hoxc11, Hoxa11, and Hoxd11; and Drosophila
Antennapedia (ANTP) are compared at the amino acid level
(31-34). Residues that are identical to ABD-B are indicated by
dashes. The positions of the N-terminal arm, helices
1-3, and C-terminal extension are indicated.
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In order to obtain the complete 5' nucleotide sequence of HOXC11, RACE
was carried out using HeLa cDNA. HeLa cDNA was chosen because
it gave a strong PCR product in PCR analysis using HOXC11-specific primers (Fig. 9). The RACE fragment was cloned, and five independent clones were sequenced. The sequence of the longest RACE product is
shown in Fig. 3 and is identical to the genomic sequence obtained with
the Genome Walker kit from CLONTECH (data not
shown). The first methionine of the longest cDNA is in a good
translational context (28) and is preceded in the genomic sequence by
an in-frame stop codon (data not shown). The deduced full-length
cDNA is 2.0 kb and includes an open reading frame, which encodes a
protein with 304 amino acids.
As shown in Fig. 4A, there are scattered regions of homology
between human HOXC11 and mouse Hoxa11 and Hoxd11 outside the homeodomain. A glycine/alanine-rich sequence (38-45%) is also present
in the three proteins just before the homeodomain. Similar regions
in transcription factors have been implicated in both transcriptional
repression (29) and activation (30). The homeodomain and C-terminal
extension of human HOXC11 are identical to that of mouse Hoxc11 (31)
and are also highly conserved with the mouse paralogous group proteins,
Hoxa11 and Hoxd11 (32, 33) (Fig. 4B). Conservation between
the homeodomains of Drosophila Abdominal-B and mammalian
HOXC11, Hoxa11, and Hoxd11 is especially evident in the recognition
helix 3 and N-terminal arm, both of which contact DNA.
Characterization of HOXC11 DNA Binding Activity--
In order to
analyze HOXC11 DNA binding activity, expression of the pF4 cDNA
insert was put under the control of a cytomegalovirus promoter.
The resulting plasmid pRC-HOX
N, encoding the C-terminal 190 amino
acids of HOXC11, was transfected into HeLa cells, which do not contain
an endogenous CE-LPH1 binding activity (10). No complex is formed using
whole-cell extract from untransfected HeLa cells in EMSA analysis (Fig.
5, lane 1), whereas a specific complex is formed with extract from transfected HeLa cells (Fig. 5,
lane 2). HOXC11 is expected to bind to similar DNA sequences as the related Drosophila Abdominal-B protein. Therefore,
the binding affinity of HOXC11 to CE-LPH1 and to an optimized
Abdominal-B binding site was compared. The CE-LPH1-protein complex
formed with extract from pRC-HOX
N transfected HeLa cells is competed to a similar extent by cold competitor CE-LPH1-17 (Fig. 5, lanes 3-5) and ABD-B-17 (Fig. 5, lanes 6-8), a related probe which has three base changes to include the optimal binding site for Abdominal-B (34). An unspecific oligonucleotide did not compete (Fig. 5, lane
10).

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Fig. 5.
EMSA analysis with end-labeled
double-stranded CE-LPH1-24 probe (25 fmol) was carried out with 2 µg
of untransfected HeLa whole cell extract (lane 1) or 2 µg
of whole cell extract prepared from HeLa cells transfected with
pRC-HOX N (lanes 2-10). Unlabeled competitor
double-stranded DNA (Fig. 1) was included in a 10-min pre-binding
reaction at 4 °C as follows: lanes 1 and 2, no
competitor; lane 3, CE-LPH1-17 (25 fmol); lane
4, CE-LPH1-17 (250 fmol); lane 5, CE-LPH1-17 (2.5 pmol); lane 6, ABD-B-17 (25 fmol); lane 7,
ABD-B-17 (250 fmol); lane 8, ABD-B-17 (2.5 pmol); lane
9, BSK-17 (2.5 pmol); lane 10, unspecific (2.5 pmol).
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The ability of elements present in the yeast reporter plasmids shown in
Fig. 2B to bind HOXC11 was analyzed by competition using
either CE-LPH1-24 (Fig. 6A)
or SIF1-24 (Fig. 6B) as probe in EMSA analysis. The binding
affinity of HOXC11 appears to be highest for SIF1-24 and CE-LPH1-24
elements, intermediate for CE-LPH1-17 and mut2 LPH elements, and low
for mut1 LPH, mut3 LPH, mut4 LPH, and unspecific elements. This result
is consistent with EMSAs carried out with the endogenous NF-LPH1
present in Caco-2 nuclear extracts (6).

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Fig. 6.
EMSA analyses with end-labeled
double-stranded CE-LPH1-24 (A) or SIF1-24 (B)
probe (25 fmol) was carried out with 2 µg of untransfected HeLa whole
cell extract (lane 1) or 2 µg of whole cell extract
prepared from HeLa cells transfected with pRC-HOX N (lanes
2-10). 2.5 pmol unlabeled competitor double-stranded DNA
(Fig. 1) was included in a 10-min pre-binding reaction at 4 °C as
follows: lanes 1 and 2, no competitor; lane
3, SIF1-24; lane 4, CE-LPH1-24; lane 5,
CE-LPH1-17; lane 6, mut1 LPH1; lane 7, mut2
LPH1; lane 8, mut3 LPH1; lane 9, mut4 LPH1;
lane 10, unspecific.
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Pattern of HOXC11 Expression--
We used Northern blot analysis
to examine the expression and size of the HOXC11 mRNA transcript
(Fig. 7). A probe from the 3'-untranslated region of HOXC11 was used to compare levels of HOXC11
expression in HeLa cells, Caco-2 cells in their undifferentiated (80%
confluent) and differentiated (1 week post-confluent) state, and small
intestine. One-week post-confluent Caco-2 cells have started to express
markers of intestinal differentiation, including LPH (7) (Figs.
8 and
9).

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Fig. 7.
Northern blot analysis of HOXC11
expression. The indicated poly(A)+ RNA (2 µg each)
were separated on an agarose/formaldehyde gel. The positions of the 18 S and 28 S ribosomal particles were determined by loading 2 µg of
total RNA in separate lanes on the same gel. The gel was blotted onto a
Hybond N+ membrane. In the top panel, the
membrane was hybridized with a DNA fragment from the 3'-untranslated
region of HOXC11; the positions of the strongest hybridizing bands are
indicated. Caco-2 UD refers to undifferentiated
(80%confluent) Caco-2 cells, Caco-2 Diff. refers to
differentiated (1 week post-confluent) Caco-2 cells. In the lower
panel, the membrane was hybridized with an actin probe. The
membrane was exposed to Biomax Kodak MS film at 70 °C for 3 days
(top panel) or 2 h (lower panel).
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Fig. 8.
Analysis of HOXC11 and LPH expression by
ribonuclease protection. In lanes 1-6, 1 µg of yeast
transfer RNA (tRNA) or the indicated poly(A)+ RNA was
hybridized overnight with a riboprobe corresponding to the 5' region of
HOXC11, subjected to RNase digestion, and loaded onto a non-denaturing
acrylamide gel. In lanes 7-12, 1 µg each of the indicated
RNA was hybridized overnight with a mixture of two riboprobes
corresponding to the 280-bp BamHI/PstI fragment
from the LPH gene and the 103-bp fragment of the cyclophilin gene. The
positions of the main protected double-stranded RNAs are indicated by
arrows. The bands marked with asterisks (*) are present in
the tRNA control lanes and probably correspond to undigested probe. The
position of dsDNA marker bands are indicated. The dried gel was exposed
to Biomax Kodak MS film at 70 °C for 2 days.
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Fig. 9.
A, PCR analysis of HOXC11 and LPH
expression. Each of the indicated cDNAs was subjected to PCR using
the indicated primer set. The PCR was carried out in a Robocycler
(Stratagene) for 1 min at 94 °C, followed by 5 cycles of 30 s
at 94 °C and 3 min at 72 °C, followed by the indicated number of
cycles of 30 s at 94 °C and 3 min at 70 °C. The G3PDH
primers amplify a 450-bp fragment of the human G3PDH housekeeping gene.
The LPH and HOXC11 primers amplify a 400-bp and a 600-bp fragment,
respectively, from regions of the LPH and HOXC11 cDNAs which
contain an intron in the genomic sequence. The PCR primers were
designed to bind to regions of HOXC11, which are poorly conserved in
other HOX gene products. M, 100-bp marker. B, PCR
analysis of HOXC11 in human fetal tissues. Each of the indicated
cDNAs was subjected to PCR as above, using G3PDH or HOXC11 primer
sets. In the bottom panel, a Southern blot of the PCR
reactions generated with the HOXC11 primer set was probed with a
radioactively labeled HOXC11-specific oligonucleotide. F.,
fetal.
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HeLa cells contain a substantial amount of a 2.1-kb transcript that
hybridizes to the HOXC11 probe. This correlates well in size with the
full-length HOXC11 cDNA determined by RACE on HeLa cDNA (2.0 kb, Fig. 3). An additional transcript of 1.7 kb, corresponding to that
reported for a hematopoietic cell line (35), is found in HeLa and also
in Caco-2 cells but not in the small intestine from a 15-year old (Fig.
7, top panel). The amount of this transcript increases upon
differentiation of Caco-2 cells, as judged by hybridization of the same
filter with an actin probe (Fig. 7, lower panel).
The same mRNAs used in Northern blot analysis were analyzed by
ribonuclease protection assay to determine the 5' end of HOXC11 mRNA (Fig. 8). An antisense riboprobe corresponding to the 5' end
of the 92B1 genomic insert gave a protected RNA band in HeLa cells
only. For unclear reasons, this protected band appears shorter than the
410 bp expected if the mRNA in HeLa cells corresponded exactly to
the riboprobe sequence. There was no protected band corresponding to
the 1.7-kb transcript observed in Northern blot analysis, probably
because of the small expected size (90 bp), which may not hybridize
under the conditions used here. The ribonuclease protection analysis
also showed that differentiated (one week post-confluent) Caco-2 cells
express LPH, although not to as high levels as small intestine (Fig. 8,
compare lanes 11 and 12, band marked
LPH). The cyclophilin probe (Cyc in Fig. 8)
hybridizes to the ubiquitously expressed cyclophilin RNA.
PCR primers were designed for LPH and HOXC11, which annealed on either
side of an intron in the genomic sequence. These were used in
qualitative PCR using cDNA templates derived from HeLa cells, human
fetal intestine (18-28 weeks), human small intestine (15 years), and
differentiated Caco-2 cells (Fig. 9A). The results confirmed
the results from the Northern and ribonuclease protection analyses, and
show additionally that human fetal intestine, which does not yet
express LPH RNA, expresses higher levels of HOXC11 than small intestine
from a 15-year-old Caucasian in which LPH expression is high (Fig.
9A, compare lanes 6 and 7 with
lanes 10 and 11). In the human fetal multiple
tissue cDNA panel (CLONTECH), expression of
HOXC11 was observed in fetal kidney, skeletal muscle, and small
intestine, whereas it was absent in all the other fetal tissues tested
(Fig. 9B).
HOXC11 Stimulates HNF1
-dependent Transcription from
the LPH Promoter--
We have previously demonstrated that the
transcription factor HNF1
binds to three sites in the 894-bp
upstream flanking region of the LPH gene.2 The proximal 227 bp of the LPH gene promoter is sufficient to drive
differentiation-dependent transcription of a reporter gene in differentiated Caco-2 cells, to a level 80% of that seen with the
894-bp LPH promoter construct.2 Thus, the LPH promoter
region from
227 to
17, which contains one HNF1
binding site
CE-LPH2c, and two potential HOXC11 binding sites, CE-LPH1a and -1c, was
used in co-transfection studies. Luciferase activity of the
pGL3-LPH227WT construct alone in undifferentiated Caco-2 cells is low
(1.5 times the level observed with pGL3-basic). As shown in Fig.
10, overexpression of the short or long
form of HOXC11 gives up to 2.7-fold activation of pGL3-LPH227WT, but no activation of pGL3-LPH227mut1ac, in which potential HOXC11 binding sites have been mutated (compare lanes 1-3 with lanes
7-9). Whereas HNF1
alone activates the wild-type LPH promoter
2-fold, co-transfection of pRC-HOX
N or pRC-HOX produces a further
7-fold or 19-fold stimulation of transcription, respectively. In the
case of pGL3-LPH227mut1ac, a lower stimulatory effect is observed
(4-fold and 10-fold, respectively). The stimulation of
HNF1
-dependent transcription by HOXC11 is much reduced
for pGL3-LPH227mut1ac,2c in which the potential binding sites for HNF1
and HOXC11 are mutated. The ability of the introduced mutations to
abrogate factor binding was confirmed in EMSAs (data not shown).

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Fig. 10.
HOXC11 stimulates
HNF1 -dependent activation of the LPH promoter.
Subconfluent Caco-2 cells were transfected with pGL3-LPH227WT, which
contains the wild-type LPH promoter from position 227 to 17 in
front of the luciferase gene, with or without the HNF1 expression
plasmid RSV-HNF. The LPH227mut1ac construct is mutated in CE-LPH1a and
-1c, and the LPH227mut1ac,2c construct has an additional mutation in
the HNF1 -binding site, CE-LPH2c. The effect of co-transfecting
expression plasmids for the short or long form of HOXC11 was tested.
Luciferase activity was corrected for variation in transfection
efficiency and is presented as -fold activation relative to
pGL3-LPH227WT alone. The means ± S.D. were calculated from three
experiments.
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DISCUSSION |
HOXC11 and Cdx-2 Bind to Proximal Regulatory Elements in LPH and
Sucrase-Isomaltase (SI)--
We have previously demonstrated that 1 kb
of the lactase gene promoter is sufficient to direct small
intestine-specific expression and post-weaning decline in transgenic
mice (3). We have focused our attention on a proximal promoter element,
CE-LPH1, that is bound by an intestine-specific factor which co-varies
with lactase expression (5). Deletion of the CE-LPH1 element in stably
transfected Caco-2 cells leads to a 64% reduction in LPH promoter
activity in differentiated cells.2 Using a yeast genetic
screen to identify factors capable of binding to CE-LPH1, the HOXC11
cDNA was isolated twice in a screen of 106 clones. This
suggests that HOXC11 is more abundantly expressed in differentiated
Caco-2 cells and/or binds the CE-LPH1 element with higher affinity,
compared with other DNA-binding proteins encoded in the cDNA
library. However, it should be noted that the cDNA used in the
library construction was subjected to a complete digestion with
NotI, resulting in the probable exclusion of some cDNAs.
Previously, a factor that binds to the related SIF1 element in the gene
for intestinal SI was isolated and identified as the homeodomain
protein Cdx-2 (8). In our present work, we have shown that both HOXC11
and Cdx-2, expressed from a single-copy plasmid in yeast cells, are
able to bind to the CE-LPH1 and SIF1 elements under in vivo
conditions, in a manner dependent on an intact TTTA(T/C) sequence.
Mutation of bases within this 5-nucleotide site, as in mut1 LPH1 and
mut4 LPH1, reduces binding of HOXC11 both in the yeast assay and in
EMSA analysis. Although binding of HOXC11 to mut3 LPH1 is intermediate
between that of mut1 LPH1 and mut2 LPH1, binding of the factors to mut3
LPH1 in yeast does not lead to growth on minimal media, due perhaps to
the all-or-none nature of the yeast screen.
HOXC11 and Cdx-2 are both homeodomain proteins. The homeodomain is a
60-amino acid stretch, which contains a helix-turn-helix motif that is
involved in DNA recognition. Many homeodomain proteins bind
preferentially to a TAAT core sequence. HOXC11 is one of 15 mammalian
Hox genes, which are related to the Drosophila
homeotic gene for Abdominal-B. The Abd-B-like Hox proteins share
several distinguishing features; they are expressed most posteriorly, their homeodomains recognize a divergent TTAT sequence (34), they do
not contain a pentapeptide motif conserved in other Hox genes, and they differ in their ability to interact with Pbx proteins (36, 37). We have demonstrated similar affinity of HOXC11 for an
optimal Abd-B sequence as for the CE-LPH1 element, which contains a
related core sequence TTAC. Two residues in the N-terminal arm of the
Abd-B homeodomain, K6 and P7, appear to play an essential role in the
divergent specificity of Abd-B compared with other Drosophila homeoproteins (34). The N-terminal arm of HOXC11 conserves one of these critical residues, the proline at position 7.
It has recently been demonstrated by binding site selection that the
Abd-B-like Hox proteins Hoxb-9, Hoxa-11, Hoxd-12, and Hox-13 bind
preferentially to a TTTAC-containing sequence (37). However, not much
is presently known about the natural binding sites for mammalian
Abd-B-type Hox proteins. The few cases where target genes have been
identified involve auto- and cross-regulatory interactions between
different Hox genes and their gene products. In the case of
the Abd-B-type proteins, HOXD9 and HOXD10 (previously called HOX4C and
HOX4D, respectively) have been shown to trans-activate the gene for
HOXC5 in cotransfection studies, mediated by a
5'-AATTTATGA-3' sequence in the HOXC5 promoter (38). In the
paralogous mouse Hoxa5 and Hoxb5 gene promoters,
the corresponding element resembles the CE-LPH1 element:
5'-AATTTACGAC-3'. Thus, Abd-B-like Hox proteins recognize a
TTTA(T/C) core sequence, which is likely to be physiologically relevant.
There have been previous reports of intestinal factors other than Cdx-2
which bind to CE-LPH1 or SIF1 elements (20, 29, 39-41). For example,
Traber et al. (41) detected a SIF1-binding protein in a
fractionated Colo-DM extract that had a lower molecular weight than
Cdx-2. We suggest that HOXC11 may account, at least in part, for these
DNA binding activities. It is also possible that the other Abd-like Hox
proteins bind to CE-LPH1 and SIF1 elements.
HOXC11 mRNA Exists in Two Forms--
Caco-2 cells contain a
1.7-kb form of HOXC11 mRNA, which corresponds in size to the
mRNA present in hematopoietic cells (35) and to the insert of the
Caco-2 cDNA library plasmid, pF4. HeLa cells contain, in addition
to the 1.7-kb mRNA, a 2.1-kb form of HOXC11 mRNA. The
full-length cDNA determined by RACE on HeLa cell cDNA was 2.0 kb. The cDNA templates for RACE may not extend all the way to the
5' end of the mRNA, due to premature pausing of the reverse
transcriptase and/or because of the removal of bases from the 5' end by
T4 DNA polymerase. However, the longest RACE sequence is preceded in
the genomic sequence by a stop codon, so the first methionine in Fig.
3, which is in a good translational context, is probably used in HeLa
cells. Although HeLa cells are likely to contain the full-length
304-amino acid HOXC11 protein, it is not clear why there is no
detectable CE-LPH1 binding activity in untransfected HeLa cell
extracts. It is possible that the endogenous factor is unable to bind
DNA due to a HeLa-specific modification. Alternatively, the conditions
used in the EMSA analysis or the presence of nonspecific DNA-binding
proteins in the crude HeLa cell extract may interfere with binding of
the endogenous HOXC11 protein to CE-LPH1 DNA.
RACE carried out on Marathon-Ready human 18-28-week fetal intestinal
cDNA suggests that the longer form of HOXC11 mRNA is also
present in fetal intestine (data not shown). The PCR data presented in
Fig. 9 show that expression of HOXC11 in human fetal tissues is limited
in its distribution, being only present in fetal intestine, kidney, and
skeletal muscle.
The short form of HOXC11 is expressed in differentiated Caco-2 cells,
which also express LPH. Both the LPH and SI proximal promoters contain
important regulatory binding sites for HNF1, a transcriptional
activator that has previously been demonstrated to be important in
hepatic and intestinal gene regulation (42-44). Although Caco2 cells
contain endogenous HNF1
, transfected pGL3-LPH227 on its own gives a
very low luciferase activity in Caco2 cells, indicating the lack of an
essential transcription factor and/or the presence of a repressor in
the undifferentiated cells. We have shown here that both short and long
forms of HOXC11 give only weak activation of a LPH-luciferase construct
on their own, but greatly enhance transcriptional activation when
co-transfected with an HNF1
expression plasmid. This
HNF1
-dependent stimulation requires an intact
HNF1
-binding site. The bifunctional protein DCoH is also known to
enhance the transcriptional activity of HNF1
. Although DCoH does not
itself contact DNA, it stabilizes HNF1
dimers and HNF1
/DNA
complexes (45, 46). The observed stimulatory effect of HOXC11 on
HNF1
may be due to a similar stabilizing effect on HNF1
DNA
binding and/or an increase in transcriptional activity of HNF1
.
Although there is a 2-fold drop in stimulation by HOXC11 and HNF1
when the HOXC11-binding sites CE-LPH1a and -1c are mutated, it is not
clear from our experiments whether HOXC11 binds to DNA in order to
stimulate HNF1
activity. HOXC11 may be tethered to DNA through an
interaction with HNF1
, or it may bind to low affinity DNA sites in
the presence of HNF1
. Future experiments will address whether HOXC11
and HNF1 interact directly with each other, and whether they co-operate
in DNA binding.
It is possible that the intestinal specific expression of LPH is
dependent, at least in part, on the overlap in tissue distribution of
both HNF1
and HOXC11. The presence of HOXC11 in tissues outside the
intestine, such as HeLa cells, fetal kidney, and fetal skeletal muscle,
suggests that it has additional functions. Homeobox genes typically
play an important role in tissue development; for example, pdx-1 (pancreatic and duodenal homeobox gene 1) is essential
for pancreatic development in mice (47). Diverse mammalian Abd-B-type HOX genes have an important role in limb and urogenital
development (32, 48-50). The expression of HOXC11 in human fetal
intestine suggests that HOXC11 plays an important role in intestinal
development.
We thank Liselotte Laustsen and Anette
Melchior Jensen for excellent technical assistance and Dr. Jørgen
Olsen and Dr. Nikolaj Spodsberg for stimulating discussions. We are
also grateful to Dr. R. Reed for the plasmids pPC86, p59.7, pRS315HIS,
and y11 and yeast strain yWAM2; Dr. M. German for providing us with the plasmid pBAT7Cdx-3; and Dr. F. Tronche for the plasmid RSV-HNF1
.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ000041.