(Received for publication, July 16, 1996, and in revised form, October 18, 1996)
From the Department of Pharmacology, Cornell University Medical College, New York, New York 10021
Homeobox genes control the spatial identity and
differentiation of tissues in the developing vertebrate embryo.
Retinoids are signaling molecules involved in the regulation of Hox
genes. We previously identified a 3 enhancer called the
RAIDR5, which contained a DR5 retinoic acid response
element (RARE) and was responsible for the retinoic acid
(RA)-associated expression of the murine Hoxa-1 gene in
teratocarcinoma cells. We demonstrate that a similar enhancer, which
contains a DR5 RARE, is located at a DNase I-hypersensitive
site 3
of the murine Hoxb-1 gene. This enhancer, the
Hoxb-1 RAIDR5, regulates the RA responsiveness of the Hoxb-1 gene and is different in location
and sequence from the RA-regulated 3
Hoxb-1 enhancers
previously described. Several DNA elements within the murine
Hoxa-1 RA-inducible RAIDR5 enhancer, including
the DR5 RARE, conserved element (CE) 1, and CE2, are conserved in the murine Hoxb-1 RAIDR5 enhancer,
the human homolog of Hoxa-1, and in the chicken
Hoxb-1 gene. Gel shifts show that the CE2 sequence
TATTTACTCA binds an RA-inducible factor, while UV cross-linking
indicates that a 170-kDa protein binds to this sequence. Thus, the
Hoxa-1 and Hoxb-1 genes possess 3
enhancers with similar sequences through which their expression and
responsiveness to endogenous retinoids are controlled.
Genetic analysis of Drosophila melanogaster led to the identification of the homeotic genes, which specify the identity of body parts during embryonic development (for reviews, see Refs. 1-4). The vertebrate homologs of these genes, the homeobox or Hox1 genes, appear in structurally related chromosomal gene clusters known as the Hox A, B, C, and D clusters, which are thought to have arisen from a single ancestral cluster by repeated duplication. The expression domains of Hox genes during embryogenesis have been suggested to create a "code" that specifies the positional fate of cells in the embryo (5, 6). Consistent with this function, experiments have demonstrated that changes in the expression of Hox genes lead to corresponding changes in tissue identity (7-9). Hox genes encode homeodomain-containing transcription factors (10), and expression of a particular Hox protein, or a combination of proteins, is thought to specify a positional identity by creating a unique pattern of gene expression (for reviews, see Refs. 2 and 11).
There is considerable evidence that retinoids such as retinoic acid (RA) play an important role in regulating Hox genes. Retinoids are required for normal vertebrate embryogenesis; the offspring of animals deficient in vitamin A have numerous developmental defects (12, 13). The addition of exogenous retinoids during vertebrate embryogenesis also leads to developmental abnormalities in zebrafish (14), Xenopus (15-18), chickens (19), hamsters (20); mice (21-24), and humans (25). Most striking is the ability of locally applied retinoids to respecify positional identity during development or regeneration of the vertebrate limb (for reviews, see Refs. 9 and 26-28). Such observations have led to the hypothesis that retinoids function as morphogens in vertebrates, specifying positional identities in a concentration-dependent fashion.
The retinoic acid receptors (RARs ,
, and
) and retinoid X
receptors (RXRs
,
, and
) are members of the steroid/thyroid/ retinoid gene family, which includes the steroid receptors, thyroid hormone receptors, the RARs, the RXRs, and the vitamin D receptor, and
are ligand-inducible regulators of transcription (for review, see Ref.
29-31). In addition to all-trans-retinoic acid,
9-cis-retinoic acid (32, 33), 4-oxoretinoic acid (34),
3,4-didehydro-RA (35), 4-oxoretinaldehyde (36), and 4-oxoretinol (37)
are activating ligands for the RARs and can affect pattern formation in
vertebrates (for review, see Ref. 38). RAR-RXR heterodimers are thought
to be the species which control the expression of RA-responsive genes
(for review, see Refs. 30 and 39). There is evidence that these
heterodimers bind either DR2 or DR5 retinoic acid-response elements (RAREs). These elements consist of two "direct repeat" (DR) sequences of AGGTCA separated by either
two or five bases (for review, see Ref. 30). Animals that lack more than one normal RAR show developmental abnormalities that correspond to
those seen in vitamin A deficiencies, definitely demonstrating the
importance of signal transduction by the retinoic acid receptors in
vertebrate development (40, 41).
Evidence that retinoids were involved in the regulation of homeobox
genes initially came from cell culture studies (42-45). In both human
and murine teratocarcinoma cells, retinoic acid can induce the
expression of many homeobox genes. This retinoic acid inducibility,
like the expression pattern of genes in the embryo, corresponds to the
order of the genes in the cluster (46, 47). Thus, the 3 most genes in
each chromosomal cluster, e.g. HOXA1 and HOXB1,
are the first to be induced by RA. This induction is transient in that
it is observed over a 12-24-h period, which is followed by a decline
in expression (44-47). Co-linear RA responsiveness has also been
demonstrated in mouse and Xenopus embryos (17, 48-50).
These observations indicate that the expression of Hox genes is
controlled in part by retinoic acid or by other retinoids that activate
the retinoic acid receptors.
The murine Hoxa-1 gene is the 3 most homeobox gene in the
"Hox a" chromosomal cluster on mouse chromosome 6 (51).
Previously, we described the identification of a retinoic
acid-responsive enhancer located approximately 4.5 kilobases 3
of the
murine Hoxa-1 gene (formerly Hox 1.6; see Scott (52) for
nomenclature). This enhancer, called the Hoxa-1
RAIDR5, was shown to be required for retinoic acid
inducibility of Hoxa-1 in cultured F9 and P19 teratocarcinoma cells, and the ~300-bp enhancer functioned in either
orientation when placed upstream of the basal TK promoter (53).
Furthermore, this enhancer function was shown to require a
DR5 RARE since point mutations within the RARE abolished RA inducibility (53). We now report the identification of very similar
enhancers located 3
of the murine and chicken Hoxb-1 (formerly called Hox 2.9) genes. The murine Hoxb-1 gene is
the 3
most homeobox gene in the "Hox b" chromosome
cluster on mouse chromosome 11 (51). We also report the identification
of a highly conserved 3
enhancer in the human HOXA1 gene.
We demonstrate that these related RAIDR5 enhancers all
mediate retinoic acid responsiveness in cultured cells and share common
sequence elements, including a DR5 RARE.
The human HOXA1
clones were isolated from a genomic library from human lymphocyte DNA
in EMBL3 obtained from Dr. Winnie Wong. Standard hybridization
conditions were modified by reduction of formamide concentration to
25%, and the probe used was the PstI-EcoRI fragment containing the 3
RA-responsive enhancer from the murine Hoxa-1 gene (53). Hoxb-1 and Hoxd-1
genomic clones were isolated from
FIX (Stratagene) and
EMBL
(constructed by Anton Berns) libraries containing 129SV genomic DNA.
The Hoxb-1 cDNA probe was provided by Dr. Joseph Grippo. The
chicken Hoxb-1 phage (54) was obtained from Dr. Gregor
Eichele. Library screening, phage purification, and DNA isolation were
by standard methods (55).
Standard methods were used to subclone DNA fragments from
genomic phage into Bluescript, pBLCAT2 (56), or pGOTKCAT. The latter is
a derivative of pGL2 (Promega) in which the luciferase gene is replaced
by the TK minimal promoter and the CAT reporter from pBLCAT2 and
includes a transcriptional terminator upstream of the polylinker to
reduce background. Transient transfections, CAT assays, and
-galactosidase assays were performed as described previously
(53). Quantitation of CAT assays was carried out using a PhosphorImager
(Molecular Dynamics).
-Galactosidase units were calculated by the
formula:
-gal units = (A420)
(100)/(volume of lysate per reaction) (time).
Conserved elements
1 and 2 were mutated by polymerase chain reaction amplification of
subcloned enhancer fragments with the following oligonucleotides,
paired with primers to flanking plasmid sequences: CE1, top strand:
5-CTGAGGATCCCGGAGGCTATTCAGATGC-3
; CE1, bottom strand:
5
-GGGATCCTCAGCTCAAAGGTGAACCCAAAGC-3
; CE2, top strand:
5
-CGCTCTGCAGAACAGTCTGAAAGGGTTG3-
; and CE2, bottom strand:
5
-TGTTCTGCAGTTGATCGGTTTGAAT-3
. These oligomers are complementary to
sequences adjacent to conserved elements 1 and 2, but their 3
tails
are noncomplementary and contain unique restriction sites. Following
polymerase chain reaction, products were cut with appropriate enzymes,
purified, and ligated. Plasmid primers were then used to amplify the
fragments containing disrupted CE1 or CE2. To generate an enhancer with
mutations in both CE1 and CE2, the process was repeated with a fragment
containing mutations in CE1, using the oligonucleotides to CE2.
Products were sequenced to confirm that the mutations in the conserved
elements were the only changes introduced. Mutated DNA fragments were
then cloned into the Hoxa-1 (1.6) lacZ minigenes (see Fig.
5). These minigenes differ from the previously described constructs
(53) in that the 3
end of the lacZ gene is joined to the
PvuII site at bp 203 of the Hoxa-1 (1.6)
cDNA. The resulting constructs include the constitutive intron of
Hoxa-1, unlike the minigenes used in previous work (53) and
lack only 44 base pairs of exon 1.
Preparation of Nuclear Extracts
Approximately 1 × 107 F9 or P19 teratocarcinoma cells, either stem (control,
undifferentiated, untreated) or retinoic acid treated for 48 h,
were washed twice with phosphate-buffered saline and resuspended in two
packed cell volumes of cell lysis buffer (10 mM Hepes, pH
7.0; 3 mM MgCl2, 40 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 5%
glycerol, 8 mg/ml aprotinin, 2 mg/ml leupeptin, 0.2% Nonidet P-40).
Cells were lysed for 5 min at 4 °C and nuclei were pelleted by
centrifugation in a microcentrifuge at 5000 rpm for 5 min. Nuclei were
resuspended in two packed nuclear volumes of extraction buffer (20 mM Hepes, pH 7.9, 1.5 mM MgCl2,
0.42 M KCl, 0.2 mM EDTA, 1 mM DTT,
0.5 mM phenylmethylsulfonyl fluoride; 25% glycerol) and
incubated for 45 min at 4 °C with gentle shaking. Samples were
centrifuged at high speed in a microcentrifuge for 10 min. Supernatants
were removed and dialyzed for 5 h at 4 °C against 50 volumes of
dialysis buffer (20 mM Hepes, pH 7.9; 0.1 M
KCl; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM phenylmethylsulfonyl fluoride; 20% glycerol). Dialyzed
samples were aliquoted and stored at 70 °C.
The sequences of
complementary CE2 oligonucleotides used as probe in the binding assays
are: sense CE2, GGCAAACCGATCAATATTTACTCAGTCTGA; and antisense CE2,
GGTCAGACTGAGTAAATATTGATCGGTTTG. The mutated CE2 oligonucleotides are
identical to those above except they contain the dinucleotide
substitutions as indicated in Fig. 6. The sequence of the AP-1
consensus oligonucleotides are: sense AP-1, AACTTTGACTCAG; and
antisense AP-1, CTGAGTCAAAGTT. Complementary nucleotides were annealed
by heating at 85 °C for 2 min, followed by successive 15 min
incubations at 65, 37, 22, and 4 °C. The double-stranded
oligonucleotide to be used as probe was end-labeled by filling in 5
overhangs with an [
-32P]dCTP using Klenow enzyme.
Binding reactions were performed in a total volume of 20 µl
containing: 0.5 ng (50,000 cpm) of probe, 5 µg of nuclear extract,
1 × binding buffer (10 mM Hepes, pH 7.9, 1 mM MgCl2, 60 mM KCl, 0.5 mM EDTA, 1 mM DTT, 10% glycerol), and 2 µg
of poly(dI-dC). Samples were incubated for 20 min at 22 °C and
separated on a 4% nondenaturing polyacrylamide gel, which was dried
and visualized by autoradiography. Supershift experiments were
performed similarly to the standard binding assay with the following
modifications. Following the 20-min 22 °C incubation, 2 µl of
either anti-Fos or anti-Jun antibody (Santa Cruz Biotechnology) was
added to the binding reaction and incubated another 2 h at 4 °C. Samples were separated on a 6% nondenaturing polyacrylamide gel at 4 °C.
Ultraviolet Cross-linking of Protein to DNA
Binding reactions were carried out in a 20-µl volume as described in the previous section. Following a 20-min incubation, the reactions were exposed to 254-nm wavelength ultraviolet light from a hand-held luminometer for 10 min at a distance of 5 cm. 20 µl of 2 × SDS-sample buffer (100 mM Tris-HCl, pH 6.8, 100 mM DTT, 4% (w/v) SDS, 0.2% (w/v) bromphenol blue, 20% (w/v) glycerol) were added to each reaction, and the samples were boiled for 5 min to denature proteins. The samples were subjected to SDS-polyacrylamide gel electrophoresis utilizing a 5% stacking gel and an 8% separating gel. A 10-kDa protein ladder (Life Technologies, Inc.) was run as a size standard. To visualize the protein standards, the gel was stained overnight in Coomassie Blue staining solution (50% (v/v), methanol 0.05% (v/v) Coomassie Brilliant Blue R-250, 10% (v/v) acetic acid, and 40% H2O) and destained for several hours in 5% (v/v) methanol, 7% (v/v) acetic acid, and 88% H2O. The gel was then dried and visualized by autoradiography.
Since we had previously identified a retinoic
acid-responsive enhancer located about 4.5 kb 3 of the start site of
transcription of the murine Hoxa-1 gene (53), we wanted to
determine whether or not this enhancer sequence was conserved in other
Hox genes located in the most 3
position of Hox chromosomal clusters
such as the murine Hoxb-1 gene, the chicken
Hoxb-1 gene, and the human HOXA1 gene. Genomic
clones for the murine Hoxa-1 and Hoxb-1 genes, the human HOXA1 gene, and the chicken Hoxb-1 gene
were isolated or obtained as described under "Experimental
Procedures." (Murine Hox genes are labeled using small letters, human
Hox genes with capital letters (52).) DNA hybridization experiments
demonstrated that the human and murine Hoxa-1 genes were
homologous not only in their transcribed regions, but also in portions
of their 3
-flanking DNA. DNA fragments containing the ~300-bp murine
Hoxa-1 retinoic acid-responsive enhancer, called
RAIDR5 (53), were used to identify homologous sequences in
the human HOXA1 gene. This fragment of human DNA was tested
for the presence of a retinoic acid-responsive enhancer by subcloning
cross-hybridizing DNA fragments into a minimal promoter-CAT vector and
transfecting these constructs into F9 or P19 teratocarcinoma cells. The
structure of the human HOXA1 gene, and the location of the
3
RA-responsive enhancer are shown in Fig. 1. One
NsiI-SacI restriction fragment mediates retinoic
acid responsiveness in transient transfection assays (Fig. 1). Thus, a
retinoic acid-responsive enhancer is present about 4.5 kb 3
of the
human HOXA1 gene, in a location very similar to that which
we previously described for the murine Hoxa-1 gene (53).
Identification of a Retinoic Acid-responsive Enhancer 3
DNase I hypersensitivity assays were used to
map a putative retinoic acid-responsive enhancer near the murine
Hoxb-1 gene using the same strategy employed to identify the
RA-responsive enhancer 3 of the Hoxa-1 gene (53). Following
RA treatment of F9 murine teratocarcinoma cells, a DNase I
hypersensitive site was detected approximately 6.5 kb 3
of the
Hoxb-1 gene (data not shown; position of the hypersensitive
site is indicated by the open arrow in Fig.
2). Minimal promoter-CAT vectors containing a series of
DNA fragments including and surrounding the location of the
RA-inducible hypersensitive site were shown to mediate responsiveness
to retinoic acid, as shown in Fig. 2. More specifically, an
NsiI-SacI fragment which includes the region of
the hypersensitive site was shown to be sufficient for RA inducibility
in the transient transfection assays in either F9 (Fig. 2) or P19 cells
(data not shown).
Identification of a Retinoic Acid-responsive Enhancer from the Chicken Hoxb-1 Gene
The chicken Hoxb-1 (2.9) genomic
phage DNA was analyzed for 3 enhancer activity by subcloning
restriction fragments of the 3
-flanking DNA and testing various
fragments for RA inducibility. In transient transfection experiments,
an approximately 2-kb BamHI-EcoRI DNA fragment
located 3
of the chicken Hoxb-1 gene was shown to confer RA
responsiveness on a minimal promoter (Fig. 3). Other 3
chicken DNA fragments lacked the ability to confer RA responsiveness (Fig. 3, and data not shown).
Comparison of the DNA Sequences of the 3
The aligned sequences of the
RA-responsive enhancers from the murine Hoxa-1, the human
HOXA1, and the murine and chicken Hoxb-1 genes
are presented in Fig. 4. First, it is striking that two different homeobox genes, Hoxa-1 and Hoxb-1,
located on different chromosomes, have such similar sequences in their
RA-inducible enhancers. Second, with respect to the mouse
Hoxa-1 and human HOXA1 3 retinoid-responsive
enhancers, the region of near identity shown in Fig. 4 is part of a
large block of conserved sequence. Furthermore, homology of the
Hoxa-1 and b-1 enhancers among different species
is particularly noteworthy, given that these DNA sequences come from
species separated by an estimated 300 million years of evolution
(57).
As expected from the ability of these enhancers to mediate RA responsiveness, all four enhancers include a DR5 (direct repeat 5) RARE. The half sites of these RAREs are precisely conserved, as is their separation by 5 base pairs. Thus, these ~300-bp enhancers are called the Hoxa-1 RAIDR5 and Hoxb-1 RAIDR5. Two other conserved elements (CE1 and CE2; boxed in Fig. 4) are also present in all four enhancers. The conservation of these sequences is equivalent to that of the RARE, suggesting that CE1 and CE2 play equally important roles in controlling the expression of the vertebrate homeobox genes. Therefore, we examined the functions of the CE1 and CE2 elements in more detail using electrophoretic mobility shift assays and transient transfection experiments.
Functional Analysis of the CE1 and CE2 in RAIDR5 EnhancerTo examine the functional importance of CE1 and CE2,
mutations were introduced that altered each nucleotide of CE1 and CE2, as described under "Experimental Procedures." In addition, a double mutation in which both CE1 and CE2 elements were disrupted and another
mutation in which the conserved RARE was specifically mutated were also
characterized. These mutations were made in the context of a
Hoxa-1 minigene/lacZ reporter construct that contains the
lacZ gene inserted into the coding sequence of Hoxa-1 as
well as 6.5 kb of 5-flanking region and about 3 kb of 3
-flanking DNA
(53) (Fig. 5). P19 teratocarcinoma cells were first
transiently transfected with these constructs, and cells were either
untreated (stem) or treated for 24 h with RA.
The results from the untreated P19 stem cells showed that mutation of
the CE1 element did not significantly change -galactosidase activity
as compared with that observed with the wild type construct (Fig. 5).
Mutation of either the CE2 element or the RARE also had no effect on
the activity of the reporter (Fig. 5). These results indicate that CE1
and CE2 do not appear to be functional elements in P19 stem cells.
The activities of all of the reporter constructs were increased by the addition of RA to P19 cells, with the exception of the construct containing a mutated RARE (Fig. 5). Mutation of CE1 resulted in an increase of approximately 3.0-fold and mutation of CE2 resulted in a 4.9-fold increase in activity, as compared to the 3.1-fold RA-associated increase in reporter activity observed with the wild type enhancer/reporter construct (Fig. 5). Mutation of both elements CE1 and CE2 resulted in an RA-associated increase in reporter activity of 3.7-fold (Fig. 5). These results indicate that the CE2 element plays a negative role in the RA responsive of the Hoxa-1 gene, since mutation of the CE2 element leads to a greater increase in the response to RA. The CE1 element does not function as a negative element in RA-treated P19 cells.
CE2 of the Hoxa-1 3Electrophoretic mobility shift
assays were performed using CE1 and CE2 sequences from the mouse
Hoxa-1 3 RAIDR5 enhancer as probes and nuclear
extracts prepared from P19 or F9 teratocarcinoma stem cells
versus P19 or F9 cells that had been treated for 48 h
with RA. No binding to the CE1 element was detected in nuclear extracts
from either stem cells or RA-treated cells (data not shown). Binding to
the CE2 element was also absent in P19 stem cells (Fig.
6, lanes 2-4). In contrast, binding to the
CE2 element was detected in RA-treated P19 cells (Fig. 6, lane
5) and F9 cells (data not shown). CE2 binding was shown to be
specific since it could be effectively competed with a 100-fold molar
excess of unlabeled CE2 oligonucleotide but could not be competed by an unrelated, nonspecific double-stranded oligonucleotide (Fig. 6, lane 6). When the CE2 element of the murine
Hoxb-1 enhancer was used as a probe, an apparently identical
binding complex was observed (data not shown). The CE2 element of the
Hoxb-1 enhancer differs from that of the Hoxa-1
CE2 element by 1 base out of 14 (Fig. 4).
Analysis of the CE2 element revealed the presence of an AP-1 site
(58-60). The AP-1 consensus site is TKANTCA where K is a G or T and N
represents any nucleotide. To determine whether AP-1 was involved in
recognition of the CE2 element, supershift assays were performed using
antibodies that recognized conserved regions of both Fos and Jun family
members. Under a variety of binding and gel buffer conditions, no
shifting of the CE2 complex was observed (data not shown), suggesting
that Fos and Jun are not present in the CE2 binding complex. To confirm
this, a consensus AP-1 recognition site was examined for its ability to
act as a competitor for the CE2 binding complex in an electrophoretic
mobility shift assay. A 100-fold molar excess of the unlabeled AP-1
consensus element did not compete for CE2 binding activity (Fig.
7, lane 10), indicating that the CE2 complex
did not contain Fos or Jun.
To determine which nucleotides in the murine Hoxa-1 CE2
element are essential for binding, a panel of CE2 oligonucleotides containing dinucleotide base mutations were examined for their ability
to compete the CE2 complex binding in an electrophoretic mobility shift
assay (Fig. 7). Mutations M1 and M7 were effective competitors and
therefore still retained the ability to bind the CE2 complex (Fig. 7,
lanes 3 and 9). Mutations M2 through M6 could not
compete for the binding complex indicating that the 10 nucleotides, TATTTACTCA, are critical to CE2 binding (Fig. 7, lanes
4-8). This sequence constitutes a novel binding site since,
although it overlaps with the AP-1 site, it contains additional
residues that are not essential for AP-1 binding. Importantly, these
ten bases are the nucleotides that are specifically conserved in the
homeobox 3 RAIDR5 enhancers of the murine
Hoxb-1, human HOXA1, and chicken Hoxb-1 genes (Fig. 4). Thus, the CE2 element is likely to
play an important role in the transcriptional regulation of the murine Hoxa-1 and human HOXA1 genes, and the murine
and chicken Hoxb-1 genes.
Brn 3.0, a pituitary-octamer-unc domain protein expressed in the pituitary gland and in the corticotroph cell line ATt-20, is involved in regulation of the proopiomelanocortin gene (61). The sequence GCATAAATAAT has been identified as a high affinity Brn 3.0 binding site. This sequence overlaps with a complementary region, TATTTA, of the CE2 element. To examine whether Brn 3.0 was involved in binding the CE2 element, a mobility shift assay was performed using nuclear extracts prepared from the pituitary ATt-20 cell line. High levels of both Brn 3.0 and the CE2 factor(s) were detected in these ATt-20 cells, but the Brn 3.0 and CE2 binding complexes migrated with different mobilities on the gel (data not shown). In addition, the Brn 3.0 site could not compete the CE2 complex and the CE2 site could not compete the Brn 3.0 complex, indicating that Brn 3.0 is not involved in binding to the CE2 element (data not shown).
To analyze further the CE2 binding complex, UV cross-linking was
performed. Binding reactions were carried out as described under
"Experimental Procedures," using nuclear extracts from P19 cells
that had been treated for 48 h with RA. Following this incubation, the samples were exposed to ultraviolet light at a wavelength of 254 nm
and at a distance of 5 cm for 10 min. The samples were then separated
on an 8% SDS-polyacrylamide gel. The probe alone is shown in Fig.
8, lane 1. In the absence of any UV
treatment, no bands are observed (lane 2). Following UV
treatment a band is observed at approximately 180 kDa (lane
3), and a smeared band is observed below 60 kDa. The 180-kDa band
is specifically competed away by a 100-fold molar excess of unlabeled
CE2 competitor (lane 4), but is unaffected by unlabeled
competitor that contains a mutation of the CE2 site (lane
5). The smear below 60 kDa is not significantly affected by
specific competitor indicating that these are nonspecific interactions.
The 180-kDa band observed includes the molecular mass of the 32 bases
of one strand of the labeled 32-base pair probe which has a molecular
mass of approximately 10 kDa (depending on which strand of the CE2
binding site is effectively cross-linked to the protein). The
cross-linked protein therefore has an approximate molecular mass of 170 kDa.
The 3
In previous work (62-66), several enhancers surrounding
the murine and human Hoxb-1 genes were analyzed, but none of
these 5 or 3
enhancers was reported to contain a DR5
RARE. However, a functional RARE 3
of the murine, chicken, and
pufferfish Hoxb-1 (2.9) genes was shown to mediate a portion
of the RA responsiveness of the Hoxb-1 gene in transgenic
animals. Marshall et al. (62) have reported that this RARE
is a functional DR2-type RARE in the murine and pufferfish
Hoxb-1 genes, while Ogura and Evans (64) have reported
similar results for the human HOXB1 gene. We have confirmed
that the mouse Hoxb-1 gene has this second RA-responsive enhancer, located 3
of this gene but much closer to the
transcribed sequence (referred to as the DR2 RARE, see Fig.
9) than the RAIDR5 enhancer described
above.
This RAIDR2 enhancer, which contains a DR2
RARE, exhibits different properties in cultured teratocarcinoma cells
when compared to the RAIDR5 enhancer we have identified in
this report. Using P19 teratocarcinoma cells, RA responsiveness of the
murine Hoxb-1 gene from this 3 RAIDR2 enhancer
could be detected (Fig. 9). However, in F9 teratocarcinoma cells these
same constructs did not mediate a response to RA, consistent with the
observed absence of a DNase I-hypersensitive site at this location in
F9 cells (Fig. 2). Thus, the murine Hoxb-1
RAIDR2 enhancer functions to induce RA responsiveness only
in some teratocarcinoma cell lines. This is a markedly different result
from that obtained with the RAIDR5 enhancers; these
function in all teratocarcinoma and embryonic stem cell lines we have
tested, including F9 and P19 teratocarcinoma cells, and CCE embryonic
stem cells (Figs. 1, 2, 3 and 5, and data not shown).
The chicken homolog of the Hoxb-1 3 proximal enhancer does
not show the same cell type variation as the cognate
enhancer from the mouse. However, while it functions in both P19 and F9 cells, in F9 cells the fold induction by RA is considerably less (Fig.
9). After examination of the sequence of this DNA fragment from the
chicken Hoxb-1, we were unable to identify a DR2
RARE identical to that previously identified by Marshall et
al. (62) or any other blocks of homology between the chicken and
its cognate fragment from the mouse Hoxb-1 gene in this
region (data not shown). Thus, this DR2 RARE appears to be
less conserved than the DR5 RARE within the
RAIDR5 enhancers identified in this report. Creation of
chimeric constructs between the 3
RAIDR2 enhancer of the
mouse Hoxb-1 and similar portions of the chicken
Hoxb-1 gene would allow us to map the sequences involved in
the cell type-specific function (P19 versus F9 cells) of the
murine Hoxb-1 RAIDR2.
Further evidence that, in cultured teratocarcinoma cells, the
RAIDR5 and RAIDR2 enhancers are functionally
distinct comes from the fact that the DR2 RARE from the
murine Hoxb-1 gene, without additional genomic sequences, is
unable to confer significant RA responsiveness on a heterologous
promoter (63, 64).2 In contrast, the
DR5 RARE (a 21-mer with 2 bases on each side of the
DR5 sequence) mediates RA responsiveness in all
teratocarcinoma cell lines tested when it is placed upstream of a basal
thymidine kinase promoter/reporter construct, even in the absence of
co-transfected receptor (53).2 Thus, the RAIDR5
enhancer 3 of the murine and chicken Hoxb-1 genes, which we
describe here, is unlikely to be redundant to other previously
described Hoxb-1 regulatory elements (62-64, 66), given the
evolutionary conservation of this enhancer and the apparent functional differences between these DR5 and
DR2 RAREs (for review, see Ref. 30).
Experiments in which sequences from Hox loci have been used to
control lacZ expression in transgenic animals have demonstrated the
complexity of Hox gene regulation (for review, see Refs. 6 and 27).
Multiple, distinct enhancer-like elements, each functioning in a stage-
and tissue-specific manner, are thought to act in combination to create
the temporal and spatial expression patterns of Hox genes. Similar
combinatory action of enhancer elements have been well documented for
the homeotic genes of Drosophila (67, 68). We previously
described the identification of one such enhancer, located at about 4.5 kb 3 of the murine Hoxa-1 gene transcription start site,
and demonstrated that this enhancer, and specifically the
DR5 RARE within it, was required for the RA responsiveness
of the Hoxa-1 gene in teratocarcinoma cells (53).
In this study we show that this RA-responsive enhancer is also present
in other homeobox genes similar in chromosomal position to
Hoxa-1 (see Fig. 10). Conservation of this
RAIDR5 enhancer sequence suggests that the DR5
RARE and the two conserved elements CE1 and CE2 serve regulatory
functions which are required for expression of both the
Hoxa-1 and Hoxb-1 genes. To date, the only RAREs
identified in the Hox clusters are the DR5 RARE of
Hoxa-1 (53, 69) (this report for the human HOXA1
gene), the DR5 RAREs of the murine and chicken
Hoxb-1 genes (this report), the DR2 RAREs in the
murine and human Hoxb-1 genes (62-64, 66), and a divergent
DR5 RARE conserved in the promoters of the human and murine
D4 genes (70, 71). Thus, while it has been shown that most Hox genes
are inducible by RA in cultured cells (46, 47), how extensive a role
direct regulation by activated retinoic acid receptors plays in the
creation of the expression patterns of other Hox genes remains to
be determined (for review, see Ref. 72).
The potential roles that the conserved elements CE1 and CE2 in the
RAIDR5 enhancer play in the regulation of Hoxa-1
and Hoxb-1 in various cell types are currently being
examined in greater depth. The data presented in this report indicates
that the CE2 binding factor(s) is a novel, previously unidentified
protein(s). The transactivation data indicate that the CE2 element
plays a negative role in the RA responsiveness of the Hoxa-1
gene. Although the level of inhibition of expression observed is small,
the repression correlates with the induction of CE2 binding activity in
P19 cells following RA treatment. In addition, CE2 binding is detected
in F9 cells, another teratocarcinoma line, and is up-regulated by RA
with the same kinetics as observed for P19 cells (data not shown).
Since the Hoxa-1 and Hoxb-1 genes are only
transiently activated by RA in murine and human teratocarcinoma cells
(44-47), the RA induction of CE2 binding activity most likely plays a
role in the repression of the RAIDR5 enhancer at later
times after RA addition. The relatively weak inhibitory effect observed
(Fig. 5) may reflect experimental limitations in using transient
transfections to perform this analysis. For instance, previous
experiments (53) demonstrated that, following RA treatment, a DNase I
hypersensitive site is found at the Hoxa-1
RAIDR5 3 enhancer, indicating a reorganization of
chromatin at this site. This change in chromatin may be essential to
the function of the CE2 element. The generation of stable cell lines
containing the Hoxa-1 minigene reporter constructs should help elucidate any role of chromatin structure on the ability of the
CE2 site to function in regulating Hoxa-1 expression.
UV treatment of binding reactions cross-links a single protein band of approximately 170 kDa to the labeled CE2 binding site. UV cross-linking in Fig. 8 was performed for 10 min. In experiments not shown, samples were also UV-treated for time periods of 30, 60, and 90 min. In these cases, the 170-kDa band was still the only band observed. These results indicate that the lack of detection of additional proteins binding to the CE2 element was not due to an insufficient amount of UV cross-linking treatment. UV treatment results in the cross-linking of only a small percentage of protein (73) that is in close proximity to the probe. Therefore, only a single protein of a multimeric protein complex binding to the CE2 element will likely acquire label. Since only one band is observed, these results indicate that the CE2 binding protein(s) recognize the CE2 site as: 1) a monomer, 2) a homodimer, or 3) a heterodimer of two proteins of identical molecular mass. It is also possible that additional protein(s) can bind to this element in other cell types.
The consensus binding site recognized by the CE2 factor(s) is
TATTTACTCA. These 10 nucleotides are identically conserved among the 3
enhancers of the murine Hoxa-1, murine Hoxb-1,
human HOXA1, and chicken Hoxb-1 genes, suggesting
that the CE2 element may play a role in regulating all of these genes.
The CE2 element overlaps with an AP-1 site, but experiments show that
neither Fos nor Jun is present in the binding complex. Although not
identical, CE2 does show some homology to other known factor binding
sites. Three elements which are similar in sequence to the CE2 binding site are those for the transcription factors Brn 3.0 and MEF-2, and the
DE-2 element of the proopiomelanocortin promoter (61, 74-76).
The 3 enhancers identified in this study are likely to be important
not only in the regulation of Hoxa-1 and Hoxb-1
genes themselves, but also in the activation of entire Hox
loci. Most of the genes in the Hoxa and Hoxb
chromosomal clusters (not all have been studied) have been shown to be
activated in response to RA in a co-linear manner with respect to time
and RA concentration (Fig. 10) (see Refs. 39 and 72 for review). There
are several models which could explain this temporal Hox
cluster activation. In one model, the Hox genes would be
activated via an RA-responsive "locus control region," similar to
that which controls the globin genes (77, 78); in this model the 3
RAIDR5 enhancer would function to regulate the RA
responsiveness of the entire chromosomal Hox gene cluster.
In a second model, the Hoxa-1 and Hoxb-1 genes would be activated directly via the RAIDR5 enhancers
analyzed in this report, and then the protein products of these genes
would activate the next 5
genes in the clusters, i.e.
Hoxa-2 and b-2, respectively. In a third model,
multiple RAREs would control the responses of Hox genes in a
manner similar to that in which stripes of gene expression are
generated in Drosophila embryos by binding sites with
differing affinities for the morphogens bicoid and hunchback (79, 80).
Although there is no direct evidence to support the validity of any one
of these three mechanisms, in the case of the first two proposed
models, the 3
RAIDR5 enhancers, identified here, are
critical to the activation of the entire Hox cluster.
Aspects of these models can be tested using cultured teratocarcinoma
and embryonic stem cells, and such experiments are currently in
progress.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) S78041[GenBank] (for murine Hoxa-1).
We thank Dr. Joe Grippo for the Hoxb-1 cDNA probe, Dr. Gregor Eichele for the chicken Hoxb-1 phage, Taryn Resnick for editorial assistance, and Drs. Anna Means and Lap Ho for critically reading the manuscript.