Pbx1-Hox heterodimers bind DNA on inseparable half-sites that permit intrinsic DNA binding specificity of the Hox
partner at nucleotides 3
' to a TAAT motif
Pbx1-Hox heterodimers bind DNA on inseparable half-sites that permit intrinsic DNA binding specificity of the Hox partner at nucleotides 3 ' to a TAAT motif
Paul S.
Knoepfler*
,
Qiang
Lu
and
Mark P.
Kamps
Department of Pathology, University of California, San Diego, School of
Medicine, 9500 Gilman Drive,
La Jolla
, CA 92093,
USA
Received March 11, 1996;
Revised and Accepted April 22, 1996
ABSTRACT
Heterodimers between the Pbx/Exd and Hox/HOM-C classes of homeodomain proteins bind regulatory elements in tissue-specific and developmentally regulated genes. In this work, we
characterize the half-site bound by both Pbx1 and Hox proteins on a prototypic element
(TGATTAAT) and determine how the orientation of the Hox protein contributes to
the DNA binding specificity of Pbx-Hox heterodimers. We demonstrate that the Hox protein binds the 3
'
TAAT sequence as its recognition core and exhibits sequence-specific binding at positions 3
'
to the TAAT core. Unfavored sequences at this position, such as two cytosines,
abrogate binding to the element. The upstream Pbx1 core sequence, TGAT, must immediately juxtapose the Hox core.
This geometry maintains the preference of Hox/HOM-C proteins for a T base at position -1, as T represents the fourth position of the Pbx1 core, and
suggests that this T base is bound by both Pbx1 and Hox proteins, Pbx1 binding
in the major grove and the Hox protein binding in the minor grove. Pbx1 also exhibits base selectivity 5'
to its TGAT recognition sequence.
INTRODUCTION
Homeobox genes regulate cell fate and segmental patterning (
1
-
4
) and encode a conserved DNA binding motif known as the homeodomain (HD;
4
). HD proteins act with great biological specificity in development and their
functional differences are largely mediated by their HDs (
5
-
8
). The vertebrate Hox proteins and
Drosophila
HOM-C proteins are a subset of HD proteins that specify structural development
along the anterior-posterior (A-P) axis (
1
,
3
,
9
) through specific recognition of DNA motifs in target genes (
10
-
13
). Hox and HOM-C proteins share structural and functional features, including the primary
sequence of their HDs, the chromosomal organization of their respective genes
and their spatial and temporal expression patterns along the A-P axis (
1
,
3
,
9
). Mutations in these genes induce specific segments to develop morphological
structures of another.
Despite the large number of different biological functions of homeoproteins,
most HDs bind DNA motifs containing a TAAT core (
12
,
14
-
16
). Within the three [alpha]-helices and flexible N-terminal arm that comprise the HD (
17
), basic residues at positions 3 and 5 bind the 5' TA, and Asp51 and a hydrophobic residue at position 47, both of which
lie in helix 3, bind the 3' A and T bases of the core, respectively (
18
,
19
). Residues in helix 3 make sequence-specific contacts 3' of the TAAT core that permit different HD proteins to discriminate
among related binding sites. When residue 50 is Lys, two consecutive cytosines
are strongly favored immediately 3' to the TAAT core (
20
,
21
), but when residue 50 is Gln two guanines are favored. Residue 50 independently
designates this specificity, because interconversion of Gln and Lys at position
50 in Bicoid or in Hox/HOM-C proteins interconverts DNA binding specificity between TAATGG and TAATCC
(
20
,
21
).
While all Hox/HOM-C proteins encode Gln at residue 50, they maintain different intrinsic
specificities for sequences positioned up to four bases 3' to the TAAT core. For example, Ubx binds TAAT
GGCT
with a 6-fold slower off-rate than TAAT
CGAC
and activates transcription 50-fold better on the TAAT
GGCT
element (
14
). By contrast, Dfd binds TAAT
GGCT
with only a 50% slower off-rate than for TAAT
CGAC
and activates transcription only 2.6-fold better via the TAAT
GGCT
element. This difference is due to the fact that Ubx prefers a C three bases 3' to the TAAT core, while Dfd prefers an A, and this specificity is
intrinsic to helix 3 (
14
). Differential binding mediated by the unstructured N-terminal arm (
14
,
22
) creates binding specificity at positions 5' to the TAAT core, however, the magnitude of these effects are smaller
than those that occur for bases 3' to the TAAT and the major effect is limited to the identity of bases at
the -1 position (
14
,
23
).
Members of the Pbx/Exd family of HD proteins cooperate with Hox proteins to
regulate target gene expression and bind DNA cooperatively with many Hox and
HOM-C proteins (
24
-
28
). The Pbx/Exd family includes the human proto-oncoprotein Pbx1 (
29
-
30
), its mammalian homologs, Pbx2 and Pbx3 (
31
),
Drosophila
Extradenticle (Exd;
32
) and
Caenorhabditis elegans
ceh-20 (
33
). The importance of Pbx1 in differentiation is also implicated by the fact that
E2A-Pbx1, an oncogenic and transcriptionally activated derivative of Pbx1
found in human pre-B cell leukemia (
29
,
30
), can block normal hematopoietic differentiation (
34
-
36
). Cooperative binding of Pbx and Hox proteins was first described on TTGATTGAT
(
26
,
37
), which was derived from selection experiments using degenerate
oligonucleotides and recombinant Pbx proteins. Heterodimer formation requires
only the HD and adjacent C-terminal 17 residues of Pbx1 (
38
) and only the HD and N-terminal sequences sufficient to include an essential pentapeptide
sequence (YPWMR) of Hox proteins (
25
,
39
,
40
). Because transfer of the Hox pentapeptide sequence onto the HD of Pbx1 causes
two molecules of Pbx1 to exhibit cooperative binding to TTGATTGAT, this element
was likely initially isolated due to binding of Pbx1 as a dimer (
38
). Thirty two genes of the Hox loci that are either known or proposed to exist
(A1-A8, B1-B8, C1-C8 and D1-D8) encode HD proteins containing variants of this
pentapeptide sequence and thus are predicted to be able to heterodimerize with
Pbx1 on appropriate DNA elements. In addition, the HD protein STF-1, which is not encoded by the Hox loci and is expressed in the pancreas
and small intestine, also contains a pentapeptide sequence (
41
) and binds TTGATTGAT cooperatively with Pbx1 (
42
).
Elements similar to TTGATTGAT are bound by Hox/Pbx heterodimers
in vivo
and are required for expression of tissue-specific genes. The Hox B1 promoter contains TGATGGAT and AGATTGAT
elements that are essential for stage-specific and tissue-specific expression and bind Hox B1 and Exd (Table
1
;
27
). In the somatostatin promoter a Pbx-STF-1 heterodimer binds TGATTAAT (
42
), a site important for expression of somatostatin in pancreatic islet-cells (
41
). This element can also be activated synergistically by STF-1 and E2A-Pbx1 (
42
). Cooperative DNA binding by Pbx and Hox proteins raises the question of how
each heterodimer partner is oriented on its half-site, whether these half-sites are separable and whether heterodimer formation alters the DNA
binding specificity of Hox proteins. In this work, we address these issues.
.
DNA probes used in these studies, including numerical designations (a comparison
of these sequences to known promoter elements is listed at the bottom)
We demonstrate that the Hox protein binds to these elements by utilizing the 3' TGAT or TAAT sequence as its recognition core, that its HD assumes the
same orientation on DNA as that of Hom-C proteins and that it exhibits sequence-specific contacts at positions 3' to sequences currently considered to comprise such elements.
The upstream Pbx1 core sequence, TGAT, must be immediately juxtaposed to the
Hox core. This geometry maintains the preference of Hox/HOM-C proteins for a T residue at position -1 (
14
) because T represents the fourth position of the Pbx1 core. Pbx1 also exhibits
additional 5' base selectivity. We suggest that an internal portion of the Pbx-Hox element is bound by both partners, Pbx1 approaching from the
major grove and the Hox protein binding in the minor grove.
MATERIALS AND METHODS
Construction of recombinant plasmids
All cDNAs were cloned in vectors pGEM 3Z F
-
, pGEM 4Z or pGEM 3Z (Promega).
Mutagenesis
Residue 50 of the HD of Hox A5 and Hox B8 cDNAs was changed to Lys using the
Muta-gene Phagemid
In Vitro
Mutagenesis kit (BioRad) according to the manufacturer's protocol. All
mutations were verified by DNA sequence analysis.
In vitro
transcription/translation
In vitro
transcription/translation was performed using the Promega TNT Coupled
Reticulocyte Lysate System in accordance with the manufacturer's protocol.
Electrophoretic mobility shift assays (EMSA)
Double-stranded oligonucleotides were labeled with [
32
P]ATP to the same specific activities by phosphorylating a common reverse
oligonucleotide that was annealed to the oligonucleotides shown in Table
1
and made double-stranded by addition of dNTPs and the Klenow fragment of DNA polymerase I.
Bound and free probe were separated by electrophoresis in native 5% acrylamide
gels formed in 0.5* TBE (27 mM Tris, 27 mM boric acid, 0.6 mM EDTA) and run in the same
buffer. Binding reactions contained 20 000 c.p.m. probe, 4 [mu]l
in vitro
translated proteins, 1 [mu]g poly(dI[middot]dC), 10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 0.1%
Nonidet P-40 and 5% glycerol. Reactions were incubated for 30 min at 23oC. For Hox A5 and Hox B8 proteins containing point mutations at
residue 50 of the HD, equivalent amounts of wild-type and mutant proteins were used in binding reactions, as determined by
labeling during transcription/translation reactions using [
35
S]methionine.
RESULTS
Sequence-specific binding of Pbx1-Hox heterodimers suggest that the Hox protein binds the 3
'
TAAT motif
The half-sites bound by Pbx1 and Hox proteins on a single element have only been
partially characterized. DNase I protection analysis suggests that Pbx1 is
bound 5' to Hox proteins (
43
). Mutation of the 5' G in TGATTGATT to A abrogates cooperative DNA binding, while mutation of
the 3' G to A maintains cooperativity, consistent with the proposal that Pbx1
binds a 5' half-site containing TGAT and that the Hox protein binds a 3' half site consisting of either TGAT or TAAT. On each
proposed half-site these core nucleotides are numerically designated 2-5, and for the combined Pbx/Hox site that we propose they are
designated 2-5 and 6-9 for Pbx1 and Hox proteins, respectively (Table
1
). Hox proteins exhibit negative selection for CC at the two positions 3' to their TAAT core and positive selection for G or T at the first 3' base (6 in the monomeric site) and either G or A at the second 3' base (7 in the monomeric site;
14
,
15
,
23
). Therefore, if the Hox protein binds the 3' TAAT or TGAT core in the Pbx1-Hox heterodimer site, positions 10 and 11 should prove crucial for
strong binding of this element by either Hox monomers or Hox/Pbx heterodimers.
To develop a model system in which we could observe the effect of sequence
alterations on the binding of both monomeric Hox proteins as well as Pbx1-Hox heterodimers, we selected the Pbx/STF-1 binding site contained in the somatostatin promoter (TGATTAAT),
which contains an optimal TAAT core. Another TAAT core sequence is encoded by
the opposite strand, however, this sequence is followed by a CA dinucleotide
which creates an unfavorable Hox monomer binding site. If changes at positions
10 and 11 of the combined Pbx1/Hox binding site altered both Hox and Hox-Pbx1 binding, it would be very unlikely that the Hox protein was bound to
the reverse TAAT core because nucleotides located 3-4 bases 5' of a TAAT site have little to no effect on Hox binding. Due to
the co-migration of the Pbx-Hox-DNA complex with a non-specific complex produced by the binding of a factor
in reticulocyte lysate, we used the larger E2A-Pbx1 fusion form of Pbx1 for these experiments. E2A-Pbx1 contains all the determinants required for cooperative DNA
binding with Hox proteins and, in a previous study, E2A-Pbx1 and Pbx1 were found to exhibit indistinguishable behavior in their
cooperative binding to DNA with Hox B7, Hox B8, Hox A5 and STF-1 (
43
). DNA probes containing GG, TA or CC at positions 10 and 11 exhibited dramatic
differences in their abilities to bind monomers of Hox B7 (lanes 5-7), Hox B8 (lanes 8-10), Hox A5 (lanes 11-13) and STF-1 (lanes 14-16) or heterodimers of E2A-Pbx1 and each Hox protein (Fig.
1
). Like Pbx1, E2A-Pbx1 failed to bind probe in the absence of a Hox protein (lane 1) and
each of the Hox proteins alone failed to form the upper complex in the absence
of E2A-Pbx1 (lanes 2-4). This suggested that the Hox protein bound the 3' TAAT sequence (positions 6-9) in a manner consistent with the sequence-specific DNA binding characteristics of Hox
proteins.
Residue 50 of the Hox HD specifies the identity of bases bound at positions 5
and 6, affirming the proposed position of the Hox protein
In the crystal structures of Eng and Ant, N51 contacts the A at position 4 and
residue 50 makes base-specific contacts at positions 6 and 7 (
17
,
18
). In Bicoid, residue 50 is Lys and specifies CC at these positions, while HD
proteins encoded by the HOM-C/Hox gene contain Gln at this residue (
20
). In order to prove that the Hox protein in the E2A-Pbx1-Hox complex was bound in the hypothesized orientation, Gln50 of Hox
A5 was converted to Lys in order to provide biochemical evidence that this
would alter binding and favor a TAATCC motif (Fig.
3
). Indeed, the K50 mutation in Hox A5 eliminated monomeric Hox A5 and E2A-Pbx1-Hox A5 binding to TGATTAAT
GG
(lane 3 versus 1) and revealed a new ability to bind TGATTAAT
CC
(lane 4 versus 2). The same shift in binding specificity was observed when an
identical analysis was performed with an oligonucleotide probe containing a
TGAT Hox core sequence instead of a TAAT core (lanes 5-8). TGAT is a common half-site variant of Pbx-Hox heterodimer binding sites (Table
1
). Mutation of Gln50 to Lys in Hox B8 produced the same effect on DNA binding
preference as observed with Hox A5 (data not shown). These experiments
demonstrate that when Hox proteins bind DNA elements with Pbx proteins they are
positioned on a 3' half-site in an orientation that permits Q50-specific contacts 3' of the core sequence.
The binding of Pbx1 is influenced by bases 5
'
to the TGAT core
Pbx1 is strongly implicated to bind DNA in the same orientation as the Hox
protein binds its 3' half-site, because fusion of the Hox pentapeptide N-terminal to Pbx1 causes it to bind the internally repeated
sequence TGATTGATT as a strong and cooperative homodimer (
38
). Thus, Pbx1 is likely to bind the upstream half-site core TGAT (bases 2-5), employing N51 to bind the invariant A at position 4
(designations in Table
1
). Because HD proteins such as Ftz, Ubx and Dfd prefer C or T located just 5' of their TAAT core site (
15
), we examined whether Pbx1 exhibited sequence specificity at this location
(position 1 in the Pbx1/Hox site) and whether it was similar or different to
that predicted for Hox proteins (Fig.
4
). Changes at position 1 had a dramatic effect on the degree of cooperative
binding with Hox proteins, A (lanes 2 and 6) being favored over T or G (lanes
1, 4, 5 and 8) and C permitting very little binding (lanes 3 and 7). By
contrast, changes at position -1 had little effect on formation of the complex containing E2A-Pbx1 and Hox B7 or Hox A5, consistent with the observation that the
-1 position has little effect on sequence-specific binding of HD proteins (lanes 9-16).
Spacing of the Pbx1 and Hox half-sites abrogates cooperative DNA binding by Pbx and Hox proteins
The cooperative binding of Pbx and Hox proteins requires an essential
pentapeptide sequence that is positioned variably from the Hox HD (
25
,
39
,
40
). In Hox B8 it resides five amino acids N-terminal to the HD, while in Hox A5 it is positioned 15 residues N-terminal to the HD. Thus, interaction of Pbx1 with this pentapeptide
sequence could be permitted for DNA elements containing spacing between the
Pbx1 and Hox half-sites. Alternatively, precise spacing geometry between Pbx and Hox half-sites may be required in order to achieve formation of the
heterodimer. To test directly whether these defined half-sites can be separated, a T, G or GG sequence was inserted between
residues 5 and 6. The T preserves the Hox preference for T at its position 1
and maintains a possible preference of Pbx1 for T at position 6. All insertions
completely abrogated formation of the E2A-Pbx1-Hox complexes, indicating that the Pbx1 and Hox half-sites cannot be separated (Fig.
5
). Failure to form heterodimers was not due to diminished binding of the Hox
protein, because insertion of a single T actually enhanced the binding of Hox
A5.
DISCUSSION
In this paper we define the binding orientation of Hox proteins in Pbx-Hox complexes and the sequence specificity of each partner at positions 5' and 3' to its half-site. The binding of E2A-Pbx1 and Hox proteins on DNA provides sequence-specific contacts in at least 11
positions (Table
1
and Fig.
6
A). Mutation of Q50 to K50 in Hox proteins altered specificity of positions 10
and 11 to CC and binding analysis on all 16 dinucleotide possibilities mirrored
the specificity of monomeric binding by Hox B7 and Hox B8 and the general
specificity of Hox and HOM-C proteins characterized by others (
14
,
44
-
46
). Thus, the Hox protein binds the TAAT core spanned by positions 6-9 using the same conformation as that used by Ant or Eng, based on
crystallographic studies (
17
,
18
). Dimer formation of Pbx with Hox A5, Hox B7 and Hox B8 on DNA thus permits the
Hox protein to exhibit its major sequence-specific contacts. In this conformation N51 would bind A at position 8 and the N-terminal arm of the HD would contact bases 5-7 (Fig.
6
B). Extension of Pbx1-Hox-DNA interactions to positions 10 and 11 is also consistent with
earlier methylation interference studies, in which a heterodimer of E2A-Pbx1 and Hox A5 exhibited partial protection of guanine 10 and 12 in the
DNA sequence TTGATTGAT
G
C
G
(positions 10 and 12 underlined;
43
). We would predict that all pentapeptide-containing Hox and HOM/C HD proteins that form heterodimers with Pbx
proteins will exhibit this same geometry on DNA.
ACKNOWLEDGEMENTS
This work was supported by Public Health Services grants 2 PO1 CA50528 and
CA56876. MPK is a PEW scholar in the Biomedical sciences. PSK is a fellow of
the Lucille P.Markey Charitable Trust.
