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Mol Cell Biol, June 1998, p. 3502-3508, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom
Received 1 December 1997/Returned for modification 2 February 1998/Accepted 25 February 1998
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The HOX11 homeobox gene was first identified through
studies of the t(7;10) and t(10;14) chromosomal translocations of acute T-cell leukemia. In addition, analysis of Hox11
/ mice
has demonstrated a critical role for this gene in murine spleen
development. A possible mode of in vivo function for the HOX11 protein
in these two situations is regulation of target genes following DNA
binding via the homeodomain, but little is known about how HOX11
regulates transcription in vivo. By performing transcriptional studies
in yeast and mammalian one-hybrid systems, a modular transcriptional
transactivation region at the NH2 terminus of HOX11 has
been functionally dissected from other parts of the protein. This
NH2-terminal region includes the previously identified short conserved Hep motif, which itself activates transcription in
one-hybrid assays. The importance of the NH2-terminal
region for the function of HOX11 in vivo was assayed by activating a HOX11-dependent gene in NIH 3T3 cells. Activation of this gene was
found to be dependent upon an intact homeodomain in HOX11, but maximal
activation was obtained only when the NH2-terminal 50 amino
acids of HOX11 was present, showing that this region of HOX11 is
important for in vivo transcriptional control of a chromosomal target
gene.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Chromosomal translocations in acute
leukemias frequently involve activation of genes encoding proteins
involved in transcription (2, 18, 19). The HOX11
homeobox gene was first identified by cloning the breakpoints of
t(7;10)(q35;q24) and t(10;14)(q24;q11) chromosomal translocations,
found in 5 to 10% of patients with acute T-cell leukemia (5, 9,
13, 15). These translocations result in the HOX11
gene, which is normally found on chromosome 10, band q24, being placed
within the transcriptional control region of the T-cell receptor 
gene on chromosome 14 or the T-cell receptor gene on chromosome
7q35 (13, 29). The abnormal expression of HOX11 in T cells
as a result of these chromosomal translocations in humans is thought to
be a key step in the progression toward malignancy (5, 9, 13,
15), a view reinforced by the demonstration of oncogenic activity
of HOX11 in transplant recipients of MSCV-HOX11-transduced bone marrow
cells (10, 11).
The HOX11 gene belongs to a family of homeodomain-encoding genes which includes the related Hox11L1 and Hox11L2 genes first characterized in the mouse (4). The homeodomain is conserved within this family, and, like other homeodomain proteins, the HOX11 family has been implicated in the regulation of cellular growth and differentiation. In mice, Hox11 is essential for generation of the spleen (3, 21), promoting survival of the splenic precursor cells (3), and null mutation of the Hox11L1 gene results in myenteric neuronal hyperplasia and megacolon (25).
Since the homeodomain is a sequence-specific DNA-binding element, most homeodomain-containing proteins are believed to function through trans regulation (direct or indirect) of specific target genes (16). Consistent with this, HOX11 is a nuclear protein and can bind to DNA in vitro (4, 26) and a fusion protein consisting of HOX11 fused to the GAL4 DNA-binding domain (GAL4-DBD) was found to transactivate various promoters in both yeast and mammalian cells (28). Three distinct domains of HOX11 were found to be necessary for transactivation by this fusion protein (28), namely, the glycine-proline-rich region at the NH2 terminus, the glutamine-rich region at the COOH terminus, and the homeodomain itself. However, the only data which pertain to functional characteristics in non-artificial reporter assays are that the NH2- and COOH-terminal regions of HOX11 appear dispensable for transforming function (11) and that introduction of HOX11 into NIH 3T3 cells results in transcription of a HOX11-dependent gene (denoted Hdg-1 [8]). Therefore, we have assessed regions of HOX11 required for optimal in vivo transactivation of the endogenous Hdg-1 gene. In this stringent assay, we show that deletions in the HOX11 homeodomain (in either the NH2-terminal arm or the third helix) prevent induction of Hdg-1 by HOX11. We also show that the NH2-terminal 50- amino-acid stretch is crucial for optimal function of the HOX11 protein in vivo.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Plasmids. The yeast expression vector for the Y1 fusion protein was constructed by PCR amplification of the HOX11 sequence corresponding to amino acids 252 to 330 and cloning into NcoI-BamHI-digested pAS-CYH2 (a modified version of pAS [6]). This intermediate construct contained a unique KpnI site preceding the HOX11 sequence, and following digestion with NcoI and KpnI, a second HOX11 PCR product corresponding to amino acids 1 to 241 was inserted to create the Y1 expression vector. Y1 protein lacks amino acids 242 to 251 of HOX11, which are replaced by glycine and threonine residues from the introduced KpnI site. Mutants Y2 to Y11 were made by PCR amplification with primers designed to amplify the indicated regions of HOX11 and the Y1 expression vector as the template. Y12 to Y17 were constructed by cloning of annealed oligonucleotides.
Mammalian expression vectors for GAL4-DBD-HOX11 fusions were also made by PCR amplification of the indicated regions of HOX11 and subsequent cloning in frame into BamHI-XbaI-digested pM1 vector (23). Mammalian expression vectors for HOX11, HOX
N50, and HOX263C were
constructed by PCR amplification of the indicated regions of
HOX11 and cloning into the XbaI site of the
pEFBOS vector (17). HOXN50 also contained a methionine
residue as the first amino acid preceding the HOX11 sequence. HOXMHEP
and HOXHEP were also made by PCR amplification of HOX11
sequence with primers designed to introduce mutations into the Hep
motif (indicated in Fig. 3). pEFBOS-HOXH3 was made by PCR with the
yeast expression vector for Y1 as the template. pEFBOS-HOXMDPA was made
as follows. A HOX11 sequence corresponding to amino acids 174 to 330 containing the mutated FPWM motif at amino acids 174 to 177 (changed to
MDPA and introducing a unique BamHI site) was PCR amplified
and cloned into the XbaI site of pEFBOS (clone
pEFBOS-ICMDPA). pEFBOS-ICMDPA contains two XbaI sites;
however, the XbaI site C terminal to the HOX11 sequence can
be blocked by dam methylation, allowing a second HOX11 PCR
product corresponding to amino acids 1 to 173 to be cloned into
XbaI-BamHI-digested pEFBOS-ICMDPA to create pEFBOS-HOXMDPA. The HOXKN mutation was made by using
HOX11 sequences inserted at the
BamHI-XhoI sites of pBluescript KS. Digestion with XbaI and BglII removed the sequence corresponding to
the first 213 codons of HOX11. This was replaced by a
PCR-generated mutant sequence containing an internal deletion of amino
acids 198 to 204. pEFBOS-HOXKN was made by PCR with this pBluescript KS-HOXKN construct as template. The sequences of all the constructs were verified.
Yeast transformation and 
-galactosidase assay.
Yeast
strain Hf7c (Clontech) was grown on yeast extract-peptone-dextrose
(YEPD) plates or in supplemented Sabouraud dextrose (SD) medium and
transformed by the lithium acetate method (22). Yeast cells
were assayed for -galactosidase activity by a modified filter assay
(24).
Transient transfections and CAT assays. Cos-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. Lipofectin reagent (Gibco-BRL) was used for transient transfections. For each 10-cm tissue culture dish, 5 µg of the reporter plasmid pG5EC and 10 µg of each pM1-based expression vector were used. A 1-µg portion of pVP65, expressing a fusion of GAL4-DBD to the VP65 activation domain, was cotransfected with pG5EC as a positive control. Chloramphenicol acetyltransferase (CAT) assays were performed 36 h after transfection.
Extract preparation and Western blotting. Yeast extracts were prepared by sodium dodecyl sulfate extraction (22). Cos-7 and NIH 3T3 cell extracts were prepared with 300 µl of lysis buffer (10 mM HEPES [pH 7.6], 0.25 M NaCl, 0.5% Nonidet P-40, 5 mM EDTA) per 107 cells. Extracts and molecular weight standards (prestained protein molecular weight standards, 14,300- to 200,000-molecular-weight range; Gibco-BRL) were electrophoresed through sodium dodecyl sulfate-15% polyacrylamide gels and transferred to a nitrocellulose membrane (Schleicher & Schuell). The membranes were blocked for 1 h in 5% Marvel (Premier Beverages) solution and incubated with either a monoclonal GAL4-DBD antiserum (Santa Cruz Biotechnology) or a polyclonal HOX11 antiserum (see below). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies prior to visualization of proteins with enhanced chemiluminescence (ECL) detection reagents (Amersham Life Science).
The HOX11 antiserum was made from bacterially synthesized HOX11 protein. The HOX11 coding sequence was cloned into the pET-15b bacterial expression vector (Novagen). Purified His-tagged HOX11 protein was prepared and used for rabbit immunization.Construction of NIH 3T3 clones. NIH 3T3 cells were maintained in DMEM containing 10% fetal calf serum. The cells were cotransfected with linearized pEFBOS-based expression plasmid (10 µg) and pMC1neopolyA selection plasmid (0.5 µg) with Lipofectin reagent. At 24 h after transfection, selective medium (DMEM containing 10% fetal calf serum and 0.5 mg of G418 per ml) was added to the cells. After approximately 12 days in selective medium, G418-resistant clones were picked and screened by Western blotting for expression of HOX11 protein or by genomic PCR for the presence of integrated pEFBOS vector (data not shown).
Northern analysis. Northern analysis was carried out as described previously (20) with 10 µg of total RNA per lane. Probes were labelled by random priming (7). The Hdg-1 cDNA probe was a 517-bp DpnII fragment of the Hdg-1 cDNA, and the mouse ATP synthase (subunit c) cDNA was a 246-bp DpnII fragment (8). Hybridization levels were quantitated with a model 300A computing densitometer (Molecular Dynamics).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The NH2-terminal region of HOX11 activates transcription in yeast. The ability of GAL4-DBD-HOX11 fusion proteins to activate the transcription of a reporter gene was tested in yeast cells. Various HOX11 sequences were fused to GAL4-DBD in the yeast expression vector pAS-CYH2 (a modified pAS [6]) and transformed into the Hf7c yeast strain (containing a lacZ reporter gene with upstream GAL4 DNA-binding sites). Although a fusion of the GAL4-DBD with the complete HOX11 sequence activated the lacZ reporter, the yeast exhibited severe growth problems (data not shown). Thereafter, we used a basic construct which contains a deletion of the third helix in the HOX11 homeodomain. This clone resulted in a detectable fusion protein with no obvious effect on yeast growth and which was still capable of transcriptional activation (Fig. 1, Y1).
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The first 50 amino acids of HOX11 can activate transcription in mammalian cells. The ability of the first 50 amino acids of HOX11 to function independently as an activation domain was confirmed in mammalian cells. Sequences corresponding to amino acids 1 to 98 of HOX11 or amino acids 1 to 50 were fused to the GAL4-DBD in the mammalian expression vector pM1 (23) (to create pM1-N98 and pM1-N50, respectively [Fig. 2]). These plasmids were transfected into Cos-7 cells together with the pG5EC reporter construct (23). While the parent vector pM1 did not activate the reporter gene, transfection of either pM1-N98 or pM1-N50 resulted in similar levels of activation of the reporter (Fig. 2A). Therefore, the first 50 amino acids of HOX11 can function independently as a transcriptional transactivation domain in mammalian cells.
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The HOX11 Hep sequence can facilitate transcriptional activation in
both yeast and mammalian cells.
Examination of the amino acid
sequence of the HOX11 NH2-terminal region for motifs which
might be important in transcription did not reveal anything other than
the previously noted Hep motif (9). The Hep sequence is
found in several homeodomain proteins and is related to the octapeptide
sequence found in many Pax proteins (1). An assessment of
its role in transactivation by HOX11 was made by preparing an internal
deletion of the Hep sequence in the expression clone pM1-N50 for
transfection of Cos-7 cells. This analysis (Fig. 2A) showed that the
deletion mutant (pM1-N50HEP) was significantly reduced (10-fold
[Fig. 2C]) in its ability to activate transcription of the
cotransfected reporter plasmid. This diminution of function apparently
occurs as a direct result of the Hep deletion and is not an effect of
protein expression levels (as judged by CAT activities normalized to
detectable protein levels [Fig. 2B; a quantitation is given in Fig.
2C]).
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Maximal activation of the endogenous Hdg-1 gene by HOX11 in NIH 3T3 cells requires the NH2-terminal 50 amino acids. The one-hybrid, fusion protein approach used above is artificial in both the context of the mutants used (i.e., fusion with the GAL4-DBD) and the transcription-responsive assay used (i.e., a transient-reporter assay). A more physiologically relevant system would be the test of mutagenesis on HOX11 functions in vivo, via activation of a gene known to be regulated by HOX11. Such a gene has recently been identified (denoted Hdg-1) by cDNA representational difference analysis (8). Hdg-1 is upregulated in NIH 3T3 cells stably expressing HOX11 protein, and this provides an in vivo assay for HOX11 functional domains. NIH 3T3 cells were stably transfected with expression vectors coding for normal HOX11 proteins or mutant forms. NIH 3T3 clones which expressed mutant HOX11 proteins were isolated (Fig. 4A), and protein expression was confirmed by Western blotting with a polyclonal anti-HOX11 antiserum (Fig. 4B and C, upper panels).
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H3) (Fig. 4B and C, lower panels,
and Fig. 4D), while deletion of the NH2-terminal arm of the
homeodomain (also required for contacting DNA [16])
resulted in low levels of Hdg-1 expression (HOXKN) (Fig.
4B and C, lower panels, and Fig. 4D). The effects seen with these
mutants indicate that an intact homeodomain is required by HOX11 to
induce Hdg-1 expression, most probably therefore acting
through DNA binding (although not necessarily binding to the
Hdg-1 gene itself).
In relation to the NH2-terminal region of HOX11, which we
found to be a modular activation domain, a deletion of the
NH2-terminal 50 amino acids of HOX11 (HOXN50) (Fig. 4A)
was found to impair Hdg-1 induction. Although HOXN50
and HOX11 protein levels were comparable (Fig. 4B and C, upper panels),
HOXN50 induced lower levels of Hdg-1 RNA than did
wild-type HOX11 (Fig. 4B and C, lower panels). Densitometric
quantitation of induced RNA levels show an approximately fourfold
reduction of Hdg-1 with HOXN50 (Fig. 4D). The
NH2-terminal 50 amino acids of HOX11 therefore appears to
be important for the optimal induction of Hdg-1 in NIH 3T3 cells, and residual levels of transcription observed after HOXN50 expression are presumably mediated by other regions of the HOX11 protein.
The role of the Hep sequence in induction of Hdg-1
expression was also assessed in NIH 3T3 clones expressing two different Hep mutants (HOXMHEP and HOXHEP [Fig. 4A]). HOXMHEP contains three
amino acid substitutions in the HOX11 Hep sequence (a mutation which
impairs the ability of the isolated Hep sequence to activate in yeast
[Y14] [Fig. 3A]), and this protein induces Hdg-1
expression at levels 2.5-fold lower than HOX11 (Fig. 4C, lower panel,
and Fig. 4D). However, the second Hep mutant, HOXHEP (containing an
internal deletion of the Hep motif) produced clonal differences. Although HOXHEP was present (as judged by Western analysis [Fig. 4C, upper panel]), some NIH 3T3 clones of HOXHEP expressed levels of Hdg-1 RNA comparable to levels induced by wild-type HOX11
(HOXHEP clones 4 and 5 [Fig. 4C]), while others did not appear to
express Hdg-1 at all (HOXHEP clones 1, 2, 3, and 6 [Fig.
4C]). No clonal variation was seen for any other HOX11 protein
studied. It is possible that for HOXHEP, conditions of growth or
specific cellular environment will explain this variation, and this is
worthy of a separate investigation. However, the present data indicate
that the Hep sequence plays a role in the transcriptional activation of
Hdg-1 mediated by the NH2-terminal region of
HOX11 in vivo.
Cooperative interactions between homeodomain proteins and Pbx/exd
proteins appear to be mediated, at least in part, by a motif, FPWM,
just upstream of the homeodomain (16). However, mutation of
this motif at amino acids 174 to 177 (HOXMDPA [Fig. 4A]) had no
effect on the ability to induce Hdg-1 expression (Fig. 4B, lower panel, and Fig. 4D). Therefore, if HOX11 does require interaction with a cofactor for Hdg-1 induction, other regions of the
protein must be involved. In addition, the COOH-terminal region of
HOX11, which was previously thought to contribute to the HOX11
activation potential (28), does not appear to be required
for Hdg-1 induction. A deletion of 67 amino acids from the
COOH terminus of HOX11 (HOX263C [Fig. 4A]) had no effect on
Hdg-1 induction (Fig. 4B, lower panel, and Fig. 4D).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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NH2-terminal activation domain and the Hep sequence. HOX11 is assumed to be an important transcriptional regulator, both in the context of genes required for spleen development in the embryo and following deregulation of the HOX11 gene by chromosomal translocations, for genes involved in the development of T-lineage tumors. It is therefore important to identify regions of HOX11 required for activation of HOX11-dependent genes in vivo. The results presented here show that the HOX11 protein can activate endogenous gene expression (directly or indirectly) in a homeodomain-dependent manner and that an activation domain at the NH2 terminus of the protein is important for this.
The NH2-terminal activation domain was initially delineated by the yeast one-hybrid assay. All fusion proteins containing the NH2-terminal 50 amino acids of HOX11 activated, including a fusion of amino acids 1 to 211 of HOX11 (data not shown), analogous to the fusion clone used in a two-hybrid assay (12). This mapping of an activation domain to the NH2-terminal 50 amino acids of HOX11 differs from the results of a previous study which identified multiple regions of HOX11 involved in transactivation, including an NH2-terminal glycine-proline-rich region of about 190 amino acids (28). In our yeast one-hybrid analysis, the glycine-proline-rich region does not appear to be important, with activation function being localized discretely to the NH2-terminal 50 amino acids. Within the NH2-terminal 50 amino acids of HOX11 is a conserved Hep sequence for which no function has yet been assigned. Our data support a possible role for the Hep sequence in transcriptional activation. First, it appears to be a necessary component of the NH2-terminal activation region, and second, the Hep sequence itself is sufficient to support activation when used in isolation in one-hybrid assays, with contributions from conserved hydrophobic residues in the core sequence. In addition, the Hep motif of Hlx and the related octapeptide motif of Pax-2 are both capable of activation when fused to the GAL4-DBD. Previously, the Pax-2 octapeptide had been shown to act in cis as a transcriptional repressor domain (14). The Pax-2 octapeptide therefore appears to have the capacity for modular transcriptional function, being capable of repression when studied in the context of the Pax-2 protein (14) and activation when studied in isolation. The Hep sequence may also act in both positive and negative transcriptional regulation.Regions of HOX11 required for Hdg-1 induction. It has become apparent that protein activation domains mapped in reporter assays may not necessarily be important for all in vivo function; e.g., studies of the GATA-1 transcription factor have shown that an obligatory activation domain mapped by standard reporter assays is dispensable for GATA-1 function in terminal erythroid cell differentiation (27). The relevance of transcriptional assays which identify protein segments with activation potential in isolation from the intact native protein is therefore at issue. Although the NH2-terminal 50 amino acids of HOX11 could function as an activation domain in one-hybrid assays, it was therefore important to evaluate the role of this region in a functional assay that provides a stringent analysis of HOX11 protein function. In particular, such an assay could provide insight into the differing results obtained from one-hybrid analyses of HOX11 (reference 28 and data presented above). The gene Hdg-1 is activated after HOX11 expression in NIH 3T3 cells (8), and this chromosomal activation provides a powerful physiological model for analysis of HOX11 functional domains. Whether the induction of Hdg-1 expression is a direct effect of HOX11 on the Hdg-1 promoter is unknown. Mapping of the Hdg-1 promoter to identify the "HOX11-responsive element" is in progress to clarify this (8a).
The induction of Hdg-1 expression in transfected NIH 3T3 cells was found to be dependent on the intact homeodomain. When the NH2-terminal 50 amino acids of HOX11 was deleted, Hdg-1 induction was significantly impaired (fourfold), in agreement with mapping of this region as an activation domain in one-hybrid reporter assays. The NH2-terminal 50 amino acids of HOX11 is therefore important for in vivo transcriptional control of a chromosomal gene. The Hep sequence, located within the NH2-terminal 50 amino acids of HOX11, may play a role in this activation of Hdg-1 transcription. However, since regions of HOX11 other than the NH2 terminus contribute to Hdg-1 induction, the in vivo role of the Hep sequence is difficult to assess. If the Hep sequence is an important component of the NH2-terminal region, one would expect Hep mutants to exhibit a reduced level of Hdg-1 induction comparable to that of the HOXN50 mutant. Consistent with this, the HOXMHEP mutant
(containing three amino acid substitutions within the conserved Hep
sequence) activates Hdg-1, with the induced level close to
that of HOXN50 (Fig. 4D).
Although the NH2-terminal 50 amino acids is important for
Hdg-1 induction, other regions of the HOX11 protein are
clearly involved. The homeodomain is essential, since deletions within this domain (either of the third helix or of the
NH2-terminal arm) result in the loss of Hdg-1
induction. Both these regions of the homeodomain are predicted to
contact DNA, and so deletions made presumably result in a loss of HOX11
DNA-binding activity. The HOX11 homeodomain has also been suggested to
contain activation function (28) and may therefore be
playing a dual role in Hdg-1 induction. In our yeast
one-hybrid analysis, we were unable to study the intact HOX11
homeodomain due to growth retardation in the presence of the functional
homeodomain, and it is therefore impossible for us to address this
question unless specific mutations which ablate the activation function
of the homeodomain while retaining DNA-binding activity can be
identified. However, the COOH-terminal glutamine-rich region of HOX11,
which previously had been thought to contain activation function
(28), did not activate in our yeast one-hybrid analysis and
also does not appear to be required for Hdg-1 induction.
Mechanism of Hdg-1 induction. The importance of the NH2-terminal 50 amino acids of HOX11 for activation of a chromosomal gene in vivo may provide insights into the mechanisms of HOX11-mediated transcriptional regulation by helping to identify interacting factors. Apart from the conserved Hep sequence, the NH2-terminal 50 amino acids does not resemble any known transcriptional regulatory domain. However, since this region is involved in Hdg-1 induction (but not necessarily by direct binding to the Hdg-1 promoter), it presumably interacts, either directly or indirectly, with a component(s) of the basal transcriptional machinery. The identification of such factors should further delineate the role of the conserved Hep sequence and help to elucidate the functional role of HOX11 in both splenogenesis and tumorigenesis.
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ACKNOWLEDGMENTS |
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N.M. was supported by an LRF fellowship, and W.G. was supported by a C. J. Martin fellowship.
We thank A. Forster for important technical help throughout this project.
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FOOTNOTES |
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* Corresponding author. Mailing address: MRC Laboratory of Molecular Biology, Hills Rd., Cambridge CB2 2QH, United Kingdom. Phone: 1223-402286. Fax: 1223-412178. E-mail: thr{at}mrc-lmb.cam.ac.uk.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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