From the Department of Pediatrics and the
§ Division of Hematology/Oncology, Northwestern University
Medical School, Developmental Systems Biology, Children's Memorial
Institute for Education and Research, Children's Memorial Hospital,
Chicago, Illinois 60614
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ABSTRACT |
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Three proteins have been identified in mammals,
GLI, GLI2, and GLI3, which share a highly conserved zinc finger domain
with Drosophila Cubitus interruptus and are believed to
function as transcription factors in the vertebrate Sonic
hedgehog-Patched signaling pathway. To understand the role GLI
plays in the Sonic hedgehog-Patched pathway and mechanisms
of GLI-induced transcriptional regulation, we have characterized its
transcriptional regulatory properties and contributions of specific
domains to transcriptional regulation. We have demonstrated that GLI
activates expression of reporter constructs in HeLa cells in a
concentration-dependent manner through the GLI consensus
binding motif and that a GAL4 binding domain-GLI fusion protein
activates reporter expression through the GAL4 DNA binding site.
GLI-induced transcriptional activation requires the carboxyl-terminal
amino acids 1020-1091, which includes an 18-amino acid region highly
similar to the -helical herpes simplex viral protein 16 activation
domain, including the consensus recognition element for the human TFIID
TATA box-binding protein-associated factor TAFII31 and
conservation of all three amino acid residues believed to contact
directly chemically complementary residues in TAFII31. The
presence of this region in the GLI activation domain provides a
mechanism for GLI-induced transcriptional regulation.
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INTRODUCTION |
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Cubitus interruptus
(Ci)1 functions as a
transcription activator in the Drosophila hedgehog-patched
signaling pathway to mediate hedgehog signaling specifying
segmentation of Drosophila embryos and patterning
of imaginal-disk outgrowth (1, 2). Drosophila cAMP response
element-binding protein (CREB)-binding protein functions as a
coactivator of Ci (2). Three homologous proteins have been identified
in mammals, including GLI, GLI2, and GLI3, which share a highly
conserved C2-H2 zinc finger domain with Ci and are believed to function as transcription factors in the vertebrate Sonic hedgehog-Patched signaling pathway, each potentially
carrying out a specific function during development (3-6). Specific as well as redundant functions have been demonstrated for GLI2 and GLI3
during mammalian skeletal development, whereas GLI but not GLI3 has
been shown to activate the HNF-3 enhancer in tissue culture (7, 8).
Sonic hedgehog-Patched signaling specifies polarity of limb
outgrowth, polarity of the developing neural tube, and somite
patterning and mutations in genes in this pathway may relate to
understanding causes of human basal cell carcinoma, holoprosencephaly,
Pallister-Hall syndrome, Greig syndrome, malignant gliomas, and
sarcomas (9-16). Because the Gli family zinc finger domain has been
shown to mediate DNA binding to the 9-bp consensus motif GACCACCCA, it
is believed that GLI, GLI2, and GLI3 bind identical or similar DNA
sequences, and understanding the specificity of function and their
specific roles in the Sonic hedgehog-Patched signaling
pathway will require an understanding of their transcriptional regulatory properties and the mechanisms responsible for their transcriptional regulation (17-19).
To begin to understand the function of GLI in the Sonic
hedgehog-Patched signaling pathway and its mechanism of action, we have studied its transcriptional regulatory properties in tissue culture and identified contributions of specific domains to
transcriptional regulation. We find that GLI activates reporter gene
transcription through the SV40 promoter and the E1b promoter in a
concentration-dependent manner. A transcription activation
domain is identified at the carboxyl terminus of the protein which
includes an 18-amino acid acidic -helix highly similar to the herpes
simplex viral protein 16 (VP16) transcription activation domain, which
targets TAFII31 and Drosophila
TAFII40 (20-22). A similar domain is present in the other
Gli family proteins. The presence of a VP16-like activation domain in
GLI provides a mechanism for transcriptional regulation.
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EXPERIMENTAL PROCEDURES |
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GLI Expression Vectors and Reporter Constructs for Cotransfection
Experiments--
pCMV-GLI was prepared by inserting the
full-length human GLI cDNA into the
HindIII/XbaI site of the pcDNA3 plasmid
(Invitrogen). pCMV-GLI()TAD was prepared by deleting a
1,409-bp AccI fragment from the 3
-end of GLI.
pCMV-GLI(
)AT was prepared by removing a 661-bp
HindIII/XhoI fragment from the 5
-end of
GLI. pM-GLI was prepared by inserting a
HincII/XbaI fragment of the human GLI
cDNA into the SmaI/XbaI site of the pM
plasmid (CLONTECH). pM-GLI515-1106 was
prepared by inserting an SmaI/XbaI fragment of
human GLI cDNA into the SmaI/XbaI
site of the pM plasmid (CLONTECH). pM-GLI1-210 was constructed by inserting polymerase chain
reaction-amplified human GLI cDNA corresponding to amino
acids 1-210 into the EcoRI/XbaI site of the pM
plasmid. pM-GLI78-686 was constructed by removing the
AccI fragment from the pM-GLI vector.
Preparation of Nested Deletions and DNA Sequencing-- DNA subfragments of pM-GLI515-1106, representing serial deletions from the carboxyl terminus, were prepared using exonuclease III/S1 nuclease digestion (23). The DNA sequence of these cDNA clones was determined using the dideoxy chain termination procedure (24).
RNase Protection Assays-- Total RNA was isolated using Tri reagent (Molecular Research Center, Inc.). 10 µg of total RNA was incubated with 1 × 105 cpm of the antisense probe. After overnight hybridization at 45 °C, unhybridized RNA was digested with an RNase A/T1 mixture. Protected RNA fragments were analyzed on a 5% denaturing polyacrylamide gel and were visualized by autoradiography.
In Vitro Translation--
In vitro translation of
pCMV-GLI was completed using nuclease-treated reticulocyte
lysate (Ambion Inc.). In vitro translation of
pCMV-GLI()TAD and pCMV-GLI(
)AT were completed
using wheat germ extract (Promega). The newly synthesized proteins were
separated on 10% SDS-polyacrylamide.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared from 2-4 × 107 cells, and the protein
concentration of each extract was determined (25, 26). 5 µg of each
nuclear extract was mixed with 2.5 µl of 2 × binding buffer
(20% glycerol (v/v), 20 mM MgCl2, 200 µM EDTA, 1 mM dithiothreitol, 100 mM KCl, 40 mM HEPES, pH 7.9, 100 µg/ml
poly(dI-dC), and 20 µM ZnSO4),
H2O, and 0 or 1 µl (1.75 pmol) of unlabeled competitor oligonucleotide (27). The mixture was incubated on ice for 5-10 min. 1 µl (8.75 fmol) of the double-stranded 32P-labeled probe
(sense strand: 5-GATCTAAGAGCTCCCGAAGACCACCCACAATGATGGTTGTATGT-3
) was added, and the mixture was incubated on ice for an additional 30 min. The samples were analyzed by electrophoresis, and retarded bands
were visualized by autoradiography.
Cell Culture and Transfections--
A total of 4 µg of DNA was
transfected in each cotransfection experiment using the GLI DNA binding
domain-containing reporters, including 1 µg of reporter plasmid, 1 µg of pSV--galactosidase (Promega), or pSV-luciferase (Promega)
transfection efficiency control plasmid, 0-1 µg of effector plasmid,
and pBluescript carrier DNA (Stratagene) in an amount to make up the
difference. For the mammalian one-hybrid experiments using the
pM-GLI-expressing effector constructs and the pG5CAT
reporter construct, a total of 4 µg of DNA was transfected in each
experiment, including 0.4 µg of reporter plasmid, 0.4 µg of
pSV-
-galactosidase (Promega) transfection efficiency control
plasmid, 0-3.125 µg of pM-GLI-expressing effector plasmid, and pBluescript carrier DNA (Stratagene) in an amount to make
up the difference. In each case the DNA was mixed with 9 µl of
LipofectAMINE reagent in 0.3 ml of Opti-MEM (Life Technologies, Inc.),
incubated for 45 min at room temperature, and then added to individual
60-mm plates containing 2 ml of Opti-MEM and 4 × 105
cells. Cells were incubated for 5 h in a CO2 incubator
before the Opti-MEM was replaced with HeLa culture media. Cells were incubated for 48 h, and lysates were prepared for CAT assays and
-galactosidase assays (Promega).
CAT Assays--
CAT assays were performed as described by the
manufacturer (Promega) with minor modifications. CAT activity was
quantitated by scintillation counter and was normalized by measuring
-galactosidase activity spectrophotometrically (Promega) or by
measuring luciferase activity (Promega) with a luminometer.
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RESULTS |
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GLI Functions as a Transcription Activator--
HeLa cells were
cotransfected with a GLI-expressing effector construct
(pCMV-GLI) and a CAT reporter (pCAT-GLI44()E),
under the control of the SV40 promoter, containing two tandem copies of
a previously identified GLI DNA binding motif,
GATCTAAGAGCTCCCGAAGACCACCCACAATGATGGTTGTATGT (17). This
binding motif, which includes a 9-bp consensus sequence (underlined)
identified in all three of the previously identified GLI DNA binding
motifs, was selected because it may contain critical binding elements
absent from the consensus 9-bp sequence. GLI mRNA was
demonstrated in the transfected cells and in the positive control
GLI- amplified D259 MG glioblastoma multiforme cell line using RNase protection analysis (Fig.
1A, lanes 1-5). No
evidence for GLI mRNA expression was found in HeLa cells
or in HeLa cells transfected with the control plasmid pcDNA3 (data
not shown). Because GLI3 has been shown to bind to the identical site
as GLI and may compete with GLI for binding sites, reverse
transcription-polymerase chain reaction was used to look for
GLI3 expression in HeLa cells. No GLI3 expression
was found in HeLa cells (data not shown). A predicted 150-kDa GLI
protein was produced off the pCMV-GLI effector using
in vitro translation (Fig. 1B). Gel mobility
shifts were used to demonstrate binding of the GLI protein to the GLI
DNA binding motif in the reporter. Nuclear protein extract isolated from HeLa cells, which do not express GLI, demonstrated no mobility shift (data not shown). Nuclear protein extract isolated from the
positive control cell line D259 MG and HeLa cells transfected with
pCMV-GLI demonstrated two shifted bands using the 44-bp GLI DNA binding motif present in the reporter construct as a probe (Fig.
1C). As increasing amounts of the GLI-expressing effector construct were cotransfected with a constant amount of the
CAT-containing reporter construct (pCAT-GLI44(
)E), CAT
activity increased more than 6-fold in a
concentration-dependent manner (Fig. 1D). To test additionally for the possibility of transcriptional repression by
GLI, pLTR-GLI was cotransfected with pCAT-GLI44.
pCAT-GLI44, which includes the SV40 enhancer, was selected
because it has higher basal CAT activity than
pCAT-GLI44(
)E, and repression might be detected more
easily from the higher baseline activity. Using pCAT-GLI44,
pLTR-GLI demonstrated only transcriptional activation with
no evidence of transcriptional repression (data not shown).
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Identification of the GLI Transcription Activation Domain--
To
understand better the mechanism of GLI-induced transcriptional
activation, experiments were conducted to identify a transcription activation domain. First, 470 amino acids were deleted from the carboxyl terminus of the GLI protein (pCMV-GLI()TAD), and
220 amino acids were deleted from the amino terminus of the GLI protein (pCMV-GLI(
)AT) (Fig.
3A). GLI(
)AT and
GLI(
)TAD mRNA were demonstrated in HeLa cells
transfected with the pCMV-GLI(
)AT and
pCMV-GLI(
)TAD effector constructs using RNase protection
analysis (Fig. 1A, lanes 6 and 7).
In vitro translation was carried out to demonstrate production of a predicted 120-kDa GLI(
)AT protein off the
pCMV-GLI(
)AT effector construct and a 100-kDa
GLI(
)TAD protein off the pCMV-GLI(
)TAD construct (Fig. 3B). Binding of the truncated proteins to
the GLI DNA binding motif in the reporter was demonstrated using gel mobility shift assays (Fig. 3, C and D). Nuclear
protein extract isolated from HeLa cells transfected with
pCMV-GLI(
)TAD demonstrated two specific shifted bands.
pCMV-GLI(
)AT, however, consistently showed only a single
shifted complex (Fig. 3D). Cotransfection of HeLa cells with
pCMV-GLI(
)TAD and pCAT-GLI44(
)E resulted in
decreased transcriptional activation compared with wild type GLI (Fig.
3E). In contrast, cotransfection with
pCMV-GLI(
)AT and pCAT-GLI44(
)E resulted in a
6-fold increase in transcriptional activation compared with controls, a
level of activation comparable to wild type GLI (Fig. 3F).
The data suggest that pCMV-GLI(
)AT may be more active than
wild type GLI at lower concentrations.
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The GLI Transcription Activation Domain Is Similar to the VP16
Activation Domain--
The region from 1020 to 1091 includes a
negatively charged -helical 18-amino acid stretch (amino acids
1037-1054) containing six aspartate or glutamate residues. The
-helical region demonstrates 50% similarity with the VP16
-helical transcription activation domain including conservation of
the FXX
(F = phenylalanine; X = any residue;
= any hydrophobic residue) general recognition element
of acidic activation domains for TAFII31 and conservation of the three residues (Asp472, Phe479, and
Leu483 in VP16, and Asp1040,
Phe1048, and Leu1052 in GLI) that are believed
to make direct contacts with TAFII31 (22) (Fig.
5). The region surrounding the GLI
-helix is proline-rich. Human GLI3 and Drosophila
Ci demonstrate conservation of the FXX
consensus
element and two of the three residues that are believed to make direct
contact with TAFII31, whereas mouse gli2 demonstrates conservation of the FXX
consensus element and one of
the three residues believed to make direct contact with
TAFII31.
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DISCUSSION |
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We have demonstrated that the oncodevelopmental gene
GLI functions as a transcriptional activator of the
heterologous promoters SV40 and E1b in HeLa cells through the GLI
consensus binding motif and the GAL4 consensus binding motif
respectively, in a concentration-dependent manner. As
increasing amounts of GLI were transfected into HeLa cells
greater CAT activity was seen. No evidence for transcription repression
was found. In-frame deletion of amino acids 686-1106 from the carboxyl
terminus of the GLI protein resulted in reduced transcriptional
activation by GLI with retention of DNA binding. This same region drove
CAT activity using a GAL4 fusion protein approach. The smallest segment
identified by our studies, which was necessary for GLI-induced
transcriptional activation, included amino acids 1020-1091. Indeed,
Ruppert et al. (28) had suggested previously that the
carboxyl-terminal acidic -helical region of GLI including amino
acids 1037-1055 may represent a transcription activation domain based
on the fact that 6 of 19 amino acids in this region are aspartate or
glutamate. Acidic activation domains of VP16, p53, and p65 have been
shown to target TAFII31, and each of these acidic
activation domains includes an FXX
motif felt to
represent the TAFII31 consensus recognition element (22). The FXX
motif is conserved in the GLI transcription
activation domain. The VP16 activation domain undergoes an induced
transition from a random coil to an
-helix upon binding to its
target protein TAFII31, and three residues believed to make
direct contacts with TAFII31 have been identified (22). The
three residues (Asp1040, Phe1048, and
Leu1052) are conserved in the GLI transcription activation
domain. These structural motifs identified in a region that we
demonstrate to function as the GLI transcription activation domain
suggests that the mechanism for GLI-induced transcriptional activation
involves TAFII31 binding. Substitution of alanine for two
of the residues believed to make direct contacts with
TAFII31 in the VP16 activation domain (Phe479
and Leu483) has been shown to reduce TAF binding affinity
and transcriptional activation (22). Substitutions in this region in
the different Gli family members may affect their TAF binding and
transcriptional regulatory properties. Conservation of
Asp1037 and Gln1035 in each of the mammalian
Gli family members in this region may be of significance for TAF
binding.
It is of interest that the VP16-like domain lies within a proline-rich
region at the carboxyl end of the GLI protein. 20% of the amino acid
residues in this region (amino acids 752-1081) are prolines. A number
of transcription factors contain proline-rich domains that appear to
function as transcription activation domains, and the proline-rich
region could contribute to transcriptional regulation by GLI (29-31).
We were able to eliminate the majority of the proline-rich domain,
however, and maintain the transcriptional activation properties of GLI,
suggesting that the VP16-like acidic -helical domain rather than the
proline-rich domain represents the critical transcriptional activation
domain of GLI.
Transcriptional activation was retained following in-frame
deletion of amino acids 2-220 from the amino terminus of the GLI protein. At low effector concentrations pCMV-GLI()AT
appeared to even enhance transcriptional activation relative to
pCMV-GLI although overall achieved a comparable level of
transcriptional activation compared with full-length
pCMV-GLI. Interestingly, amino acids 68-135 have been
identified previously as a region of similarity between Gli family
proteins showing 66% amino acid identity between GLI (amino acids
68-135) and GLI3 (amino acids 279-348) (28). Included within this
region of GLI (amino acids 67-82) is a conserved domain that has been
shown to be functionally significant in Caenorhabditis
elegans TRA-1 and has been called the GF or "gain of function"
region. Mutations in this region abolish negative regulation of TRA-1
activity (32). Based on the TRA-1 data, conservation of the region in
GLI and GLI3, and our current functional experiments, it is possible
that the "GF region" at the amino-terminal end of GLI and its
immediate vicinity represents an inhibitory domain in GLI as well. Also
unique to GLI(
)AT was the fact that it consistently demonstrated a
single shifted complex on gel mobility shift assay compared with wild type GLI, which consistently demonstrated two shifted bands. Two shifted protein complexes may result from partial proteolysis, differing states of protein phosphorylation or glycosylation, or
possibly from protein interactions. Removal of the AT region may affect
any of these processes.
It has been suggested that mammalian Gli family members function in the
Sonic hedgehog-Patched pathway similarly to Ci in the
Drosophila hedgehog-patched signaling pathway (15, 33). Decapentaplegic (dpp) represents one of the downstream
targets of Ci in this pathway (33). It is believed that low
concentrations of Ci repress dpp expression and high
concentrations of Ci induce expression of dpp with Ci acting
as a repressor or activator of transcription depending on its
concentration (33). Recently it has been demonstrated that the 155-kDa
Ci protein may be cleaved, resulting in a 75-kDa form that includes the
amino terminus through the zinc finger domain but lacks the entire
carboxyl end, which includes the transcription activation domain (34).
This 75-kDa form of Ci then functions as a transcription repressor.
Using our in vitro assay conditions we can provide no
evidence that GLI functions as a transcription repressor over a wide
range of concentrations. It is possible that the three mammalian Gli
genes, GLI, GLI2, and GLI3 function
cooperatively to carry out comparable functions to ci in the
mammalian Sonic hedgehog-Patched signaling pathway. Indeed,
it has been shown recently that GLI activates the HNF-3 enhancer
whereas GLI3 represses the HNF-3
enhancer (8). Differences in TAF
binding or binding affinity could potentially contribute to these
different properties. Alternatively it is possible that the three
mammalian genes have evolved to add increased layers of complexity to
transcriptional regulation in the mammalian Sonic
hedgehog-Patched signaling pathway.
Transcription factors are generally considered to be modular, made up
of several functional domains. The GLI protein represents an
interesting example with each functional module conserved through different evolutionary lines. The GLI protein contains a
conserved zinc finger domain found in Drosophila
ci and C. elegans tra-1, a conserved 3-untranslated
region translation control element conserved in C. elegans
tra-2, and now a conserved transcription activation domain found
in herpes simplex virus VP16 (35).
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ACKNOWLEDGEMENTS |
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We thank K. Kinzler for supplying the full-length human GLI cDNA (pLTR-GLI) and the polyclonal GLI antibody.
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FOOTNOTES |
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* This work was supported by Public Health Service Grants CA-64395 from the NCI and HD-28992 from the NICHHD, National Institutes of Health, and by the Ronald McDonald House Charities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Children's Memorial Hospital, Division of Hematology/Oncology Box 30, 2300 Children's Plaza, Chicago, IL 60614.
1 The abbreviations used in this paper are: Ci, Cubitus interruptus; bp, base pair; VP, viral protein; TAF, TATA box-binding protein-associated factor; CMV, cytomegalovirus; TAD, transcription activation domain; AT, amino terminus; CAT, chloramphenicol acetyltransferase.
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REFERENCES |
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