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Human Molecular Genetics Advance Access originally published online on June 30, 2005
Human Molecular Genetics 2005 14(15):2181-2188; doi:10.1093/hmg/ddi222
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

A previously unidentified amino-terminal domain regulates transcriptional activity of wild-type and disease-associated human GLI2

Erich Roessler1,{dagger}, Alexandre N. Ermilov2,{dagger}, Dorothy Katherine Grange3, Aiqin Wang2, Marina Grachtchouk2, Andrzej A. Dlugosz2 and Maximilian Muenke1,*

1Medical Genetics Branch, NHGRI, NIH, Bethesda, MD 20892-3717, USA, 2Department of Dermatology and Comprehensive Cancer Center, University of Michigan, Ypsilanti, MI, USA and 3Department of Pediatrics, Genetics and Genomic Medicine, Washington University, USA

* To whom correspondence should be addressed at: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, 35 Convent Drive—MSC 3717, Building 35, Room 1B-203, Bethesda, MD 20892-3717, USA. Tel: +1 3015947487 or 3014028167, Fax:+1 3014807876; Email: mmuenke{at}nhgri.nih.gov

Received May 9, 2005; Revised June 8, 2005; Accepted June 16, 2005

GenBank accession no. DQ086814


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Zinc finger-containing Gli proteins mediate responsiveness to Hedgehog (Hh) signaling, with Gli2 acting as the major transcriptional activator in this pathway in mice. The discovery of disease-associated mutations points to a critical role for GLI2 in human Hh signaling as well. Here, we show that human GLI2 contains previously undescribed 5' sequence, extending the amino-terminus an additional 328 amino acids. In vitro, transcriptional activity of full-length GLI2 is up to 30 times lower than that of GLI2{Delta}N (previously thought to represent the entire GLI2 protein), revealing the presence of an amino-terminal repressor domain in the full-length protein. GLI2{Delta}N also exhibits potent transcriptional activity in vivo: overexpression in mouse skin leads to the formation of Hh-independent epithelial downgrowths resembling basal cell carcinomas, which in humans are associated with constitutive Hh signaling. The discovery of this additional, functionally relevant GLI2 sequence led us to re-examine several pathogenic human GLI2 mutants, now containing the entire amino-terminal domain. On the basis of the functional domains affected by the mutations, mutant GLI2 proteins exhibited either loss-of-function or dominant-negative activity. Moreover, deletion of the amino-terminus abrogated dominant-negative activity of mutant GLI2, revealing that this domain is required for transcriptional repressor activity of pathogenic GLI2. Our results establish the presence of an amino-terminal transcriptional repressor domain that plays a critical role in modulating the function of wild-type GLI2 and is essential for dominant-negative activity of a GLI2 mutant associated with human disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Properly regulated Hedgehog (Hh) signaling plays crucial roles in a variety of conserved embryonic processes underlying human malformation syndromes, whereas uncontrolled Hh pathway activation is associated with several neoplasms (1Go,2Go). In humans, genetic perturbations in Hh signaling are associated with numerous congential malformations or syndromes, such as holoprosencephaly, crainiofacial clefting associated with pan-hypopituitarism and polydactyly, Greig Syndrome, Pallister-Hall Syndrome, several distinct polydactyly malformations as well as a cancer predisposition syndrome, Gorlin's Syndrome. Human malignancies linked to deregulated Hh signaling include basal cell carcinoma, medulloblastoma, small cell lung cancer, several gastrointestinal malignancies and prostate cancer. Therefore, continued analysis of the Hh signaling pathway is essential to our understanding of the pathogenesis of these conditions.

During physiologic Hh signaling, secreted Hh proteins bind and antagonize the receptor Ptch on target cells, allowing the activation of the signaling effector Smo (3Go). Smo operates through several cytoplasmic proteins to bring about changes in gene expression via Gli transcription factors. In Drosophila, the single Gli orthologue Ci functions as either a transcriptional repressor or Hh-dependent activator (4Go), whereas in higher eukaryotes, Gli activity reflects the combined repressor and activator functions of all Gli proteins (Gli1, Gli2 and Gli3) present within the cell (5Go,6Go). Detailed analysis of various mouse mutants has led to several conclusions regarding Gli protein function in vivo: Gli1 is not required for normal Hh signal transduction; Gli2 acts primarily as a transcriptional activator but may have some repressor activity and Gli3 generally functions as a repressor but can be a weak activator in certain cellular contexts [(7Go) and references therein]. In general, Gli2 appears to be the primary transcriptional activator mediating Hh responses in mice (8Go–16Go). In contrast, Gli1 appears to be the primary effector in zebrafish, with Gli2 playing minor roles (17Go), indicating functional divergence of Gli protein function among vertebrate species.

Both the basal level of Gli transcriptional activity and its modulation during Hh signaling are controlled by functional domains that have been characterized using a variety of experimental approaches. All wild-type Gli proteins contain carboxy-terminal activation domains and a more centrally located zinc finger DNA-binding domain (Fig. 1A). Ci and Gli3 (mouse and human) also contain amino-terminal domains that are involved in transcriptional repression in several physiological settings. Thus, in the absence of Hh, Ci functions as a potent transcriptional repressor due to proteolytic cleavage and accumulation of protein with intact DNA-binding zinc fingers and amino-terminus, but missing the carboxy-terminal transactivation domain (18Go). Similarly, Gli3 appears to exist primarily as a truncated repressor missing the carboxy-terminus, with transcriptionally active full-length Gli3 accumulating only in cells responding to a Hh signal (19Go–21Go). In contrast to Gli3, mouse and human Gli1 proteins do not contain amino-terminal repressor domains and appear to function only as transcriptional activators.



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  		Figure 1. Functional domains of Gli proteins and human GLI2 gene structure. (A) All full-length Gli proteins contain a carboxy-terminal domain required for transcriptional activity and a zinc finger domain which binds DNA in a sequence-specific manner. Mouse and human Gli3 contain an amino-terminal domain, not present in Gli1 of either species, which is involved in transcriptional repression. Note discrepancy between mouse Gli2 and previously described human GLI2, now designated GLI2*, showing presence of an amino-terminal repressor only in the mouse protein. Complete human GLI2 protein described in this report (dashed box) contains an amino-terminal repressor functionally homologous to that described in mouse Gli2. (B) The generally accepted human GLI2{alpha} isoform (now designated GLI2* or GLI2{Delta}N) consists of eight coding exons (1–8, in red) preceded by two non-coding exons (–1 and 0) as well as 98 bp identical to the HTLV-1 Tax gene. This HTLV-1 sequence is not present in normal genomic DNA and presumably arose by retroviral insertion in the HTLV-1 adult T-cell leukemia cell line (HUT102) from which all six GLI2/THP isoforms were isolated. Hypothetical translation of the coding region predicts a 1258 amino acid protein lacking an NH2-terminal repressor domain [GLI2* in (A)]. (C) RT–PCR analysis of commercial poly-A RNA (Clonetech) using primers anchored in exons 0 and 1 identified a primary product that encodes four additional exons (blue) highly homologous to the mouse Gli2 gene and extends the human GLI2 reading frame by 328 amino acids. The inclusion of these four exons shifts the predicted initiator methionine to exon 0, similar to the amino-terminus of the mouse and other vertebrate genes. A revised full-length form of human GLI2 containing an amino-terminal MYC-tag in pCS2 was generated and used in all subsequent studies.

 
Gli2, like Gli3, appears to be capable of functioning either as a transcriptional activator or repressor, depending on cellular context and species. Although reliable antibodies for detecting endogenous Gli2 protein are not available, carboxy-terminal deletion mutants of mouse Gli2 have been created which mimic the naturally occurring repressor forms of Gli3 and Ci in their ability to block transcriptional activity (22Go). Moreover, processing of full-length mouse Gli2 to a putative transcriptional repressor form has been reported both in cultured cells (23Go) and in vivo, following ectopic expression in Drosophila (24Go). On the other hand, genetically engineered deletion of the mouse Gli2 amino-terminus leads to a constitutively active protein that induces target gene expression in a Hh-independent manner (15Go,22Go). Together, these data point to an important regulatory function for the Gli2 amino-terminus. In the absence of a Hh signal, full-length Gli2 is a relatively weak transcriptional activator; in cells responding to Hh, proteolytic removal of the amino-terminal repressor, or its functional inactivation via another mechanism, appears to be required for full Gli2 activator function.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Interestingly, although the human GLI2 cDNA has been reported in previous studies (25Go,26Go), sequence encoding an amino-terminal repressor domain was not described (Fig. 1A and B). We now report the cloning of previously unidentified GLI2 sequence encoding an additional 328 amino-terminal amino acids. Functional characterization of this domain reveals that it operates as a repressor of wild-type GLI2 function and is required for dominant-negative activity of disease-associated human GLI2.

Isolation of the full-length human GLI2 cDNA
As recent database entries suggested that additional GLI2 sequence might exist, we directly examined this question by RT–PCR using poly-A RNA (Clontech) from normal individuals. As described in Fig. 1C, we detected four additional coding exons that extend the previously described human GLI2 protein by 328 amino acids, which are highly homologous to the amino-terminal repression domains of Gli2 genes from other vertebrate species. For the sake of clarity, we will refer to previously described human GLI2 as GLI2* or GLI2{Delta}N, to reflect the finding that amino-terminal sequence was not included in these earlier reports.

Comparisons between full-length GLI2 and GLI2{Delta}N
We performed luciferase-based reporter assays in cultured cells to compare the transcriptional activity of GLI2* (GLI2{Delta}N) with that of full-length GLI2 or a full-length chimera containing the amino-terminus of mouse Gli2 (m/hGLI2) (Fig. 2A). Although GLI2 and m/hGLI2 efficiently stimulated reporter activity, they were substantially (up to 30-fold) less active than GLI2{Delta}N in assays using three different Gli-responsive reporters (Fig. 2 and data not shown). Similar results were obtained in mesenchymal C3H 10T1/2 cells (Fig. 2B) and epithelial WT7 keratinocytes (Fig. 2D and E). The amino-terminus of human GLI2 thus appears to contain a domain with transcriptional repressor activity similar to that previously described in mouse Gli2, and mouse and human Gli3 (26Go). As an independent measure of Gli activity, Hh pathway-mediated induction of the osteogenic marker alkaline phosphatase was measured in cultures of C3H 10T1/2 cells. In keeping with the results of luciferase-based reporter assays, GLI2{Delta}N was much more effective than GLI2 or m/hGLI2 at inducing alkaline phosphatase activity in this bioassay (Fig. 2C). In addition, transgenic mice expressing GLI2{Delta}N in skin produced basal cell carcinoma-like downgrowths originating directly from epidermis (Fig. 2F), indicating that this protein is capable of activating cell proliferation in a Hh-independent manner similar to mouse Gli2{Delta}N and activated SMO but not full-length Gli2 (15Go,27Go,28Go).



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  		Figure 2. Transcriptional activity of full-length and amino-terminal truncated GLI2. (A) GLI2 proteins used for assays in (B–F). GLI2* or GLI2{Delta}N is used to designate what was previously considered full-length GLI2. m/hGLI2 is a chimera containing the amino-terminal repressor from mouse Gli2 (hatched box) fused to GLI2{Delta}N. (B, D, E) Luciferase-based reporter assays using indicated Gli-responsive constructs (see Materials and Methods) in C3H 10T1/2 fibroblasts and epithelial cells (WT7 keratinocytes). Transcriptional activity of GLI2{Delta}N measured using an 8xGliBS-luc reporter (B and D) is up to ~30-fold higher than that of full-length GLI2 or full-length chimeric m/hGLI2. A Gli-responsive K17 reporter (35Go) showed less striking differences in activity of GLI2 proteins but negligible baseline activity (E) (data not shown). (C) Expression of the Hh/Gli-inducible osteogenic differentiation marker alkaline phosphatase in C3H 10T1/2 cells was markedly higher in response to transfection with GLI2{Delta}N than GLI2 or chimeric m/hGLI2. (F) K5-GLI2{Delta}N transgenic mice (lower panel) develop numerous basal cell carcinoma-like epithelial downgrowths originating directly from the epidermis. The same length of skin from a control littermate (upper panel) contains a single hair follicle.

 
Analysis of mutant forms of full-length GLI2
Our previous studies had identified mutations in the human GLI2 gene associated with ventral craniofacial, pituitary, limb and/or brain anomalies (26Go), but functional analysis of these mutants was performed with GLI2 proteins missing the unidentified amino-terminus. Given the importance of this domain in wild-type GLI2 activity (Fig. 2), we re-examined the behavior of these pathogenic human GLI2 mutants compared to wild-type GLI2, this time including the complete amino-terminus (Fig. 3). We included an additional GLI2 mutant, designated mutB in this paper, which we cloned from a previously described family (29Go) with multiple clinical features (Fig. 4) in common with other patients with GLI2 mutations (26Go).



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  		Figure 3. Predicted domain structure and activity of wild-type GLI2 and GLI2 variants from patients with craniofacial, pituitary, limb and/or brain anomalies (26Go,29Go). (A) On the basis of data in this report, summary depicting structure and activity of wild-type GLI2 and GLI2 mutants. mutB was isolated from a family that was previously described (29Go) as having bilateral post-axial polydactyly, cryptorchidism, microphallus, empty sella on CT scan and pan-hypopituitarism. The remaining GLI2 mutants were described in our earlier study (26Go) (original plasmid 2=mutA in this report; plasmid 5=mutC; plasmid 8=mutD; plasmid 16=mutE; plasmids 4,15=mutF), but not previously studied in full-length form. (B and C) Transcriptional activity assays using the K17-luc Gli reporter (35Go) and indicated GLI2 plasmids transfected into WT7 keratinocytes alone (B) or in combination (C). Modest transcriptional activity of mutA compared with full-length GLI2 (B), with undetectable activity of mutB–F. (C) Gli reporter activity in cells cotransfected with equal amounts of wild-type GLI2 and either control plasmid (bar 2), wild-type GLI2 plasmid (bar 3) or mutant GLI2 plasmids (bars 4–9). MutB and mutC exhibited dominant-negative activity (compare bars 5 and 6 with bar 2). With the exception of mutA, cotransfection of the remaining GLI2 mutants with wild-type GLI2 had either no detectable activity or significantly lower activity than cotransfection with wild-type GLI2 (compare bars 7–9 with bars 2 and 3).

 


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  		Figure 4. A three generational family segregating a truncating CAG to TAG mutation (3768C>T, see mutB) in human GLI2. Individual II-2 was patient 2 in a previous clinical report describing the association of polydactyly and hypopituitarism (29Go). He exhibited bilateral post-axial polydactyly (blue), well-developed extra digits with bone and nails, pan-hypopituitarism (orange), empty sella on CT scan at 2 years of age, bilateral cryptorchidism and microphallus. His brother and father manifested post-axial polydactyly (in blue) without other significant findings. His nephew (III-5) was born at 35 weeks and displayed pan-hypopituitarism (orange) and bilateral post-axial polydactyly (blue) at birth. Aplasia of the pituitary was confirmed by MRI scan. He had neonatal respiratory distress, surgical repair of a patent ductus arteriosus and microphallus with small testes. His endocrine problems responded well to hormone replacement. The asterisk (*) denotes the heterozygous 3768 C>T sequence change.

 
We first examined the intrinsic transcriptional activity of each mutant (Fig. 3B). With the exception of mutant mutA which is missing exon 5 (a theoretical alternatively spliced form not demonstrated experimentally), transcriptional activity of GLI2 mutants was undetectable using two different Gli-responsive reporters (Fig. 3B and data not shown). The residual activity of mutA is likely due to the fact that this is the only mutant possessing an intact carboxy-terminal transactivation domain. The reduced activity of mutA, compared with wild-type GLI2, is likely the result of impaired DNA-binding properties (30Go) due to a potential splicing event which removes Zinc finger 4 but retains the proper reading frame encoding Zinc finger 5. In keeping with the results of our reporter assays, GLI2{Delta}NmutA (plasmid 2 in our previous report) was also a weak activator of skin tumor formation in frog embryo injection studies (26Go). Collectively, the loss of transcriptional activity of all GLI2 mutants missing an intact carboxy-terminus (Fig. 3A) supports the concept that this region contains an essential transcriptional activator.

MutB and mutC have dominant-negative properties
We next performed cotransfection studies to examine the overall Gli activity when GLI2 mutants were expressed together with wild-type GLI2. Responses fell into two general classes: strong, dominant-negative inhibition of wild-type GLI2 activity and modest stimulatory activity compared with GLI2 alone (Fig. 3A and C). Only those mutants with intact zinc fingers 4 and 5 (mutB and mutC), previously implicated in efficient DNA binding (30Go), exhibited potent GLI2 inhibitory activity. mutD did not appreciably alter Gli reporter activity when cotransfected with wild-type GLI2 (Fig. 3C, bar 7), suggesting that this mutant is inactive; whereas cotransfection of mutA led to an ~2-fold higher activity levels (Fig. 3C, bar 4), similar to those obtained with wild-type GLI2+ wild-type GLI2 cotransfection (Fig. 3C, bar 3). Interestingly, cotransfection of mutE or mutF with wild-type GLI2 (Fig. 3C, bars 8 and 9) led to modestly enhanced reporter activity compared with an equivalent amount of GLI2 alone (Fig. 3C, bar 2). Because these mutants have no intrinsic transcriptional activity (Fig. 3B) and are unlikely to bind DNA due to the absence of zinc fingers 4 and 5 (Fig. 3A), these results suggest that altered forms of GLI2 can influence wild-type GLI2 function indirectly, perhaps by competing with cellular factors involved in modulating repressor-domain function. Transmodulation of other GLI proteins by mutant GLI2 could account for the unanticipated finding of polydactyly, normally associated with heightened Hh signaling activity, in several of our families (26Go,29Go). The inability of mutD to enhance Gli activity may be related to the fact that it retains zinc fingers 1–4 and may therefore have sufficient DNA-binding activity to exhibit a low level of transcriptional repression.

Dominant-negative activity resides in the amino-terminus of GLI2
The results of these cotransfection experiments were in striking contrast to findings we previously reported using the amino-terminally truncated versions of these mutants, none of which exhibited dominant-negative activity (26Go). We performed additional cotransfection assays to directly test whether the newly described repressor domain is involved in dominant-negative activity of disease-associated human GLI2 mutants. Cotransfection of wild-type GLI2 with mutC resulted in a dose-dependent inhibition of Gli reporter activity, with a >90% inhibition when equal amounts of the two effector plasmids were used (Fig. 5B). In striking contrast, cotransfection of wild-type GLI2 plasmid with {Delta}NmutC plasmid (missing the amino-terminal repressor), resulted in a negligible reduction in reporter activity compared with wild-type GLI2 plasmid alone. Immunoblot analysis confirmed that the loss of dominant-negative activity was not due to reduced expression of {Delta}NmutC protein compared with wild-type GLI2. On the contrary, {Delta}NmutC accumulated to levels several-fold higher than either wild-type GLI2 or mutC (Fig. 5C).



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  		Figure 5. Dominant-negative activity of pathogenic GLI2 is dependent on the amino-terminus. (A) Domain structure of wild-type and mutant GLI2 proteins. mutC is missing the carboxy-terminal transactivation domain but contains an intact zinc finger DNA-binding domain and amino-terminal repressor; {Delta}NmutC is also missing the amino-terminus. (B) Cotransfection studies showing dose-dependent inhibition of wild-type GLI2 activity by mutC. Removal of the amino-terminal repressor in {Delta}NmutC leads to nearly complete loss of Gli inhibitory activity, despite the presence of an intact DNA-binding domain. (C) Immunoblot analysis verifying the expression of MYC-tagged wild-type GLI2 and GLI2 mutants in lysates used for reporter assay in (B), normalized for ß-galactosidase. A relatively low level of mutC is needed for potent dominant-negative inhibition of wild-type GLI2 activity (lane 5). Despite the robust expression of {Delta}NmutC (lane 6), absence of the amino-terminal repressor in this mutant leads to loss of dominant-negative activity [bar 6 in (B)].

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our studies reveal a strong inhibitory effect of the newly described GLI2 amino-terminus on transcriptional activity of wild-type GLI2 and establish that this domain is essential for dominant-negative activity of a human GLI2 mutant associated with developmental abnormalities in humans (26Go). These results support the notion that the Gli2 amino-terminus has a conserved role in repressing transcriptional activity in humans, as it does in other vertebrates. They also provide an explanation for the unexpectedly high activity of human GLI2 (actually GLI2{Delta}N) reported in previous expression studies (31Go,32Go).

Currently, haploinsufficiency for GLI2 is associated with disease in three families, whereas two additional families also show dominant-negative properties in the mutated GLI2 protein, reducing transcriptional activity even further. Although the number of identified cases is too small for extensive genotype–phenotype correlations, the variable penetrance is striking, in particular the presence of polydactyly in isolation. Even mice with targeted disruption of Gli2 do not have digit abnormalities, unless Gli1 is also deleted. Furthermore, mice heterozygous for targeted disruption of Gli2 are reportedly healthy and fertile. This emphasizes both the similarities and the differences between vertebrate species in their relative utilization of the different Gli proteins.

On the other hand, our findings raise the interesting possibility that gain-of-function alleles of GLI2 also exist, in which pathogenic mutations may lead to loss of transcriptional repressor activity and consequent enhancement of Gli function. It now will be possible to perform mutational analysis of the GLI2 repressor domain in selected human cancers to determine whether somatic changes are associated with tumorigenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Sequencing, molecular cloning and clinical studies
Once the direct sequencing of the RT–PCR product cloned into pCR2.1 (Invitrogen) established the full-length GLI2 reading frame, a replacement cassette was designed with a unique 5' EcoR1site and the 3' unique Fse1 site within exon 2. This cassette was sub-cloned into the wild-type human GLI2 construct in pCS2(MT) as well as mutant forms previously described (26Go). An additional new GLI2 mutant (mutB in Fig. 3) was identified in a three generation family segregating apparent autosomal dominant hypopituitartism and/or post-axial polydactyly (29Go). All available affected individuals in each of three generations carried a heterozygous 3768 C>T mutation causing premature termination (revised full-length gene) at amino acid residue 1256. This truncated protein encodes an intact amino-terminus and Zinc finger domain but lacks an intact carboxy-terminal activator domain. All constructs were verified by direct sequencing using ABI3100 instrumentation (Applied Biosystems, Foster City, CA, USA), protocols and reagents (ABI PRISM® Big DyeTMTerminator version 3.1). Site-directed mutagenesis was performed under contract with Transponics (York, PA, USA) using a minor modification of the TransformerR mutagenesis protocol (Promega). All molecular studies performed on human subjects DNA samples conformed to the ethical guidelines of the NHGRI IRB with informed consent. The full-length human GLI2 cDNA has been assigned GenBank accession no. DQ086814.

Cell culture
C3H 10T1/2 cells were grown in Dulbecco's Minimum Essential Medium (MEM) supplemented with glutamine and 10% fetal bovine serum (FBS) (Invitrogen), in a 37° incubator at 5% CO2. The WT7 keratinocyte cell line arose spontaneously following repeated passaging of epidermal keratinocytes isolated from newborn mice according to standard procedures (33Go). WT7 cells are grown in modified S-MEM (Sigma-Aldrich) containing 10% FBS and 1 ng/ml keratinocyte growth factor (PeproTech). FBS for keratinocyte culture was a mixture of Ca2+-depleted FBS (33Go) and untreated FBS, in proportions yielding a final medium Ca2+ concentration of 0.05 mM for optimal keratinocyte growth.

Reporter assays
Lipofectamine plus (Invitrogen) was used according to the manufacturer's guidelines for all transfections, which were performed in six-well plates. When testing transcriptional activity of individual GLI plasmids, 0.7 µg of effector plasmid was used; when cotransfecting two GLI plasmids, we used 0.4 µg of each. Reporter plasmids, used at 0.4 µg/well, included 8xGliBS-luc and m8xGliBS-luc (34Go); BCL2prom (1.9 kb) and BCL2prom (cont) (32Go) and K17-luc, which contains 1950 bp of the Gli-responsive Keratin 17 promoter (35Go) cloned into pGL3-basic (Promega). A control plasmid (pSV-lacZ, 0.1 µg) was included in each reaction to normalize for transfection efficiency. Two days after transfection, cells were harvested in CCLR buffer (Promega) and luminescence measured in a Monolight 3010 luminometer. Assay results were normalized to ß-galactosidase activity, determined using a colorimetric assay. For in situ assessment of alkaline phosphatase activity in C3H 10T1/2 cells, cultures were fixed 24 h after transfection and an alkaline phosphatase Vectastain kit (Vector Labs) was used to visualize enzyme activity.

Immunoblot analysis
Immunoblotting studies were performed using cell lysates prepared for luciferase assay, following addition of Laemmli sample buffer, denaturation, SDS–polyacrylamide gel electrophoresis and transfer to PVDF membrane. Lysate volumes were adjusted based on results of colorimetric ß-galactoside activity to normalize for transfection efficiency. Filters were incubated with monoclonal anti-MYC antibody 9E10 to detect MYC-tagged GLI2 proteins and anti-ß-galactosidase antibody as a control. Visualization was performed using secondary antibodies (Jackson Immunoresearch) conjugated to horseradish peroxidase, followed by incubation with ECL reagent (Pierce) for chemiluminscent detection.

Production of transgenic mice
Expression of GLI2{Delta}N was targeted to skin using a transgenic cassette containing 5.3 kb of bovine K5 promoter, which is active in proliferative basal cells in epidermis and several other stratified epithelia and the outer root sheath of hair follicles (36Go). DNA was injected into (C57BL/6 X SJL)F2 oocytes and transferred to pseudopregnant females by personnel in the University of Michigan Transgenic Animal Model Core.


   ACKNOWLEDGEMENTS
 
We are grateful to the families who participated in these studies and the DIR NHGRI for their support (E.R. and M.M.). We thank Drs Pierre Coulombe, Jose Jorcano, Fritz Aberger and Hiroshi Sasaki for providing reagents and Drs Brian Bonish and Mark Hutchin for performing preliminary experiments. We appreciate constructive comments on the manuscript from Drs Sarah Millar, Eric Fearon, Mikhail Nikiforov and Marisol Soengas. Support was provided by NIH grants CA87837 and AR45973 (A.A.D.).

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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