Post-transcriptional Regulation of the GLI1 Oncogene by the Expression of Alternative 5' Untranslated Regions*

Xue-Qing Wang and Joseph A RothnagelDagger

From the Department of Biochemistry and the Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia

Received for publication, June 15, 2000, and in revised form, October 10, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The oncogene GLI1 is involved in the formation of basal cell carcinoma and other tumor types as a result of the aberrant signaling of the Sonic hedgehog-Patched pathway. In this study, we have identified alternative GLI1 transcripts that differ in their 5' untranslated regions (UTRs) and are generated by exon skipping. These are denoted alpha -UTR, beta -UTR, and gamma -UTR according to the number of noncoding exons possessed (three, two, and one, respectively). The alpha - and beta -UTR forms represent the major Gli1 transcripts expressed in mouse tissues, whereas the gamma -UTR is present at relatively low levels but is markedly induced in mouse skin treated with 12-O-tetradecanoylphorbol 13-acetate. Transcripts corresponding to the murine beta  and gamma  forms were identified in human tissues, but significantly, only the gamma -UTR form was present in basal cell carcinomas and in proliferating cultures of a keratinocyte cell line. Flow cytometry analysis determined that the gamma -UTR variant expresses a heterologous reporter gene 14-23-fold higher than the alpha -UTR and 5-13-fold higher than the beta -UTR in a variety of cell types. Because expression of the gamma -UTR variant correlates with proliferation, consistent with a role for GLI1 in growth promotion, up-regulation of GLI1 expression through skipping of 5' noncoding exons may be an important tumorigenic mechanism.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GLI1 was originally isolated as a highly amplified gene in a malignant glioma (1) and subsequently implicated in the development of other tumor types, including liposarcoma, rhabdomyosarcoma, osteosarcoma, and astrocytoma (2, 3). Later it was shown that GLI1 encodes a transcription factor that is a downstream nuclear component of the Sonic hedgehog-Patched signaling pathway (4-6). This pathway is evolutionarily conserved and found to operate in a number of tissues during vertebrate development and especially in regions involving mesoderm-ectoderm interactions (7-10). Intercellular signaling by this pathway is initiated when Sonic hedgehog (a secreted protein) binds to Patched (a cell surface transmembrane protein), resulting in the activation of GLI1 in the nucleus and subsequent expression of target genes. Overexpression of Sonic hedgehog has been shown to up-regulate Gli1 in chick limb buds and in the epidermal ectoderm of frog embryos (11, 12), whereas Gli1 expression is undetectable in Sonic hedgehog null mouse embryos (13, 14), confirming that Sonic hedgehog signaling regulates Gli1 expression.

The discovery of Patched mutations in familial and sporadic forms of basal cell carcinoma (BCC),1 the most common skin cancer, has associated aberrant signaling of the Sonic hedgehog-Patched pathway with the formation of these tumors (15-17). The genetic data are supported by experimental evidence showing that overexpression of Sonic hedgehog and other components of this pathway results in the induction of BCCs in transgenic mice and transgenic human skin (18-20). Overexpression of GLI1 in the epidermis of transgenic animal models produces BCC-like lesions (21, 22) and will transform rodent epithelial cells in cooperation with adenovirus EIA (23), indicating that unregulated expression of GLI1 is oncogenic. Recent studies have shown that GLI1 expression is greatly increased in BCCs but not in the surrounding normal tissue, consistent with a central role in tumor formation (21, 24, 25).

In addition to GLI1, two other isoforms have been identified in vertebrates (termed GLI2 and GLI3), each encoded by a separate gene (8, 10). The GLI genes are highly expressed during development, and their expression profiles correlate with organogenesis but show only low-level expression in most adult tissues (7, 9, 10). In the skin, GLI1 expression is readily observed in the epidermal compartment of the developing hair follicle, whereas GLI2 and GLI3 transcripts are detected in the surrounding mesenchyme (10, 21, 25). The role of each GLI in mediating the Sonic hedgehog signal is not yet clear, but recent gene ablation studies have shown overlapping roles and indicated some functional redundancy (26, 27). A number of studies have indicated that GLI1 is a transcriptional activator, whereas GLI2 and GLI3 can act as both activators and repressors depending on specific post-translational modifications (28-30). Interestingly, GLI2 and GLI3 are now thought to regulate GLI1 transcription directly by binding to the GLI1 promoter (28, 30).

The 5' untranslation region (UTR) has a large influence on translation and plays a key role in post-transcriptional gene regulation (31-36). The efficiency of translation initiation is largely governed by the composition and structure of the 5' UTR of the mRNA, which is determined by both its length and its sequence. Extensive secondary structures and one or two small upstream open reading frames (uORFs) within a 5' UTR can profoundly inhibit protein translation. Most highly expressed mRNAs have relatively short (20-100 nucleotides) 5' UTRs that lack uORFs and extensive secondary structures (for review, see Ref. 31). In contrast, mRNAs encoding oncoproteins, growth factors, transcription factors, and other regulatory proteins are poorly translated and often have long, highly structured 5' UTRs with multiple upstream ATGs (32, 35, 36). In this study, we have identified alternative 5' UTRs of GLI1 transcripts in mouse and human tissues, which are generated by exon skipping and have marked differences in translation efficiency. Until now, post-transcriptional regulation of GLI1 has been inferred (37), but a precise mechanism has not been determined. Our results suggest that post-transcriptional regulation of GLI1 is mediated by exon skipping and show an association of the most efficiently translated 5' UTR transcript with BCC.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), 5' Rapid Amplification of cDNA Ends (RACE), and PCR-- Skin, brain, heart, kidney, liver, lung, muscle, stomach, spleen, testis, and tongue were obtained from various strains of neonatal, juvenile, and adult mice. Total RNA was isolated from these tissues using TRI Reagent (Molecular Research Center). First-strand cDNA was synthesized from 5 µg of total RNA primed with oligo(dT)16 (PerkinElmer) or random hexamers (CLONTECH) using Superscript II reverse transcriptase (Life Technologies) in a total volume of 20 µl. One-tenth by volume of the cDNA was used as the template for subsequent PCR reactions. Primer pairs (listed in Table I) for mouse GLI1 corresponding to sequences within exon 1 (mGliF1) and exon 2 (mGliR2) and for human GLI1 corresponding to exon 1 (hGliF1) and 2 (hGliR2) sequences were used. Each PCR reaction was repeated at least three times with different RNA preparations and included negative controls for each set of reactions. For 5' RACE, we used a cDNA template that was generated using the primer RACE1 and C tailed using terminal transferase according to the manufacturer's protocol (Life Technologies). PCR was performed using the RACE anchor and adapter primers (Life Technologies) and the GLI1-specific nested primer RACE2. Mouse genomic DNA was amplified using primers derived from exon 1a (mGliF1a) and exon 2 (mGliR2) sequences. All PCR products were separated on 0.8-2% agarose gels and visualized with ethidium bromide. Separated fragments were purified using QIAEX II (Qiagen) and sequenced directly using the Big Dye termination kit and automated fluorescent sequencing on an ABI-Prism 377 DNA sequencer (PerkinElmer).

12-O-Tetradecanoylphorbol 13-acetate (TPA) Treatment of Mouse Skin-- Neonatal and 7-day-old mice (Swiss outbred) were treated with 20-50 µl of 100 µg/ml TPA (Sigma) topically applied to back skin. Mice were killed at different time points (0, 3, 8, and 24 h) after application, and total skin RNA was prepared for RT-PCR as described above.

5' UTR-Green Fluorescent Protein (GFP) Constructs and Functional Analysis of 5' UTRs-- Each of the three alternative 5' UTRs of mouse GLI1 was generated by RT-PCR using primers mGliF1Nhe and mGliQR2Age (Table II) that contain NheI and AgeI restriction sites, respectively. PCR products were gel purified and cloned into the pGEM-T Easy vector (Promega). The four ATG codons of the alpha -UTR were mutated sequentially using the QuikChange site-directed mutageneis kit (Stratagene) and complimentary primers mGliMF1-4 and mGliMR1-4 (Table II). The gamma -UTR sequence was multimerized using primers mGliF1Bam and mGliR2Bgl that contain BamHI and BglII sites, respectively. The amplified fragment was cloned into pGEM-T Easy and sequenced, and the insert was released by digestion with BamHI and BglII. The purified fragment was ligated in the presence of both restriction enzymes to form similarly oriented concatomers that were then used as templates for PCR amplification using mGLiF1Nhe and mGliR2Age. The products of this reaction were sized on an agarose gel and the band corresponding to four copies of the gamma -UTR cloned into the pGEM-T Easy vector. All inserts were verified by sequence analysis, released from the pGEM-T Easy vector by restriction digestion with NheI and AgeI, and subcloned into the corresponding sites of the GFP expression vector pEGFP-N1 (CLONTECH).

HaCaT, a human keratinocyte cell line (38), Cos-1 (39), and BHK-21 (40) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, ampicillin, and streptomycin (Life Technologies). Primary mouse skin fibroblasts were obtained from newborn Swiss mice using an established protocol. Briefly, skin was removed, washed in PBS, and incubated in 2.5% dispase (Life Technologies) for 24 h at 4 °C. The dermis was separated from the epidermis and incubated in 0.2% collagenase (Sigma) at 37 °C for 1 h. Cells were then pelleted, washed in PBS, and cultured in Dulbecco's modified Eagle's medium as described above. Primary fibroblasts were used within the first 2 weeks of culturing.

Transient transfection of GFP constructs was performed using LipofectAMINE Plus reagent (Life Technologies) according to the manufacture's instructions. Cells were seeded on round, glass coverslips in 24-well (fluorescence microscope study) or 6-well (flow cytometry study) plates 24 h before transfection and incubated at 37 °C to a density of ~50-70% confluence. Cultures were washed twice in serum-free media and then incubated in DNA-LipofectAMINE Plus complexes in OPTI-MEM (Life Technologies) for 3 h at 37 °C. Dulbecco's modified Eagle's medium containing serum was then added to the culture. One day later, cells were fixed for microscopy or harvested for flow cytometry analysis. Twenty thousand cells per sample were analyzed on a FACSCalibur (Becton Dickinson) cell sorter, using CELLQuest software (Becton Dickinson).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Alternative Mouse GLI1 5' UTRs-- To identify the transcriptional start site of the mouse GLI1 gene, 5' RACE was performed on total skin RNA from a BALB/c mouse, and the resulting PCR product was sequenced directly. This analysis showed that sequences around the translation start codon and at the beginning of the transcript were identical to the published mouse GLI1 sequence obtained from F9 cells (41). However, the RACE product also contained an additional 119 bases, not present in the published sequence, located at the splice junction between exons II and III as numbered by Liu et al. (41). We named this new 5' UTR variant alpha -UTR and the published sequence beta -UTR (Fig. 1A). To search for other possible splicing variants, RT-PCR was conducted using a forward primer located at exon 1 and a reverse primer that hybridizes immediately 5' of the GLI1 ATG codon (Table I). This PCR revealed a much smaller product, which was sequenced directly and found to consist of only one 5' noncoding exon. This alternative mouse GLI1 variant corresponds in both size and sequence to the published human transcript obtained from a glioma cell line (7, 41) and was denoted the gamma -UTR variant (Fig. 1A).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   Sequence, exon composition, and pre-mRNA structure of the alternative 5' UTRs of mouse Gli1. A, sequence alignment of the three alternative GLI1 5' UTR variants (denoted alpha -, beta -, and gamma -UTR) expressed in mouse. The novel 119-base pair sequence of exon 1a is shown in bold lowercase letters. The ATG codons denoting the beginning of uORFs are underlined, and the main ORF encoding Gli1 is shown in bold uppercase letters. The intron-exon boundaries are indicated by arrows. B, schematic showing the exon composition of the alternative 5' UTRs and the organization of the pre-mRNA from which they are derived. Exons are denoted by open boxes, and introns are denoted by solid lines with intron size shown. The translation start site (ATG) of the main ORF is located in exon 2 and indicated by a bent arrow.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Primer sequences 1

To determine how these alternative transcripts were generated, we examined the genomic organization of this region. Mouse genomic DNA was amplified using the primer pair mGliF1a and mGliR2 (Table I). Sequence analysis of the 2.8-kilobase product identified the novel 119-nucleotide sequence within the alpha -UTR variant as an authentic exon (which we denoted exon 1b) that is flanked by 2.5 kilobases (intron Ia) and 115 bases (intron Ib) of intervening sequences (Fig. 1B). The identification of this additional noncoding exon required a change in the nomenclature used in the earlier study by Lui et al. (41), with exon II becoming exon 1a and exon III (which encodes the translation start site) renumbered exon 2 (see Fig. 1B).

Expression of Mouse GLI1 5' UTRs-- The expression of the alternative 5' UTR variants was determined in neonatal, juvenile, and adult tissues from various mouse strains by RT-PCR (Fig. 2). This analysis revealed that the UTR variants had no particular tissue-specific expression pattern but did show marked strain-specific differences. In all tissues examined the larger UTR forms (alpha - and beta -UTR) predominate, whereas the gamma -UTR appears as a minor amplified product (Fig. 2A). In some strains, such as BALB/c, DBA, and C57BL/6, the alpha -UTR variant was the major form, whereas in CD1 and SV129 strains, the beta -UTR form was the predominant transcript (Fig. 2B). In Swiss outbred mice, expression of the alpha - and beta -UTR was heterogeneous, with some animals expressing both forms and others expressing only one (data not shown). In a given individual, the expression profile of the two larger transcripts was identical in all tissues examined irrespective of the strain used (Fig. 2A). We also followed the expression of the UTR variants in postnatal skin development and found that the apparent levels of all GLI1 transcripts were reduced with increasing age and that the gamma -UTR transcript was not detected at all in adult skin (data not shown).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   The expression of alternative GLI1 5' UTRs is not tissue specific but does show strain variation. RT-PCR was performed on mRNA isolated from brain (Br), liver (Li), Lung (Lu), skin (Sk), stomach (St), and tongue (To) tissues of a postnatal mouse (A). A signal for the beta - and gamma -UTR variants is also present in all tissues examined but is barely discernible. B, in BALB/c, DBA, and C57Bl/6 strains, the alpha -UTR variant is the major transcript (BALB/c is shown in lane 1), whereas the beta -UTR variant is the dominant transcript in CD-1 and SV129 strains (CD-1 is shown in lane 2). M, DNA marker.

To evaluate whether the expression of these 5' UTR variants correlated with proliferative status, newborn and 7-day-old animals were treated with TPA topically applied to back skin. These experiments revealed that expression of the larger UTR transcripts was maximally reduced at 3 h after application, whereas expression of the gamma  form was increased (Fig. 3). Reduced expression of the alpha - and beta -UTRs was still evident, albeit not as marked, 24 h after application (data not shown). Because acute TPA treatment results in increased mitotic activity of basal layer keratinocytes (42), these data indicate an association of the gamma -UTR transcript with proliferation and the alpha - and beta -UTRs with differentiation.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 3.   Expression of the GLI1 5' UTR variants is altered by TPA treatment. The expression of the gamma -UTR transcript is increased, whereas expression of the alpha -UTR is reduced, in TPA-treated skin (+) relative to control skin (-). M, DNA size marker.

Identification of Alternative Human GLI1 5' UTRs-- We next searched for alternative human GLI1 transcripts in newborn foreskin by RT-PCR with primers derived from exons 1 and 2 (Table I) of the published sequence (7). Two PCR products were generated and sequenced, with the smaller fragment corresponding to the published sequence (7) and the larger containing an additional 144 bases located at the splice junction of exons 1 and 2 (Fig. 4A). The larger transcript was termed beta -UTR, and the smaller sequence was termed the gamma -UTR variant (Fig. 4, A and B). Notably, the novel 144-nucleotide sequence found within the human beta -UTR has significant homology with mouse exon 1a but is 30 bases larger and contains two additional ATG codons (Fig. 4C).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Sequence, exon composition, and pre-mRNA structure of alternative human GLI1 5' UTRs. A, sequence alignment of the alternative GLI1 5' UTR variants identified in human tissues (denoted beta - and gamma -UTR). The novel 144-base pair sequence of exon 1a is shown in bold lowercase letters. The ATG codons denoting the beginning of uORFs are underlined, and the main ORF encoding GLI1 is shown in bold uppercase letters. The intron-exon boundaries are indicated by arrows. B, schematic showing the exon composition of the alternative 5' UTRs and the organization of the pre-mRNA from which they are derived. Exons are denoted by open boxes, and introns are denoted by solid lines with intron size shown. The translation start site (ATG) of the main ORF is located in exon 2 and indicated by a bent arrow. C, comparison of human exon 1a sequences with mouse exon 1a. *, identical bases.

The beta - and gamma -UTRs were also present in a brain sample, and their identity was confirmed by sequence analysis (Fig. 5). The expression of the beta - and gamma -UTRs was further examined in HaCaT cells and seven BCC samples. We found that the gamma  transcript was present in proliferating cultures of HaCaT cells and all BBC samples, but in contrast to foreskin keratinocytes and brain tissue, we were unable to amplify the beta  transcript from these mRNAs (Fig. 5). Therefore, the gamma -UTR transcript may represent the major variant expressed by proliferating cells in human tissues as well.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5.   Detection of human GLI1 5' UTR variants by RT-PCR. RT-PCR results from skin, brain, the HaCaT human keratinocyte cell line (HaC), two BCC biopsies, and a no-RNA control (dH2O) are shown. M, DNA size marker.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Primer sequences 2

Functional Analysis of the GLI1 5' UTRs-- The 5' UTR is known to regulate gene expression by influencing the efficiency of translation. An examination of the GLI1 5' UTR variants revealed three small uORFs in mouse alpha -UTR, two in human beta -UTR, one that overlaps the GLI1 ORF in the mouse beta -UTR, and none in the gamma -UTRs (Table III). The secondary structure for each 5' UTR was analyzed using the RNA folding prediction program MFOLD (43). This program predicted extensive secondary structures in the longer UTRs, with calculated free energy values of -72.7 to -94.5 kcal/mol for the mouse alpha -UTR, -65 kcal/mol for the human beta -UTR, and -55 kcal/mol for the mouse beta -UTR. The human and mouse gamma -UTRs were predicted not to form stable secondary structures and to have free energy values of -12 kcal/mol (Table III). The presence of uORFs and stable secondary structures in the larger UTRs suggests that they will be less efficiently translated than the gamma  variant, which lacks these features.


                              
View this table:
[in this window]
[in a new window]
 
Table III
Theoretical analysis of GLI1 5' UTRs

To test the above prediction in vivo, the three mouse GLI1 5' UTR fragments were cloned upstream of a GFP reporter gene (Fig. 6A), and the constructs were transiently transfected into HaCaT, Cos-1, and BHK-21 mammalian cell lines and primary mouse skin fibroblasts. The difference in GFP fluorescence produced by these constructs was striking (Fig. 6B), with the alpha -UTR construct producing the lowest fluorescence levels, the beta -UTR construct producing intermediate levels, and the gamma  variant producing the highest levels (even higher than the GFP vector control). Importantly, there was no apparent difference in the transfection efficiency of these constructs for a given cell type, showing that the increase in the number of brightly fluorescing cells transfected with the gamma  form is caused by an increase in GFP production.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   The Gli1 5' UTR variants differentially express a GFP reporter construct in transfected cells. A, diagram showing the GFP constructs used in the transfection studies. The mouse Gli1 5' UTR sequences were cloned upstream of the GFP ORF as indicated. pEGFP-N1, parent GFP construct. B, GFP fluorescence observed in Cos-1 cells transfected with the GFP expression vector alone (GFP) and alpha -UTR-GFP (alpha ), beta -UTR-GFP (beta ), and gamma -UTR-GFP (gamma ) constructs. Transfection efficiency was determined by counting GFP-expressing cells, which revealed that each construct was transfected with the same efficiency (within experimental limits).

To quantify these results, cells were also subjected to flow cytometry analysis (Fig. 7A). The advantages of this technique over enzymic reporter assays are that GFP levels are determined in individual cells using a large number of cells (20,000 cells/construct), and untransfected cells are discarded from the calculations, thus removing any bias attributable to transfection efficiencies. This analysis revealed that the alpha - and beta -UTRs expressed the reporter 60-90% lower than the GFP vector control, whereas the gamma -UTR produced 2-3-fold enhancement of expression (Fig. 7B). Comparison of expression levels between the three variants reveals a 14-23-fold increase in GFP production by the mouse gamma -UTR construct over the alpha -UTR (gamma /alpha ) and a 5-13-fold increase over the beta -UTR (gamma /beta ; Fig. 7C). The greatest differences in GFP intensities were seen in HaCaT cells. These data show that the gamma -UTR facilitates expression of a heterologous protein, whereas the alpha - and beta -UTRs significantly suppress protein production.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Flow cytometry analysis of 5' UTR-GFP constructs in transfected cells. A, fluorescence intensity histogram compiled from the analysis of 20,000 cells per sample for each construct (see Fig. 6) in transfected Cos-1 cells. The peak closest to the vertical axis is attributable to untransfected cells (negative cells), which show low fluorescent intensities because of autofluorescence. The second peak represents fluorescence from transfected cells that express GFP. The four histograms representing each GFP construct were merged to allow direct comparison of fluorescence intensities. This analysis revealed that the gamma -UTR construct produced the highest GFP intensities (mean value, 1.045 × 103) and the alpha -UTR produced the lowest (mean value, 6.2 × 101). B, graphical representation of GFP intensities of the 5' UTR variants relative to the empty GFP vector (defined as 100) in transfected BHK-21 (BHK), Cos-1, and HaCaT cells. C, graphical representation of the ratios of GFP intensities among 5' UTR variants in the cell lines indicated. The gamma -UTR/alpha -UTR (black-square gamma /alpha ) and gamma -UTR/beta -UTR ( gamma /beta ) ratios are shown for constructs transfected into BHK-21 cells (BHK), Cos-1 cells, primary mouse skin fibroblasts (MSF), and HaCaT cells. The largest differences in GFP fluorescence between the gamma -UTR and the alpha - and beta -UTR variants were observed in HaCaT cells. The values shown are the averages of three independent transfection and cell sorting experiments.

To determine whether this suppression is caused by the presence of uORFs or some other property of the longer UTRs, such as increased secondary structure, we made two additional constructs. The uORFs of the alpha -UTR variant were removed by mutating all four upstream ATG codons to TTG. Significantly, the mutant alpha -UTR construct expressed the reporter at levels that approached that of the gamma -UTR construct (Fig. 8). To ascertain whether the length of the UTR sequence could influence expression levels, we produced a gamma -UTR multimer containing four copies of this sequence in the same orientation. The gamma -UTR multimer, which contained 314 base pairs compared with the 307 base pairs of the alpha - variant, produced GFP levels that were only marginally lower than a single copy of the gamma -UTR (Fig. 8). Taken together, these data show that the uORFs of the Gli1 UTRs play a major role in the suppression of protein production.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 8.   Flow cytometry analysis of mutant 5' UTR-GFP constructs in transfected cells. A graphical representation of GFP intensities of wild-type alpha -UTR (alpha ), mutant alpha -UTR (alpha mut), wild-type gamma -UTR (gamma ), and the 4-mer gamma -UTR (gamma x4) constructs relative to the empty GFP vector in Cos-1 cells is shown. The values shown are the averages of three independent transfection and cell-sorting experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The expression of alternative mRNAs from a single gene is an important mechanism for gene regulation and for the generation of functionally different proteins (44, 45). In this study, we have identified alternative GLI1 transcripts that differ in their 5' UTRs and are generated by exon skipping. The longer transcripts (alpha  and beta  in mouse and beta  in human) were present in all normal tissues examined, whereas the shortest transcript (gamma -UTR) was present in neonatal tissues but rarely detected in adult tissues. Significantly, the gamma  form is the only variant found in BCCs and in a proliferating human keratinocyte cell line. Moreover, expression of the gamma  form could be induced by TPA treatment of mouse skin. We have found that the GLI1 5' UTRs determine the expression levels of a heterologous coding sequence in transfected cells, with the gamma  form associated with the highest levels of expression and the alpha - and beta -UTRs associated with significantly lower levels. These findings suggest that GLI1 levels may be regulated through the use of alternative 5' UTRs and predict that an unregulated increase in the gamma  transcript over the alpha  and beta  forms may be tumorigenic.

We initially examined several tissues from various strains of mice, both neonatal and adult, for evidence of tissue-specific expression of the GLI1 alternative transcripts. We found that expression of these transcripts did not show tissue specificity, but the levels of the alpha  and beta  transcripts (relative to each other) did vary between strains. Because BALB/c mice predominantly express the alpha  form and CD1 mice predominantly express the beta  form, we sequenced the intron-exon boundaries of this region in these strains and found them to be identical (data not shown), suggesting that the expression differences between strains was not caused by polymorphic variation at these sites. Whether these strain differences were caused by polymorphic differences in intronic cis-acting elements or by differences in transacting factors remains to be determined. We did not observe any transcripts equivalent to the mouse alpha -UTR in the human tissues examined, and we were unable to find sequences equivalent to mouse exon 1b in the human gene, suggesting that humans do not have the capacity to express an alpha -UTR variant, although the presence of three upstream ATG codons in exon 1a of the human message compared with just one in mouse, together with the increased stability of the human beta -UTR (Table III), appears to compensate for the lack of exon 1b sequences in the human sequence. However, we cannot exclude the possibility that expression of the alpha -UTR variant may be idiosyncratic in human as it is in mice because of the small sample size that was available in this study.

The expression of the 5' UTR variants did show an apparent correlation with proliferative status. The longest transcripts are expressed in normal tissues but not in BCCs or in a human keratinocyte cell line, whereas the shortest transcript is the only variant present in BCCs and in HaCaT cells. In addition, expression of the gamma  form was induced by TPA treatment of mouse skin, whereas the levels of the longer transcripts were concomitantly reduced. These observations suggest an association of the shortest 5' UTR with actively proliferating keratinocytes and the alpha  and beta  transcripts with quiescent cells. The association of the most actively translated transcript with proliferation fits neatly with the observation of increased GLI1 expression in BCCs and with experimental in vivo data showing overexpression of GLI1 resulting in BCC-like lesions (21, 22, 24, 25). The concomitant reduction in alpha  and beta  transcripts with increased levels of the gamma  form after TPA treatment is consistent with a post-transcriptional modification of a single pre-mRNA species. Although the precise mechanism is unknown, the activity of transacting factors that control alternative splicing decisions have been shown to be modulated by mitogens such as TPA (46-49). It can also be reasoned that alternative splicing of the GLI1 message may be regulated by Sonic hedgehog signaling. We are currently testing the possibility that unregulated signaling of the Sonic hedgehog-Patched pathway (as occurs in BCCs) results in the generation of increased levels of the gamma  form through changes in the composition of splicing factors bound to the nascent GLI1 pre-mRNA. It is also conceivable that tumor-causing mutations could occur in the splice sites of upstream noncoding exons that would enhance the formation of the gamma  form. Alternatively, mutations that alter the regulation of factors involved in splice site selection may also contribute to tumor formation. In addition, GLI1 has been shown to bind RNA directly at a conserved motif that is distinct from the GLI DNA binding cis-element and to regulate translation of the bound mRNA (37, 50). Because these putative GLI binding motifs are present in the pre-mRNA sequence of GLI1, an intriguing possibility is that GLI1 itself or a related protein may determine exon selection. Two recent reports have indicated that GLI2 and GLI3 transcriptionally regulate GLI1 by binding directly to promoter sequences, but the same data do not preclude up-regulation of GLI1 via a post-transcriptional mechanism by these factors as well (28, 30).

Our results suggest that the alpha - and beta -UTR variants contain potent translation inhibitors and are in agreement with other studies showing that 5' UTRs with uORFs and stable secondary structures are features of poorly translated mRNAs (31-36). Furthermore, we have shown that the uORFs present in the alpha - and beta -UTRs play an important role in mediating this inhibition. The increased GFP levels observed for the gamma  form may be wholly attributable to the lack of uORFs, because increasing the length of the gamma -UTR variant 4-fold only marginally decreased GFP levels. However, it is also possible that the gamma -UTR transcript may be more stable than the longer variants and consequently is able to produce more product per transcript than the alpha - and beta -UTR variants. Several studies have shown that impaired translation can initiate mRNA decay and that both stem-loop structures and uORFs within 5' UTRs can modulate mRNA stability (51). Regardless of whether these sequences modulate translation efficiency or mRNA stability (or both), the retention or skipping of 5' noncoding exons generates alternative UTRs that differentially express a heterologous reporter, and it is assumed of GLI1 as well. In conclusion, this study suggests that alternative splicing is an important mechanism in the regulation of this gene, which may in part contribute to the up-regulation of GLI1 observed in BCCs.


    ACKNOWLEDGEMENTS

We thank Dr. Norbert Fusenig for the HaCaT cells, Dr. Peter Dodd for the human brain mRNA sample, and Dr. Anthony Dicker for human foreskin biopsies and dissected BCC samples. We thank Betsy Hung and Lexie Friend for assistance with cell culture.


    FOOTNOTES

* This work was supported by the Queensland Cancer Fund and the University of Queensland Cancer Research Fund.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.

Dagger Supported by a Wellcome Trust Senior Research Fellowship in Medical Research (Australia). To whom correspondence should be addressed. Tel.: 61-7-3365-4629; Fax: 61-7-3365-4699; E-mail: josephr@biosci.uq.edu.au.

Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M005191200


    ABBREVIATIONS

The abbreviations used are: BCC, basal cell carcinoma; GFP, green fluorescent protein; UTR, untranslated region; uORF, upstream open reading frame; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA ends; mGli, mouse Gli; TPA, 12-O-tetradecanoylphorbol 13-acetate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Kinzler, K. W., Bigner, S. H., Bigner, D. D., Trent, J. M., Law, M. L., O'Brien, S. J., Wong, A. J., and Vogelstein, B. (1987) Science 236, 70-73[Medline] [Order article via Infotrieve]
2. Roberts, W. M., Douglass, E. C., Peiper, S. C., Houghton, P. J., and Look, A. T. (1989) Cancer Res. 49, 5407-5413[Abstract]
3. Stein, U., Eder, C., Karsten, U., Haensch, W., Walther, W., and Schlag, P. M. (1999) Cancer Res. 59, 1890-1895[Abstract/Free Full Text]
4. Ingham, P. W. (1998) EMBO J. 17, 3505-3511[Abstract/Free Full Text]
5. Johnson, R. L., and Scott, M. P. (1998) Curr. Opin. Genet. Dev. 8, 450-456[CrossRef][Medline] [Order article via Infotrieve]
6. Ruiz i Altaba, A. (1999) Nat. Cell Biol. 1, 147-148
7. Kinzler, K. W., Bigner, S. H., Ruppert, J. M., and Vogelstein, B. (1988) Nature 332, 371-374[CrossRef][Medline] [Order article via Infotrieve]
8. Ruppert, J. M., Kinzler, K. W., Wong, A. J., Bigner, S. H., Kao, F. T., Law, M. L., Seuanez, H. N., O'Brien, S. J., and Vogelstein, B. (1988) Mol. Cell. Biol. 8, 3104-3113[Medline] [Order article via Infotrieve]
9. Walterhouse, D., Ahmed, M., Slusarski, D., Kalamaras, J., Boucher, D., Holmgren, R., and Iannaccone, P. (1993) Dev. Dyn. 196, 91-102[Medline] [Order article via Infotrieve]
10. Hui, C. C., Slusarski, D., Platt, K. A., Holmgren, R., and Joyner, A. L. (1994) Dev. Biol. 162, 402-413[CrossRef][Medline] [Order article via Infotrieve]
11. Marigo, V., Johnson, R. L., Vortkamp, A., and Tabin, C. J. (1996) Dev. Biol. 180, 273-283[CrossRef][Medline] [Order article via Infotrieve]
12. Lee, J., Platt, K. A., Censullo, P., and Ruiz i Altaba, A. (1997) Development 124, 2537-2552[Abstract/Free Full Text]
13. St-Jacques, B., Dassule, H. R., Karavanova, I., Botchkarev, V. A., Li, J., Danielian, P. S., McMahon, J. A., Lewis, P. M., Paus, R., and McMahon, A. P. (1998) Curr. Biol. 8, 1058-1068[Medline] [Order article via Infotrieve]
14. Chiang, C., Swan, R. Z., Grachtchouk, M., Bolinger, M., Litingtung, Y., Robertson, E. K., Cooper, M. K., Gaffield, W., Westphal, H., Beachy, P. A., and Dlugosz, A. A. (1999) Dev. Biol. 205, 1-9[CrossRef][Medline] [Order article via Infotrieve]
15. Gailani, M. R., Stahle-Backdahl, M., Leffell, D. J., Glynn, M., Zaphiropoulos, P. G., Pressman, C., Unden, A. B., Dean, M., Brash, D. E., Bale, A. E., and Toftgard, R. (1996) Nat. Genet. 14, 78-81[Medline] [Order article via Infotrieve]
16. Hahn, H., Wicking, C., Zaphiropoulous, P. G., Gailani, M. R., Shanley, S., Chidambaram, A., Vorechovsky, I., Holmberg, E., Unden, A. B., Gillies, S., Negus, K., Smyth, I., Pressman, C., Leffell, D. J., Gerrard, B., Goldstein, A. M., Dean, M., Toftgard, R., Chenevix-Trench, G., Wainwright, B., and Bale, A. E. (1996) Cell 85, 841-851[Medline] [Order article via Infotrieve]
17. Johnson, R. L., Rothman, A. L., Xie, J., Goodrich, L. V., Bare, J. W., Bonifas, J. M., Quinn, A. G., Myers, R. M., Cox, D. R., Epstein, E. H., Jr., and Scott, M. P. (1996) Science 272, 1668-1671[Abstract]
18. Fan, H., Oro, A. E., Scott, M. P., and Khavari, P. A. (1997) Nat. Med. 3, 788-792[Medline] [Order article via Infotrieve]
19. Oro, A. E., Higgins, K. M., Hu, Z., Bonifas, J. M., Epstein, E. H., Jr., and Scott, M. P. (1997) Science 276, 817-821[Abstract/Free Full Text]
20. Xie, J. M., Murone, M., Luoh, S. M., Ryan, A., Gu, Q. M., Zhang, C. H., Bonifas, J. M., Lam, C. W., Hynes, M., Goddard, A., Rosenthal, A., Epstein, E. H., Jr., and de Sauvage, F. J. (1998) Nature 391, 90-92[CrossRef][Medline] [Order article via Infotrieve]
21. Dahmane, N., Lee, J., Robins, P., Heller, P., and Ruiz i Altaba, A. (1997) Nature 389, 876-881[CrossRef][Medline] [Order article via Infotrieve]
22. Nilsson, M., Unden, A. B., Krause, D., Malmqwist, U., Raza, K., Zaphiropoulos, P. G., and Toftgard, R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3438-3443[Abstract/Free Full Text]
23. Ruppert, J. M., Vogelstein, B., and Kinzler, K. W. (1991) Mol. Cell. Biol. 11, 1724-1728[Medline] [Order article via Infotrieve]
24. Reifenberger, J., Wolter, M., Weber, R. G., Megahed, M., Ruzicka, T., Lichter, P., and Reifenberger, G. (1998) Cancer Res. 58, 1798-1803[Abstract]
25. Ghali, L., Wong, S. T., Green, J., Tidman, N., and Quinn, A. G. (1999) J. Invest. Dermatol. 113, 595-599[Abstract/Free Full Text]
26. Motoyama, J., Liu, J., Mo, R., Ding, Q., Post, M., and Hui, C. C. (1998) Nat. Genet. 20, 54-57[CrossRef][Medline] [Order article via Infotrieve]
27. Park, H. L., Bai, C., Platt, K. A., Maise, M. P., Beeghly, A., Hui, C. C., Nakashima, M., and Joyner, A. L. (2000) Development 127, 1593-1605[Abstract/Free Full Text]
28. Dai, P., Akimaru, H., Tanaka, Y., Maekawa, T., Nakafuku, M., and Ishii, S. (1999) J. Biol. Chem. 274, 8143-8152[Abstract/Free Full Text]
29. Ruiz, I, and Altaba, A. (1999) Development 126, 3205-3216[Abstract/Free Full Text]
30. Sasaki, H., Nishizaki, Y., Hui, C. C., Nakafuku, M., and Kondoh, H. (1999) Development 126, 3915-3924[Abstract/Free Full Text]
31. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148[Abstract]
32. Kozak, M. (1991) J. Cell Biol. 115, 887-903[Abstract]
33. Sonenberg, N. (1994) Curr. Opin. Genet. Dev. 4, 310-315[Medline] [Order article via Infotrieve]
34. Kozak, M. (1996) Mamm. Genome 7, 563-574[CrossRef][Medline] [Order article via Infotrieve]
35. van der Velden, A. W., and Thomas, A. A. (1999) Int. J. Biochem. Cell Biol. 31, 87-106[CrossRef][Medline] [Order article via Infotrieve]
36. Willis, A. E. (1999) Int. J. Biochem. Cell Biol. 31, 73-86[CrossRef][Medline] [Order article via Infotrieve]
37. Graves, L. E., Segal, S., and Goodwin, E. B. (1999) Nature 399, 802-805[CrossRef][Medline] [Order article via Infotrieve]
38. Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., and Fusenig, N. E. (1988) J. Cell Biol. 106, 761-771[Abstract]
39. Gluzman, Y. (1981) Cell 23, 175-182[Medline] [Order article via Infotrieve]
40. Ferrari, M. (1962) Virology 16, 147-151
41. Liu, C. Z., Yang, J. T., Yoon, J. W., Villavicencio, E., Pfendler, K., Walterhouse, D., and Iannaccone, P. (1998) Gene (Amst.) 209, 1-11[CrossRef][Medline] [Order article via Infotrieve]
42. Heyden, A., Lutzow-Holm, C., Clausen, O. P., Thrane, E. V., Brandtzaeg, P., Roop, D. R., Yuspa, S. H., and Huitfeldt, H. S. (1994) Differentiation 57, 187-193[CrossRef][Medline] [Order article via Infotrieve]
43. Zuker, M. (1989) Science 244, 48-52[Medline] [Order article via Infotrieve]
44. Ayoubi, T. A., and Van De Ven, W. J. (1996) FASEB J. 10, 453-460[Abstract/Free Full Text]
45. Lopez, A. J. (1998) Annu. Rev. Genet. 32, 279-305[CrossRef][Medline] [Order article via Infotrieve]
46. Darville, M. I., and Rousseau, G. G. (1997) Nucleic Acids Res. 25, 2759-2765[Abstract/Free Full Text]
47. Du, K., Leu, J. I., Peng, Y., and Taub, R. (1998) J. Biol. Chem. 273, 35208-35215[Abstract/Free Full Text]
48. Levanon, D., Bernstein, Y., Negreanu, V., Ghozi, M. C., Bar-Am, I., Aloya, R., Goldenberg, D., Lotem, J., and Groner, Y. (1996) DNA Cell Biol. 15, 175-185[Medline] [Order article via Infotrieve]
49. Screaton, G. R., Caceres, J. F., Mayeda, A., Bell, M. V., Plebanski, M., Jackson, D. G., Bell, J. I., and Krainer, A. R. (1995) EMBO J. 14, 4336-4349[Abstract]
50. Jan, E., Yoon, J. W., Walterhouse, D., Iannaccone, P., and Goodwin, E. B. (1997) EMBO J. 16, 6301-6313[Abstract/Free Full Text]
51. Jacobson, A., and Peltz, S. W. (1996) Annu. Rev. Biochem. 65, 693-739[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.