Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional Gli-binding site
Introduction
Patched (ptc) was first identified as a segment polarity gene in Drosophila melanogaster and ptc has been shown to be one of the key players in the hedgehog (HH)-signaling pathway, a pathway shown to be of outmost importance for patterning, proliferation and differentiation in almost every tissue during development (for a recent review, see McMahon et al., 2003).
In Drosophila, when the lipid-modified HH binds ptc, the inhibitory effect on smoothened (smo), a seven-transmembrane protein with strong resemblance to a G-protein-coupled receptor is relieved. The active smo protein leads, by an unknown mechanism, to the dissociation of a multiprotein complex from microtubules. The multiprotein complex contains the serine–threonine kinase fused (fu), suppressor of fused [su(fu)] regulating nuclear–cytoplasmic shuttling of cubitus interruptus (ci), the kinesin-related protein costal2 (cos-2) and the zinc finger containing transcription factor cubitus interruptus (ci) that activates downstream target genes like wingless (wg), the TGF-B family member decapentaplegic (dpp) and ptc itself.
The HH-signaling pathway in mammals is less studied and more complex than in Drosophila. Mammals have three different types of hedgehog proteins, Sonic hedgehog (SHH), Indian hedgehog (IHH) and Desert hedgehog (DHH), two ptc homologues, PTCH1 and PTCH2 Carpenter et al., 1998, Zaphiropoulos et al., 1999, one smo homologue SMOH, and three ci homologues GLI1, GLI2 and GLI3 (see Ruiz i Altaba et al., 2002 and references therein). Ci functions both as a repressor and an activator depending on regulated proteolysis resulting in different protein isoforms. GLI3 and GLI2 are possibly processed in a similar manner, whereas GLI1 works mainly as an activator (Ruiz i Altaba, 1999). Interestingly, Gli1 and Gli2 have been shown to be able to induce tumors when overexpressed in experimental systems (see Ruiz i Altaba et al., 2002 and references therein). In addition, GLI1 is consistently overexpressed in human basal cell carcinoma (BCC; Dahmane et al., 1997).
The human counterpart to Drosophila ptc, PTCH1, was identified as the gene underlying the development of the Nevoid Basal Cell Carcinoma Syndrome (NBCCS or Gorlin's syndrome; Hahn et al., 1996b, Johnson et al., 1996). NBCCS is an autosomal dominant disorder characterized by predisposition to multiple basal cell carcinoma (BCC) and other tumors. PTCH1 has been shown to be mutated, and mutant mRNA was constitutively expressed, as an indication of active ongoing HH-signaling, not just in familiar BCC but also in sporadic BCC and other tumor types like medulloblastoma and primitive neuroectodermal tumors (see Ruiz i Altaba et al., 2002 and references therein).
The human PTCH1 gene spans 34 kb of DNA and is located on chromosome 9q22.3. It contains three alternative 5′ ends, exon 1, exon 1A and exon 1B, followed by 22 coding exons common to all three transcripts Hahn et al., 1996a, Hahn et al., 1996b, Johnson et al., 1996. So far, only exon 1B has been definitely shown to code for protein (Kogerman et al., 2002). Human exon 1B is homologous to sequences present in Drosophila, rodent, frog and fish, whereas sequence similarities to exon 1 and exon 1A have as of yet only been reported for mice (Kogerman et al., 2002). Exon 1B and exon 3 contain one ATG start codon each, while exon 1A only contains putative alternative start codons (CTG). Exon 1 contains an in-frame stop codon (TGA) but all PTCH1 variant mRNAs contain open reading frames in-frame with exon 2 (Kogerman et al., 2002). Functional assays using different PTCH1 isoforms in combination with SHH and SMOH show that all three PTCH1 isoforms can inhibit the activity of SHH and interact with SMOH, but only the exon 1B isoform is able to fully inhibit SMOH activity (Kogerman et al., 2002).
The biological function of PTCH1 may be more complex than previously thought due to the three alternative first exons and the three different transcripts that are expressed. Presence of alternative exons suggests the existence of more than one promoter, and alternative regulatory elements driving the expression of PTCH1. The expression of exon 1B transcripts appears to be lower relative to exon 1 and exon 1A transcripts in epidermis, thymus, prostate, spleen, testis, colon, ovary and intestine (Kogerman et al., 2002). Interestingly, analysis of the expression levels in nodular BCCs showed a preferential increase in the expression of exon 1B (Kogerman et al., 2002). The selective upregulation of exon 1B transcripts in BCC implies that expression of this transcript is regulated via different mechanisms compared to the other two transcripts.
In order to better understand how the three PTCH1 isoforms are regulated, promoter and transcription factor binding studies were performed. We now report the presence of three functional promoters, one in front of each of the three alternative first exons, and that expression of exon 1B is regulated by the Gli transcription factors as well as SHH and SMOH. We additionally demonstrate that all three Gli proteins, GLI1, Gli2 and GLI3, are capable of binding to the consensus Gli-binding site present upstream of exon 1B, and that all transcriptional effects due to activation of the HH-signaling pathway appear to be mediated via this single functional Gli-binding site.
Section snippets
Constructs used in promoter and binding studies
Constructs were made from the 4.3-kb fragment of the PTCH1 5′ regulatory region as previously described (Hahn et al., 1996a) either by restriction digestion or by polymerase chain reaction (PCR; Table 1). PCR reactions were performed using either Taq polymerase (Promega, Madison, USA) or the Advantage GC cDNA PCR kit (Clontech, Franklin Lakes, USA) depending on the GC-richness in the area that was amplified. The sizes of the PCR products were analyzed on 1–2% agarose gels and ligated into the
Analysis of the PTCH1 gene 5′ flanking region
To determine putative elements controlling the expression of the PTCH1 gene, we analyzed the sequence region containing all three alternative first exons for the presence of known regulatory elements (Fig. 1). The transcriptional start site for exon 1B located 373 bp upstream of the ATG is taken as position +1 (Johnson et al., 1996). The transcriptional start sites for exon 1 and exon 1A are positioned 1313 and 1535 bp downstream of the exon 1B transcriptional start site Hahn et al., 1996a,
Discussion
Correctly regulated expression of the different proteins involved in the HH-signaling pathway is of great biological importance. Lack of expression of many of the proteins involved in HH-signaling during embryogenesis leads to lethality, whereas altered expression after birth sometimes leads to tumor formation. PTCH1 is an HH receptor and often found to be mutated in both sporadic and familial BCC as well as in the germ line in NBCCS patients implying an essential nonredundant role in the
Acknowledgements
We wish to thank Fahimeh Ranamah for PTCH1 primers and advice on the analysis of PTCH1 mRNA variants. We also want to thank Dr D.C. Hughes for the cDNA clone of murine Gli2, Dr. A.P. McMahon for cDNA clone of human SHH and Dr Karl-Heinz Grzeschik for critical review of the manuscript. The project was supported by grants from the Swedish Cancer Fund, the Swedish Research Council and Pharmacia.
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