Supplementary Figure S1; Supplementary Tables S1-S3.

PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

Abstract The p53, p63 and p73 family of sequence-specific transcription factors share similar biochemical properties and structural features resulting in the recognition of DNA with identical amino acids and similar binding affinity. However, loss of individual members results in very different phenotypes in mice as well as humans. We have addressed functional relationships between p53 family proteins using a combination of mutants, target response elements (REs) and sequence-specific transactivation assays based in yeast and human cells. p53 alleles exhibiting enhanced transcriptional activation and altered promoter selectivity were previously identified in the L1 and L3 loop of the DNA binding domain of the protein. Included was S121F that results in defective p21 induction and confers a strong apoptotic phenotype in mammalian cells. Guided by result with p53-S121F, -T123A and -S240N mutants, the corresponding p63 and p73 alleles were investigated for ability to drive transactivation at over fifty p53 REs in isogenic yeast reporter strains. The phenotypes observed with p53 alleles were reproduced with the p63- and p73- alleles. Focusing on p73-S139F (corresponding to p53-S121F) transactivation at many RE sequences, we identify and experimentally confirm an RE target sequence code that predicts enhanced or reduced transactivation potentials. The correlation is particularly strong for induction at p53 targets within promoters of genes associated with apoptosis. Furthermore, p73 S139F showed an enhanced DNA-binding cooperativity in vitro, and loop L1 shows conformational changes in the crystal structure of the protein-DNA complex. The altered function phenotype was confirmed not only in gene reporter assays but also for endogenous gene expression in the p53 null HCT116 human cell line, suggesting that target recognition can predict relative changes in transcription rates at different loci. Based on the correlation between RE code and associated gene functions, we propose the selection during evolution of an RE sequence code within target promoters that can affect intrinsic conformational flexibility / DNA binding affinity of p53 proteins thereby modulating in vivo selectivity, specifically favoring the activation of apoptotic target genes Citation Format: Yari Ciribilli, Paola Monti, Alessandra Bisio, H. T. Nguyen, A S. Ethayathulla, Micheal A. Resnick, Hector Viadiu, Gilberto Fronza, Alberto Inga. Mutations in the p53 gene family reveal conservation of structure-function in the L1 and L3 loops and a response element code for transcriptional specificity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2013-2316

Pediatric Blood & CancerVolume 52, Issue 2 p. 303-304 Letter to the Editor Identification of a novel TP53 germline mutation in a large Italian Li—Fraumeni syndrome Family Valeria Capra MD, Corresponding Author Valeria Capra MD [email protected] U.O. Neurochirurgia, Istituto G. Gaslini, Genoa, ItalyU.O. Neurochirurgia, Istituto G. Gaslini, Largo G. Gaslini 5, 16148 Genoa, Italy.===Search for more papers by this authorAlessandro Consales MD, Alessandro Consales MD U.O. Neurochirurgia, Istituto G. Gaslini, Genoa, ItalySearch for more papers by this authorPaola Nozza MD, Paola Nozza MD Department of Pathologic Anatomy, G. Gaslini Children's Hospital, Genoa, ItalySearch for more papers by this authorPaola Monti PhD, Paola Monti PhD Molecular Mutagenesis and DNA Repair Unit, Department of Epidemiology and Prevention, National Cancer Research Institute (IST), Genoa, ItalySearch for more papers by this authorAlberto Inga PhD, Alberto Inga PhD Molecular Mutagenesis and DNA Repair Unit, Department of Epidemiology and Prevention, National Cancer Research Institute (IST), Genoa, ItalySearch for more papers by this authorGilberto Fronza PhD, Gilberto Fronza PhD Molecular Mutagenesis and DNA Repair Unit, Department of Epidemiology and Prevention, National Cancer Research Institute (IST), Genoa, ItalySearch for more papers by this author Valeria Capra MD, Corresponding Author Valeria Capra MD [email protected] U.O. Neurochirurgia, Istituto G. Gaslini, Genoa, ItalyU.O. Neurochirurgia, Istituto G. Gaslini, Largo G. Gaslini 5, 16148 Genoa, Italy.===Search for more papers by this authorAlessandro Consales MD, Alessandro Consales MD U.O. Neurochirurgia, Istituto G. Gaslini, Genoa, ItalySearch for more papers by this authorPaola Nozza MD, Paola Nozza MD Department of Pathologic Anatomy, G. Gaslini Children's Hospital, Genoa, ItalySearch for more papers by this authorPaola Monti PhD, Paola Monti PhD Molecular Mutagenesis and DNA Repair Unit, Department of Epidemiology and Prevention, National Cancer Research Institute (IST), Genoa, ItalySearch for more papers by this authorAlberto Inga PhD, Alberto Inga PhD Molecular Mutagenesis and DNA Repair Unit, Department of Epidemiology and Prevention, National Cancer Research Institute (IST), Genoa, ItalySearch for more papers by this authorGilberto Fronza PhD, Gilberto Fronza PhD Molecular Mutagenesis and DNA Repair Unit, Department of Epidemiology and Prevention, National Cancer Research Institute (IST), Genoa, ItalySearch for more papers by this author First published: 05 December 2008 https://doi.org/10.1002/pbc.21796Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume52, Issue2February 2009Pages 303-304 RelatedInformation