Co-ordinator report, JDRF-Telethon Project IDDM locus analysis in T1DM- Identification and characterization of functional polymorphisms , 2006-07

Final Report - Task A. Genetic Studies. Search for sequence polymorphisms in the ICOS region. Type1 Diabetes (T1D) is a polygenic disease caused by the immune-mediated destruction of pancreatic insulin-secreting-cells. Genetic studies of human T1D indicate the presence of a disease locus in a restricted region on chromosome 2q33 (IDDM12) and in the orthologous region of murine chromosome 1. CD28, CTLA4 and ICOS genes, coding for lymphocyte co-stimulatory receptors, map in the IDDM12 locus. Our previous results suggest the presence of two independent disease variants in the IDDM12 region on chromosome 2q33, one located in the CTLA4 region (CT60) and a second one in the ICOS region (IcosMicro). The markers of the two genes associated are not in linkage disequilibrium with each other but both show significant association with the disease. The LD substantially drops in the intron I region of the ICOS gene. We therefore undertook a search for new variants in the downstream region of the ICOS termination site to be tested for association with T1DM. For the analysis of the region comprised between ICOS gene and d2s2189 microsatellite (150 kb) we selected 12 SNPs, already described in the NCBI SNP database. These SNPs were selected for being located in the sequences that were at least 80% identical to the orthologous mouse region over a 100 bp. The underlying rationale is that essential regulatory elements are conserved among mammals, suggesting that cross-species sequence comparisons should identify them. Results SNP1 rs4522587 was not in H-W Equilibrium in our population and it was eliminated from the study. SNP3 rs1188425, SNP7 rs7593004 and SNP9 rs10173852 were not polymorphic in a pre-screening of 50 T1D cases and 50 random controls. SNP2 rs10497874, SNP5 rs12615668, SNP6 rs933985, SNP8 rs1896284, SNP11 rs16840733 and SNP12 rs16840740 did not show any trend of association with the disease in the pre-screening study. SNP4 rs2353320 and SNP10 rs1559934 that showed a trend of association with T1D in the case-control pre-screening were analysed in a panel of 111 T1D patients and 195 random controls. Only for SNP10 rs1559934 we have confirmed the significant association with T1D (Table I) To confirm this result we performed the TDT analysis for the SNP 4 and 10, on our panel of 174 T1D family trios. The TDT analysis has not confirmed the case-control results. Nevertheless a putative alternative promoter of human genes maps between rs1559934 (SNP10) and d6s2189 and we are now analyzing another SNP in this region. New task. Foxp3 5'UTR polymorphism In the last few years, the role of the regulatory CD4+CD25+ T cells (Treg) in autoimmune diseases has been well proved. FOXP3 is a master gene for the generation and function of the Treg cells, that are essential in the maintenance of immune self-tolerance (1,2). FOXP3 human gene, on chromosome Xp11.23, and the mouse orthologue Foxp3/Scurfy encode a member of a family of transcription factors, characterised by a forkhead box (fox)/winged-helix domain (FKH), localised at the C-terminus of the protein. It has been demonstrated that this domain is required for the nuclear localisation and DNA binding of the protein (3). Scurfy mutant mice have an X-linked recessive mutation in the FOXP3 gene that is responsible for the production of a functionally inactive protein lacking the DNA-binding domain, FKH. These mice have a lymphoproliferative disorder and hemizygous males die at 3 weeks from birth (4). In humans, FOXP3 mutations cause X-linked autoimmune disregulation syndrome known as IPEX (Immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome) characterised by neonatal onset insulin-dependent diabetes, infections, diarrhoea, dermatitis, thyroiditis and anemia (5). Moreover, it has been demonstrated that a microsatellite polymorphism in the promoter/enhancer region of the FOXP3 gene is associated with T1D in the Japanese population (6). Instead, no association has been reported between several polymorphisms in coding exons and surrounding intron-exon boundaries of the FOXP3 gene and T1D in the Sardinian population (7). We selected a SNP C/T located in the 5'-UTR Intron-1 of the FOXP3 gene (Foxp3C/T SNP) for T1D-association studies. Interestingly, we observed by data base analysis that the SNP is located in a putative heptameric non-canonical consensus site for AP-1 (c-Jun) binding: an important transcription factor regulating cell proliferation. Next, we typed 165 T1D families (81 with a male proband and 84 with a female proband) for the SNP. We found a not significant increase of T allele transmission to affected male sibs (T allele transmitted = 14%, T allele untransmitted = 9%) and a significant distortion of transmission of the T allele to the affected female sibs (T allele transmitted = 18%, T allele untransmitted = 7%; p = 0.02). To confirm this finding we then performed a case-control-study and we found a significant association of the T allele with the disease in the male patients (Table II). Considering these very interesting association data, we decide to analyze if this polymorphism may have a role on FOXP3 gene regulation. AP-1 (activator protein-1) is a transcription factor involved in the regulation of cell proliferation, cell-cycle progression, in cell migration and differentiation and apoptosis (8-10). AP-1 proteins belong to the bZIP family of transcriptional activators and are key mediators in the pathogenesis of cancer and other disease states. This transcription factor is composed of members of the Jun and Fos proto-oncogene families. The Jun proteins (c-Jun, JunB and JunD) can dimerize among themselves or heterodimerize with Fos family proteins which includes c-Fos, FosB, Fra-1 and Fra-2. AP-1 proteins bind as dimers to a DNA sequence called the AP-1 DNA-binding site or AP-1 site, with the consensus sequence 5'-TGACTCA-3' (11). The AP-1 consensus sequence is a DNA binding site for the transcription factor c-Jun. c-Jun interacts directly with specific target DNA sequences to regulate gene expression. To investigate DNA:protein interactions, a human c-Jun recombinant protein was subjected to EMSA using as probe three double-stranded oligonucleotides containing either the AP1 canonical consensus (5'-TGACTCA-3') or the AP-1 putative consensus FOXP3/C (5'-TGACCGA-3') and FOXP3/T (5'-TGATCGA-3'). In the FOXP3 Intron-1 sequence, these two AP-1 putative consensuses are partial overlapped with a highly conserved STAT-binding site (TTC(N3)GAA). Therefore, the double-stranded oligonucleotides, used for the AP-1 putative consensus, contain both the AP-1 putative consensus and the STAT consensus binding motif. The binding reaction occurs in a specific binding buffer containing an optimum concentration of poly d(I-C) for c-Jun DNA binding to prevent unspecific binding of proteins to the probe oligonucleotides (Binghui L., 1992). c-Jun recombinant protein binds the probe oligonucleotides AP-1/STAT-FOXP3/C, containing AP-1 putative site FOXP3/C, even if the shift is very much smaller than the AP1 canonical consensus used as positive control, while there is no shift with oligonucleotides AP-1/STAT-FOXP3/T, containing AP-1 putative site FOXP3/T. AP-1 activity is composed primarily of Jun and Fos heterodimers and secondarily of Jun homodimers. To confirm the binding of Jun protein to the DNA fragments, we used a nuclear extract isolated from HeLa cells in which AP-1 activity derived primarily from the Jun homodimers (12). In this case, an evident shift is observed both with the oligonucleotides AP1/STAT-FOXP3/C and AP-1/STAT-FOXP3/T. To check if the observed shifted bands were specific for AP-1 protein complex, an excess unlabeled competitor oligonucleotides containing the AP-1 element (AP-1 consensus, AP-1/STAT-FOXP3/C or AP-1/STAT-FOXP3/T, as specific competitor, or AP-2 consensus, as aspecific competitor) was added to the reaction before addition of the labelled target oligonucleotides. AP-1 canonical consensus competed for binding both with AP-1/STAT-FOXP3/C and AP-1/STAT-FOXP3/T putative sites, but this competition is more evident with the AP-1/STAT-FOXP3/C putative site. Using increased amounts of nuclear extract, an increased shift is observed with both putative consensus but, all things being equal, the shift is major with the consensus AP-1/STAT-FOXP3/T than consensus AP-1/STAT-FOXP3/C. In accordance with these data, in competitive experiments, using increased amounts of unlabeled competitor oligonucleotides AP-1/STAT-FOXP3/C or AP-1/STAT-FOXP3/T, and AP-1 consensus like probe, we observed a more evident competition of AP-1/STAT-FOXP3/T than AP-1/STAT-FOXP3/C. These data suggest that both AP-1/STAT-FOXP3/C and AP-1/STAT-FOXP3/T contain a legitimate AP-1 binding site but, probably, the consensus AP-1/STAT-FOXP3/T binds a different AP-1 protein complex better than the consensus AP-1/STAT-FOXP3/C. To check if the DNA-protein interaction of the consensus AP-1FOXP3/C and AP-1FOXP3/T with the protein nuclear extract was influenced by STAT consensus, we have used two double-stranded oligonucleotides containing only the AP-1 putative sites. In this case, we observed an evident shift only with consensus AP-1FOXP3/C and a very light shift with the consensus AP-1FOXP3/T. Moreover, in competitive experiments, using increased amounts of unlabeled competitor oligonucleotides AP-1FOXP3/C or AP-1FOXP3/T, and AP-1 consensus like probe, we observed a more evident competition of AP-1FOXP3/C than AP-1FOXP3/T. These different results obtained with oligonucleotides AP-1/STAT-FOXP3/C or T and AP-1FOXP3/C or T, suggest that STAT-binding site influences, in some way, the interaction of the AP-1 protein complex with the two AP-1 putative consensus sites in FOXP3 gene. Using HeLa nuclear extract, the AP-1 protein complex formed with two AP-1 putative site is slightly smaller than that formed with AP-1 canonical consensus. Probably, this derived from the different composition of the two protein complexes. Task B. Role of the CTLA4 gene 3' UTR (AT)n repeat and the CT60 polymorphism on mRNA stability and targeting. Task B1. Role of the 3' UTR of CTLA4 including the (AT)n repeat on mRNA stability and targeting. We demonstrated that the 3' UTR of CTLA4 regulates Luciferase reporter gene expression; confers instability to the human CTLA4 mRNA and influences its translation efficiency. Finally, a contribution of the autoimmune diseases-associated (AT)n repeat polymorphic alleles in gene expression was observed (Malquori et al., BBA 2007). Here we summarise the results obtained. The 3'UTR of human CTLA4 decreases the expression of the Luciferase reporter gene. To determine the effect of the 3'-UTR on CTLA4 gene expression, reporter gene constructs were produced by inserting DNA encoding various regions of the 3'-UTR into the pGL3c vector. Each construct contained the luciferase coding sequence under the control of the SV40 promoter and enhancer elements, followed by the 3'UTR of luciferase and the SV40 late poly(A) signal. The reporter constructs differed only in the regions of the 3'UTR of CTLA4 that were inserted 5' to the late poly (A) signal of SV40. Regions of the 3'-UTR of CTLA4 investigated included a full-length 3'-UTR sequence (nucleotides 672-1812), with variations in the (AT)n polymorphic repeat, i.e.: a short repeat, 6AT; a medium repeat, 16AT and a long one, 24AT. They correspond to the alleles present in the human population, whose distribution in autoimmune disease have been investigated in several genetic studies. Moreover an artificial construct lacking the AT repeat and indicated as ?AT, not present in the human population, was produced. Finally, two macro deletions from the distal and proximal ends of the 3'-UTR, called respectively ?3' (nucleotides 673-1187) and ?5' (nucleotides 1236-1812) were analysed. Since all reporter constructs contained identical promoter elements, differences in luciferase activity reflect mainly differences in regulation as a result of post-transcriptional events. We used two human cell lines to study the 3'UTR of CTLA4 in luciferase reporter assays: HEK 293T, an epithelial embryonic kidney cell line and Jurkat, a leukemic T cell line. Reporter gene constructs were transiently transfected along with the pRL-vector, encoding the luciferase of Renilla reniformis, next the cells incubated for 24h were assayed for luciferase activity. Firefly luciferase activity driven by the pGL3c vectors were normalised by the renilla luciferase activity driven by the pRL vector using the Dual-Luciferase Reporter (DRLTM) Assay System. Luciferase activity (ratio of firely/renilla luciferase) measured in cells transfected with the pGL3c vector lacking any chimeric 3'UTR sequences was designated as 100%. In HEK 293T cells, inserting the full length 3'UTR of CTLA4 in the firely luciferase vector resulted in a marked decrease in luciferase activity, independently from the length of the (AT)n repeat (p<0,0001), suggesting the presence of negative regulatory element(s) within the 3'UTR of the CTLA4 message. The results in HEK 293T cells, while indicating the presence of strong negative element(s) in the distal region of the 3'UTR (nucleotides 1188-1812), do not allow us to identify this as the unique regulatory region. We would like to postulate that the proximal region of the 3'-UTR, that by itself does not appear to have an effect, cooperates with the distal region probably by a secondary mRNA structure constrain that it is lost in the two separated fragments or by affecting the trans-acting factor(s) recruitment. Moreover the (AT)n repeat length contributed to the regulation of luciferase gene expression. While the short (AT)n repeat ?AT and the 6AT mutants decreased the luciferase activity by 60%, the medium and long repeats, 16AT and 24AT, decreased the reporter activity by 70%. It should be noted that a high number of observations were necessary to achieve statistically significant data, in some instances up to thirty. This was not surprising given the small relative risk conveyed by longer (AT)n alleles (odds ratio ~1.3) in disease-susceptibility genetic studies. The 16AT was statistically different from the short 6AT construct (p<0.0001), while no difference was observed between the 16AT and the 24AT. The gene reporter analyses were also conducted in transiently transfected human Jurkat T cells and essentially similar results were obtained even if more pronounced. Moreover the ?3' and ?5' macrodeletions both decreased the luciferase activity suggesting that negative regulatory elements were present in both regions. The data thus indicate the possibility that cis-acting element(s) mapping to the proximal region of the 3'UTR can bind tissue-specific trans-acting factor(s) not or insufficiently expressed in HEK 293T cells, where the proximal region of the 3'UTR by itself did not influence the luciferase activity. Finally, the data suggest that negative regulatory element(s) located in the proximal and distal regions of the 3'UTR are both required to regulate luciferase gene expression. The 3' UTR of human CTLA4 decreases the expression of the CTLA4 gene. To investigate the effect of the 3' UTR on CTLA4 gene expression, the 3'UTR mutants, previously described, where subcloned 3' to the ORF of CTLA4; all constructs contained the cytomegalovirus (CMV) promoter sequence, the CTLA4 ORF and the bovine growth hormone (BGH) poly(A) signal. A Flag sequence was inserted by PCR into the CTLA4 ORF, 3' to the nucleotide sequence encoding the leader peptide and 5' to the sequence encoding the extracellular region of CTLA-4 in order to obtain an N-terminal Tag in the CTLA-4 mature protein, once the leader peptide is cleaved. The CTLA4 (ORF-3'UTR) constructs were transiently transfected in HEK 293T cells with a pcDNA3-HA-actinin expression vector. After 24h, cell extracts were separated on a 12% SDS-PAGE and analysed by western blotting, with Tag specific antibodies. Insertion of the 3'-UTR of CTLA4 resulted in a marked reduction of CTLA-4 expression; moreover, a slight effect of the (AT)n repeat length was observed, where lysates of cells transfected with the medium and long alleles (16AT and 24AT) constructs showed lower Flag signals than the one transfected with the 6AT. The results demonstrate that the 3'-UTR of CTLA4 decreases the expression not only of the reporter luciferase gene but also of its own gene. The 3'UTR regulates the mRNA steady-state levels of CTLA4. Changes in CTLA-4 protein expression could be due to either alterations in message stability or to altered rates of mRNA translation. To investigate these two possibilities, we measured the steady state levels of CTLA4 mRNAs using Northern blot analyses. CTLA4 mRNA levels were normalised with the levels of the Neomycin resistance gene, both transcripts being under the control of the pcDNA3 expression vector, driven respectively by the CMV and the SV40 promoters. If the decreased CTLA-4 protein expression observed in western blotting experiments was due to decreased message stability, then we would observe comparable changes in message levels. If decreased CTLA-4 protein expression were due to inhibition of translation, then we would expect to measure no change or a disproportionate change in CTLA4 mRNA levels. The results indicate that protein expression levels correlate with variations in steady state mRNA levels and suggest that the 3'UTR regulates CTLA-4 expression through message stability. However, we were expecting more pronounced differences in the steady state mRNA levels of cells transfected with the full length 3'UTR constructs. Indeed, the differences observed by western blotting, even if not quantitative, and the variations in luciferase activity were in the range of 70-80%. 3'UTR sequences regulate CTLA4 mRNA stability. To confirm that changes in steady-state mRNA levels reflect altered message stability, we directly measured message degradation in HEK 293T cells transiently transfected with the CTLA4 3'UTR mutants. Upon transfection the cells were treated with Actinomycin D (ActD) and CTLA4 mRNAs were measured by Northern blotting at various time points upon transcription inhibition. The mRNA were normalised to GAPDH mRNA levels. We observed that CTLA4 message without any 3'UTR sequences displayed a half-life of 8h. The full-length 3'UTR insertion resulted in a dramatic decrease in mRNA stability with an half-life of 3-4 hours, whereas no major differences in mRNA stability were observed between the transcripts containing the (AT)n mutants. We also noticed that upon ActD treatment two transcripts of different length were detected in cells transfected with the full length 3'UTR-CTLA4 constructs. The two bands probably reflect different poly(A) tail lengths of the same transcript. Moreover, the data are not in complete agreement with the steady-state mRNA measurement in particular for the 3'UTR deletion mutants, however it should be considered that in these experiments we are measuring the effect of a single regulatory event while in the steady-state analysis a transcription regulation cannot be excluded. 3'UTR sequences regulate CTLA4 mRNA translation rate. To evaluate a possible contribution of the 3'UTR sequences in the regulation of translational efficiency, we tested the ability of the 3'UTR to alter message expression using in vitro translation assays. Plasmids containing the different CTLA4-3'UTR sequences were linearised by restriction enzymes at the 3' end of the cloned genes and used in in vitro transcription/translation reactions with the T7 RNA polymerase, rabbit reticulocyte lysates and 35S-methionine. The proteins were separated by SDS-PAGE and the radioactive signals analysed by PhosphorImager. The full-length 3'UTR sequence mediated a marked inhibition of translation compared to the CTLA4 control vector lacking any 3'UTR sequences, whereas the ?3' and ?5' UTR regions had a milder effect. Interestingly, the profile is similar to the one observed in the luciferase reporter assays and in the western blotting analyses, supporting the hypothesis that the 3'UTR mediates also translational inhibition. We excluded the possibility that the 3'UTR, in our in vitro experimental conditions, was influencing the stability of the transcripts in the 90 min assay by Northern blot analysis of the reactions. In conclusion we demonstrated that the 3'UTR of CTLA4 contains sequences that regulate gene expression at the post-transcriptional level, influencing both mRNA stability and translation efficiency. The regulated binding of trans-acting factors to the 3'UTR of CTLA4 in T cell subsets might be relevant in maintaining immune tolerance and immune homeostasis. Moreover, we demonstrate a contribution of the autoimmune disease-associated (AT)n repeat polymorphic alleles in the expression of luciferase reporter gene and CTLA4. The following studies planned in Task B1 are in progress: a)The analysis of the effect of the 3'UTR on putative compartimentalization of the mRNA is under investigation. We have set up in situ hybridization studies on CTLA4 3'UTR transfected Hek293T cells to evaluate the localisation of the mRNA and preliminary studies indicate an effect of the 3'UTR on mRNA subcellullar localisation. We are planning to derive stable cell lines expressing CTLA4 mutants to further investigate these novel and interesting observations. b) Crosslinking experiments with in vitro transcribed overlapping RNA fragments (300 nucleotide) spanning the full length 3' UTR of CTLA4 and cytosolic or nuclear Hek293T cell lysates were performed. We observed by SDS-PAGE and phophorImager analysis that the length of the (AU)n repeat influences the binding affinity of unknown trans-acting factors. Further experiments will be required to characterise these factors. Task B2. Role of the CT60 polymorphism on mRNA stability and translation efficiency. We have produced by PCR, plasmid expression constructs with various combinations of the (AT)n repeat and the CT60 polymorphic SNP to evaluate the contribution of the CT60 on mRNA stability and CTLA-4 protein expression. Briefly Hek293T cells were transfected with 4 expression plasmids pCDNA3-FlagCTLA4(ORF)-3'UTR(AT)6/24-CT60G/A and RNA and proteins were extracted and characterised by Northern Blot and Western blot analysis. The results indicate that the region of the CTLA4 locus located 3' to the poly(A) signal downregulates the expression of both mRNA and protein, while the CT60 polymorphic site, at least in our experimental conditions, does not appear to contribute to either mRNA stability or protein expression (L. Malquori, L. Carsetti and G. Ruberti, Biochim Biophys Acta. 2008 Jan;1779(1):60-5. Epub 2007 Dec 3.).

Project Abstract - T1DM is a polygenic disease caused by the immune-mediated destruction of pancreatic insulin-secreting-cells. Increasing evidence both in humans and in animal models has shown that defect(s) in immunoregulation underlie autoimmune diabetes, similarly to other immune-mediated disorders. T cell activation results from the integration of signals generated through the T cell receptor with those from additional positive and negative regulatory pathways. Disruption of this balance leads to a defective immune response or alternative over-activation of the system as observed in several human diseases. Genetic studies of human T1DM indicate the presence of a disease locus in a restricted region on chromosome 2q33 (IDDM12) and in the orthologous region of murine chromosome 1. CD28, CTLA4 and ICOS genes, coding for lymphocyte co-stimulatory receptors, map in IDDM12 locus. Data obtained by our group in nuclear families and in a case-control study show an association with T1DM of a series of markers in the IDDM12 region. These appear to belong to two separate association groups, one centered on the CTLA4 locus and the other on the ICOS locus. The specific aims of this project are to: 1. Identify and functionally characterize the disease variation in the ICOS region (Task A); 2. Study the functional role of the T1DM-associated CTLA4 3' UTR (AT)n repeat and the CT60 polymorphis on mRNA stability and targeting (Task B).