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. 2011 Dec;54(12):3078-82.
doi: 10.1007/s00125-011-2290-6. Epub 2011 Sep 14.

An Alternative Polyadenylation Signal in TCF7L2 Generates Isoforms That Inhibit T Cell Factor/Lymphoid-Enhancer Factor (TCF/LEF)-dependent Target Genes

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Free PMC article

An Alternative Polyadenylation Signal in TCF7L2 Generates Isoforms That Inhibit T Cell Factor/Lymphoid-Enhancer Factor (TCF/LEF)-dependent Target Genes

J M Locke et al. Diabetologia. .
Free PMC article

Erratum in

  • Diabetologia. 2011 Dec;54(12):3170

Abstract

Aims/hypothesis: Intronic single nucleotide polymorphisms within the transcription factor 7-like 2 (TCF7L2) gene are associated with risk of type 2 diabetes. It is widely hypothesised that the predisposing variation is involved in cis-regulation of TCF7L2 activity. The aim of this study was to seek evidence for the existence of novel TCF7L2 isoforms encoded within the type 2 diabetes-associated genomic region.

Methods: We searched expressed sequence tag (EST) databases for novel TCF7L2 transcripts and sought to validate the function and integrity of any isoforms found using a combination of RT-PCR, western blotting and reporter gene techniques.

Results: Analysis of EST databases suggested the presence of an alternative polyadenylation site located in intron 4 of TCF7L2. We used 3' rapid amplification of cDNA ends and real-time PCR to validate the integrity of this polyadenylation signal and show its wide use across human tissues. Western blotting results are consistent with the use of this polyadenylation signal to generate novel protein isoforms. The alternative polyadenylation signal results in the production of isoforms that retain the β-catenin binding domain but do not possess the high-mobility group box DNA-binding domain. Promoter-reporter gene assays suggest that these isoforms inhibit TCF7L2-dependent target genes by sequestering β-catenin.

Conclusions/interpretation: We have identified a novel polyadenylation signal within TCF7L2 that can result in the production of isoforms that act to repress TCF/LEF-dependent target genes. These findings may provide new insights into the association of TCF7L2 with susceptibility to type 2 diabetes.

Figures

Fig. 1
Fig. 1
Identification of an alternative polyadenylation signal within intron 4 of TCF7L2 widely used in human tissues. a Schematic depicting the location of the alternative polyadenylation signal within the human TCF7L2 gene. The dark grey rectangle represents the isoform-specific coding region, and the light grey rectangle the isoform-specific 3′ untranslated region, generated by the use of the novel polyadenylation signal. Cis sequences probably involved in 3′ end formation are detailed with boxes encompassing the polyadenylation sequence and U-rich element. The arrow indicates the cleavage site. Positions of the amplicons for TaqMan analysis are shown. b The relative use of the alternative polyadenylation site across a selection of human tissues involved in the pathogenesis of type 2 diabetes. Relative expression was determined using custom TaqMan assays that amplify the isoform-specific coding region and intronic sequences downstream of the alternative cleavage site. c The relative expression of full-length TCF7L2 expression across human tissues. Expression was normalised using the GeNorm algorithm, which selected B2M and GUSB as the two most stable housekeeping genes of those measured. All expression values are relative to that of one kidney sample. Numbers in brackets indicate the number of separate RNA samples on which expression was measured. Data are presented as the mean and standard error
Fig. 2
Fig. 2
The use of the alternative polyadenylation signal generates protein isoforms with the ability to inhibit TCF/LEF-dependent transcription. a TCF7L2 protein production in human adult pancreas, small intestine and HeLa cells using a pAb. The specificity of the antibody–protein interaction at 20 kDa is proven by a blocking peptide (+BP) experiment and use of a monoclonal TCF7L2 antibody (mAb) predicted to bind to the truncated isoform. Fibrillarin was used as a loading control. b An isoform generated by the use of the alternative polyadenylation signal inhibits TCF/LEF-dependent target genes by sequestering β-catenin. MIN6 cells were co-transfected with firefly and Renilla luciferase reporter genes and expression vectors as indicated. The β-catenin (β-cat) construct encodes a constitutively active form of this protein. Firefly luciferase production was driven by a promoter containing three copies of the consensus TCF/LEF-binding motif, CCTTTGATC. Renilla luciferase was measured to correct for differences in transfection efficiency. Reporter gene activities were determined 48 h post-transfection. Bars represent relative luciferase activities compared with control transfected with empty vector and β-catenin. Mean values and standard deviations from five independent experiments are given. Western blotting was performed to determine TCF7L2 and tubulin protein production in transfected samples. **p < 0.01

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