Fig 2: 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 3: PAA promoted TPH-1 expression to regulate the protein stability of ß-catenin and ß-catenin-mediated transcription. (a) The protein expression and relative quantitative data of ß-catenin and TCF4 in UUO mice. (b) The protein expression and relative quantitative data of GSK-3ß and Axin1 in UUO mice. (c) The protein expression and relative quantitative data of ß-catenin and TCF4 in NRK-52E cells. (d) The protein expression and relative quantitative data of GSK-3ß and Axin1 in NRK-52E cells. Blue, sham or CTL group; gray, TPH-1 shRNA-transfected or PAA-treated group; black, UUO or TGF-ß1-treated group; purple or red, UUO + TPH-1 shRNA or TGF-ß1 + PAA group.**p < 0.01 compared with sham-operated or CTL group (n = 6).##p < 0.01 compared with UUO or TGF-ß1-treated group (n = 6).Dot presented the single data results in the bar graph.CTL, control; GSK, glycogen synthase kinase 3ß; PAA, poricoic acid A; TGF, transforming growth factor; TPH-1, tryptophan hydroxylase-1; UUO, unilateral ureteral obstruction.

Fig 4: OL differentiation defects in the brain of Tcf7l2?HMG mice lacking the DNA-binding HMG domain.(a) Schematic diagram shows domains in Tcf7l2 (upper) and Cre-mediated excision (lower) of the floxed exon 11, which encodes the DNA-binding HMG domain. NLS: Nuclear localization sequence. (b) Upper panel, expression of Tcf7l2 exon 11 in the mRNAs of OPCs isolated from control (Ctrl) and Tcf7l2?HMG brains at P2 was assayed by qRT–PCR (n=3 animals per genotype). Lower panel: western blot analysis of the spinal cords from control and Tcf7l2?HMG mice at P10 with anti-Tcf7l2 (amino-terminal epitope) and glyceraldehydes 3-phosphate dehydrogenase (GAPDH; loading control). (c) Expression of Mbp and Plp1 in cortices from control and Tcf7l2?HMG mice by in situ hybridization. Arrows indicate the cerebral white matter in coronal sections, except P60 Mbp in sagittal sections. (d) MBP immunostaining in the cortices from control and Tcf7l2?HMG mice at P14 assayed by immunohistochemistry. (e) Expression of membrane-anchored enhanced GFP (EGFP) driven by a CNP promoter in the cortices from control and Tcf7l2?HMG mice carrying the CNP-mGFP transgene at P14. (f) Quantification of Plp1+ OLs (per mm2) in the cortex of control and Tcf7l2?HMG at P60; n=3 animals per genotype. (g) The cortices of Ctrl and Tcf7l2?HMG mice at P14 were immunostained with anti-PDGFRa antibody. (h) PDGFRa+ OPC numbers were quantified per mm2; n=3 animals per genotype. (i) BrdU incorporation in the cortex of control and Tcf7l2?HMG mice carrying PDGFRa-GFP reporter and pulse labelled with BrdU for 2 h at P14. (j) BrdU+/PDGFRa+ OPC cell numbers were quantified per mm2; n=3 animals per genotype. (k,l) Electron microscopy of the optic nerves (OP) and corpus callosum (CC) of control and Tcf7l2?HMG mice at P14 and P60. (m) The percentages of myelinated axons in the OP and CC at P14 and P60; n=3 animals per genotype. Data are presented as mean±s.e.m. ***P<0.001; Student's t-test. Scale bars, 100 µm (c,d), 50 µm (e,g–j) and 2 µm (k,l).

Fig 5: Tcf7l2 coordinates with Sox10 to regulate OL terminal differentiation.(a) De novo motif analysis identified Tcf7l2/Tcf4, Sox10-binding motifs as most significant binding motifs in Tcf7l2-binding regions in mOLs. (b) ChIP-seq binding profiles of Sox10 around Tcf7l2 peak summits in mOLs. (c) Heatmap of the signal intensities from ChIP-seq assays of Tcf7l2 and Sox10 across Tcf7l2-binding sites (±1 kb) called in mOLs. (d) Sox10 co-immunoprecipitated with Tcf7l2 in mOLs. (e) Visualization of Tcf7l2-binding profiles in OPCs, iOLs and mOLs on representative myelin gene loci (Mbp, Myrf, Olig1,Ugt8 and Zfp191). Sox10/Tcf7l2 co-occupancy (highlighted) in mOLs was also shown. (f) qRT–PCR assay for Mbp, Cnp expression in OPCs transfected with expression vectors for Tcf7l2, Sox10 or both; n=3 independent experiments. (g) The expression vector carrying Flag-Tcf7l2 was co-transfected with HA-Sox10 and a varied amount of Myc-Kaiso in 293T cells for 48 h. Lysates were co-immunoprecipitated with anti-Flag-Tcf7l2 and subjected to western blot analysis. Glyceraldehy 3-phosphate dehydrogense (GAPDH) as a loading control. (h) Model of Tcf7l2 regulation of OL differentiation through sequential interactions with Kaiso and Sox10 to promote stepwise OL lineage differentiation. At the onset of OPC differentiation, Tcf7l2 binds Kaiso to inhibit Wnt signalling activation and subsequently associates with Sox10 to promote OL maturation. Data are presented as mean±s.e.m. *P<0.05, **P<0.01 and ***P<0.001; analysis of variance (ANOVA) with Newman–Keuls multiple comparison test.