Fig. 2: Y337H abolishes TCF7L2 DNA binding ability and impairs pup USVs.
a The genome structure of mouse Tcf7l2. The alternatively spliced exons were marked in red. The ENU-induced nonsynonymous mutation identified in family #30 (c.T1019C, p.Y337H, ENSMUST00000111656.7) is located in Tcf7l2 exon 10 that encodes part of HMG box. DNA chromatogram illustrates the mutation (Y337H/ + ) (lower). b The sequence conservation of TCF7L2 HMG box and its neighboring residues. *, Y337, W365, Y376, and Y377; O, M335 and M338; red ribbon, alpha helix segments. c The Y337 and neighboring residues were adapted into previously reported HMG box/DNA interface. The hydrophobic core formed by Y337, W365, Y376, and Y377 is highlighted. d The expression of TCF7L2 in wildtype (+/+) and mutant (Y337H) midbrain from 4-month old mice. GAPDH, as a loading control. TCF7L2 appeared two major bands (Short and Long) in thalamus (upper) and statistic analysis of the relative expression level of these two bands (lower) in + /+ and Tcf7l2Y337H/+ mice. The anti-TCF7L2 antibody used here was generated by a synthetic peptide corresponding to sequences in HMG box. *, the major smaller band (Short). e–j HMG/DNA binding ability measured by EMSA. WT and the various mutant TCF7L2 HMG proteins fused with MBP-tag were purified by anti-MBP beads (e). k Representative spectrogram of USVs produced by + /+ and Tcf7l2Y337H/+ pups detected by MUPET. l–o The key features of USVs produced by + /+ and Tcf7l2Y337H/+ pups at P7. The value are presented as mean ± SD. In d, n = 4 (+/+ and Y337H/ + ); in l–o, +/+, n = 7, Y337H/ + , n = 8; t-test, SPSS. N.S., no significant difference.
