Fig. 8: O-GlcNAcylation of BNC2 and TEAD4 regulates their stability and chromatin binding.

A LX-2 cells were treated with 2 µM Thiamet-G for 24 h before BNC2 and TEAD4 immunoprecipitations followed by HCD mass spectrometry. HCD-MS/MS spectra of peptides covering the T455 and S490 O-GlcNAcylated sites in BNC2 and the S69 and S99 O-GlcNAcylated sites in TEAD4 are shown. The identified O-GlcNAcylated amino-acid residues are indicated in red in the peptide sequence. B, C LX-2 cells were transfected for 24 h with a control plasmid (mock), or plasmids encoding wild-type (WT) or mutated versions of 3x-FLAG-BNC2 (B) and Myc-TEAD4 (C). Sub-cellular fractionation was performed to obtain cytosolic and chromatin fractions used for western blot or simple western immunoassays using anti-FLAG (B) or anti-Myc (C) antibodies. The presented images are representative of three biologically independent experiments. Bar graphs show relative levels of chromatin-bound 3xFLAG-BNC2 (B) and Myc-TEAD4 (C). LMNA was used as protein loading control. Fold changes (FC) relative to WT (arbitrarily set to 1) are shown. Two-sided one-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log₂ FC between individual mutants and control was statistically different from 0. MW, molecular weight markers. D, E LX-2 cells were transfected as in B, C and cells were treated or not with 50 µg/mL cycloheximide (CHX) for an additional 24 h. Extracts were analyzed by western blot or simple western immunoassays using anti-FLAG (D), anti-Myc (E) or anti-CCND1 antibodies. The presented images are representative of 4 biologically independent experiments. Bar graphs show quantification of 3xFLAG-BNC2 and Myc-TEAD4 levels. HSP90 was used as protein loading control. Bar graphs show relative protein decrease induced by CHX which were calculated by subtracting the log₂ FC CHX / control of double mutants by that of WT proteins. Unpaired t-test was used to assess the statistical significance of differences in log₂ FC CHX / control between double mutants and WT proteins. MW, molecular weight markers. For all panels, the bar graphs show means + SD together with individual biological replicates.