Fig. 6: Multi-omic analyses reveal a role for O-GlcNAcylation in controlling the myofibroblastic transcriptional regulatory landscape.

A Identification of transcriptional regulatory regions decommissioned upon treatment with OGT inhibitor OSMI-1 (referred to as OGTi) in LX-2 cells using mining of H3K27ac ChIP-seq data. B Characterization of O-GlcNAcylation-dependent transcriptional regulatory regions. Average H3K27ac ChIP-seq (n = 4 biologically independent experiments) and CoP-seq signals from LX-2 cells treated or not with 50 µM OGTi for 24 h. Heatmaps show the signals in ±2.5 kb windows around the center of regions identified as displaying a significant loss of H3K27ac in OGTi-treated cells (n = 3,507 regions). C Association between transcriptional regulatory regions losing H3K27ac (from B) and deregulated genes (RNA-seq from Fig. 4) in OGTi-treated LX-2 cells displayed as log₂ Odds ratio. See “Materials and methods” section for the procedure used to assign H3K27ac regions to genes. Two-sided Fisher’s exact test was used to assess the statistical significance of the biased association with down- or upregulated genes. D Identification of TRs bound to O-GlcNAc-dependent regulatory regions (i.e. comparison of a database of TR cistromes with regions showing significant loss of H3K27ac in OGTi-treated cells) was performed along with identification of O-GlcNAcylated TRs in LX-2 cells using a click chemistry-based approach coupled to mass spectrometry as described in the “Materials and methods” section (n = 3 biologically independent experiments). E. TRs whose cistrome significantly overlaps with regions losing H3K27ac upon OGTi treatment of LX-2 cells were retrieved as detailed in the “Materials and methods” section and are reported here. Those identified in the LX-2 O-GlcNAcome were further highlighted in blue. F The 16 O-GlcNAcylated TRs (from E) were ranked according to their specificity of expression in MF-HSCs defined as log₂ fold changes (log₂ FC) in MF-HSCs compared to average expression in 112 other human non-MFs primary cell types. G. Specific expression of BNC2, TEAD4, YAP1 and TEAD1 in MFs is shown using log₂ FC in MF-HSCs or MFs (n = 13) compared to average expression in 112 other human non-MFs primary cell types. H LX-2 cells were transfected with 20 nM siTEAD4 or siControl and cells were harvested 72 h later. Western blot assays (left panel) and TEAD4, COL1A1 and COL3A1 protein levels quantifications (middle panel) are shown. HSP90 was used as protein loading control. The presented images are representative of 3 biologically independent experiments. MW, molecular weight markers. The right graph shows RT-qPCR data (n = 3 biologically independent experiments). Log₂ FC between siTEAD4 and siControl conditions are shown. The bar graph shows means + SD together with individual biological replicates. Two-sided one-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log₂ fold changes (log₂ FC) were statistically different from 0. I. Heatmaps show the average BNC2, TEAD4 (LX-2 cells) as well as YAP1 (IMR90 cells) ChIP-seq signals in ±5 kb windows around the center of regions identified as displaying a significant loss of H3K27ac in OGTi-treated cells (n = 3507 regions from B). J Nuclear extracts from LX-2 cells were subjected to immunoprecipitation with an antibody against TEAD4 (ab58310, Abcam). Input and immunoprecipitated materials were analyzed by simple western immunoassay using antibodies directed against BNC2, TEAD4, and YAP1. The presented data are representative of two biologically independent experiments.