Journal: Nature Communications

Article Title: Functional genomics uncovers the transcription factor BNC2 as required for myofibroblastic activation in fibrosis

doi: 10.1038/s41467-022-33063-9

Figure Lengend Snippet: A Input and BNC2 ChIP-seq signals together with CoP-seq and H3K27ac ChIP-seq signals from LX2 cells were visualized at the BNC2 cistrome (1479 binding sites). Heatmaps at the bottom show the signals in 5 kb regions centered on the BNC2 peaks. Average signals are plotted on top of the heatmaps. B The BNC2 cistrome was used to search for proteins with similar genomic binding profiles in CistromeDB and the top 20 hits were ranked according to the GIGGLE similarity score. Proteins involved in TGFβ signaling (SMADs) and Hippo/YAP1 (TEAD and YAP1) were highlighted in red and green, respectively. C Analyses performed as in A to monitor SMAD3 and YAP1 ChIP-seq signals from MFs at the BNC2 cistrome. D Transcriptional regulators specifically identified in BNC2 RIME but not IgG control RIME were clustered according to the percent protein coverage in individual biological replicates ( n = 3 biologically independent experiments; anti-BNC2 antibody 55220-1-AP, Proteintech). Percent coverage obtained in an additional BNC2 RIME experiment using the anti-BNC2 antibody HPA018525 (Sigma-Aldrich) is shown on the right. E Nuclear extracts from LX2 cells expressing recombinant BNC2 and YAP1 were subjected to immunoprecipitation with an antibody against BNC2 (55220-1-AP, Proteintech). Immunoprecipitated material was analyzed by western blot using antibodies directed against BNC2 or YAP1. The presented data are representative of two biologically independent experiments. MW, molecular weight markers. F , G The Integrated Genome Browser (IGB) was used to visualize ChIP-seq profiles for BNC2 (black track, LX2 cells), SMAD3 (red track, LX2 cells), YAP1 (green track, IMR90 cells), and H3K27ac (blue track, MF-HSCs) at the COL1A1 ( F ) and BNC2 ( G ) genes.

Article Snippet: LX2 cells were transfected for 48 h with a plasmid coding for human BNC2 (custom construction, E-Zyvec) and a plasmid coding for constitutively active nuclear YAP1 (YAP1-S127A, Addgene).

Techniques: ChIP-sequencing, Binding Assay, Control, Expressing, Recombinant, Immunoprecipitation, Western Blot, Molecular Weight

Journal: Nature Communications

Article Title: Functional genomics uncovers the transcription factor BNC2 as required for myofibroblastic activation in fibrosis

doi: 10.1038/s41467-022-33063-9

Figure Lengend Snippet: A RT-qPCR data showing changes in gene expression upon murine primary Q-HSCs spontaneous in vitro activation into MF-HSCs ( n = 6 biologically independent experiments). Log 2 fold changes (FC) between MF-HSCs (6 days of culture) and Q-HSCs (1 day of culture) are shown. B RT-qPCR data showing changes in gene expression upon YAP1 inhibition in MF-HSCs. Isolated mouse primary HSCs were treated with 1 µM verteporfin or vehicle for 6 days before being harvested [ n = 3 ( Yap1 ), 4 ( Bnc2 and Ctgf ), or 5 ( Col1a1 and Acta2 ) biologically independent experiments]. Log 2 FC between verteporfin and vehicle-treated cells are shown. C RT-qPCR data showing changes in gene expression upon Yap1 silencing in MF-HSCs ( n = 3 biologically independent experiments). Log 2 FC between siYAP1 and siCTRL-transfected cells are shown. D RT-qPCR data showing differences in gene expression in mouse primary HSCs grown for 9 days in 3D (spheroids) or in 2D [ n = 3 ( Bnc2 ) or 4 ( Col1a1 , Acta2 , Yap1 , Ctgf ) biologically independent experiments). Log 2 FC between cells grown in 3D and 2D are shown. B – D Expression of Ctgf , an established YAP1 target, was assessed. E RT-qPCR data showing changes in gene expression induced by treatment of MF-HSCs with TGFβ (1 ng/mL) for 24 h ( n = 4 biologically independent experiments). Log 2 FC between cells treated with TGFβ and vehicle are shown. F RT-qPCR data showing changes in gene expression induced by treatment of EMS404 MF-HSCs with the indicated TGFβ signaling inhibitors for 24 h ( n = 3 biologically independent experiments). Log 2 FC between cells treated with TGFβ signaling inhibitors and vehicle are shown. In all panels, bar graphs show means ± SD. Statistical significance was assessed using two-sided one-sample t test with Benjamini–Hochberg correction for multiple testing to determine if the mean log 2 FC was statistically different from 0. * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: LX2 cells were transfected for 48 h with a plasmid coding for human BNC2 (custom construction, E-Zyvec) and a plasmid coding for constitutively active nuclear YAP1 (YAP1-S127A, Addgene).

Techniques: Quantitative RT-PCR, Gene Expression, In Vitro, Activation Assay, Inhibition, Isolation, Transfection, Expressing