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. 2021 Aug 26;12:5136. doi: 10.1038/s41467-021-25370-4

Fig. 7. Combined pioneering activity of T-BRA and TALE proteins remodels the chromatin, making it accessible at Msgn1 regulatory regions, thereby directing WNT transcriptional response.

Fig. 7

a ChIP-qPCR analysis showing specific recruitment of T-BRA to the LEF1-binding site of the Msgn1 regulatory region at 12 and 24 h. T-BRA binding is maintained in Pbx-DKO cells (yellow). b Heatmap displaying relative expression of selected differentially regulated genes in WT (black), Pbx-DKO (yellow) and pMsgn1-mut cells (blue) at 24 h (RNA-seq, P ≤ 0.05, fold-change ≥1.5). pMsgn1-mut allele carries point mutations in the PBX/HOX-1-binding site. Representative PSM and NMP markers are indicated. n = 2 biological replicates. Significance was assessed by DESeq2 on the basis of two-sided Wald test with Benjamini–Hochberg adjusted P-values. c Representative immunofluorescence staining for T-BRA (white), SOX2 (red) and TBX6 (green) at 48 h of differentiation reveals expansion of NMPs (T-BRApos;SOX2pos, dashed white lines) and reduction of PSM (TBX6pos) in pMsgn1-mut cells. Scale bar: 50 µm. d ChIP-qPCR analysis on the Msgn1 promoter displays PBX1 recruitment at the PBX/HOX-1-binding site in WT (black), but not in Pbx-DKO (yellow) nor in pMsgn1-mut cells (blue). Similarly, ChIP-qPCR analyses with LEF1 and T-BRA antibodies highlight specific binding of LEF1 and T-BRA to the LEF1-binding region in WT cells. While T-BRA is recruited to the Msgn1 promoter in Pbx-DKO and pMsgn1-mut cells, LEF1 binding is lost in both. Data are mean ± s.e.m. (n = 2 biological replicates) in a and d. e Chromatin accessibility analysis (ATAC-seq) of the Msgn1 locus in WT, Pbx-DKO and pMsgn1-mut cells along PSM differentiation displays specific open regions overlapping with the PBX (light blue) and LEF1-binding sites (brown) in WT cells at 12 and 24 h. In Pbx-DKO cells, accessibility is strongly reduced at the PBX/HOX-1-binding region. Additionally, accessibility of the Msgn1 locus is severely impaired in both LEF1 and PBX/HOX-1-binding regions in pMsgn1-mut cells, confirming the role of the PBX complexes as modulators of chromatin accessibility on WNT-responsive elements. Threshold of vertical viewing range of data based on RPKM values is specified.