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

Fig. 6. Sequential recruitment of PBX/HOX-1 complexes to Msgn1 promoter changes the chromatin landscape and activates transcription.

Fig. 6

a In situ hybridisation shows loss of Msgn1 expression in E8.5 Pbx-com tailbuds. Scale bar: 100 µm. b Relative Msgn1 mRNA expression measured by RT-qPCR, revealing dynamic expression during PSM differentiation and strong downregulation in Pbx-DKO cells (yellow). c UCSC genome browser snapshot of the murine Msgn1 locus showing conservation across vertebrates. PBX1 (light blue) and LEF1 (brown) are recruited to Msgn1 promoter at 12–24 h. LEF1 binding is lost in Pbx-DKO at any time-points. d Pseudotime kinetics of lineage markers along PSM differentiation. Prep1 is expressed before PS induction (marked by T-Bra), while Pbx1, Meis2, Hox-1 are transcribed within the same time-window of Msgn1. e Dot plot analysis of scRNA-seq data of paraxial mesoderm-related clusters showing enriched expression of Pbx, Meis2, Hox-1 in MPCs at 24 h. f ChIP-qPCR analyses of TALE proteins at indicated time-points uncover the peak of PREP1 binding at 12 h and PBX1/MEIS2 at 24 h. g Top: schematics of Msgn1 locus and formation of the PBX/MEIS2/HOX-1 complex. Bottom: Msgn1-P2 oligonucleotide sequence (50 bp in the middle of PBX1 ChIP-seq peak) containing WT (upper case) and mutated (lower case) PBX/HOX-1-binding site (red). h EMSA with in vitro-translated TALE proteins and Msgn1-P2 oligonucleotide. The composition of each binding reaction is indicated. A ternary complex (TC) is formed only when PBX, MEIS2 and HOXA1 are mixed together (lines 4–6). TC is co-migrating with a complex present in MPCs nuclear extracts (line 8). The oligonucleotide with single-base substitutions in the PBX/HOX-binding site (Msgn1-P2-M) abrogates all complex formation (lines 12–14). LYS, reticulocyte lysate (lines 7 and 15). i ATAC-seq of Msgn1 locus in WT and Pbx-DKO cells along differentiation reveals specific opening of PBX1 and LEF1-binding sites. ATAC-seq signals in WT mimic Msgn1 transcriptional profile, with maximum accessibility and expression at 24 h. In Pbx-DKO cells, accessibility to Msgn1 is reduced in the LEF1-binding and the PBX/HOX-1-binding regions. Threshold of vertical viewing range of data based on RPKM values is indicated in c and i. Data are mean ± s.e.m. (n = 2 biological replicates) in b and f.