Fig. 3: In vivo analysis of the mesodermal Ubx BioID-interactome.

a STRING-based reconstruction of the interaction network of all proteins identified as close-proximity partners of Ubx^WT in the mesodermal tissue by targeted BioID. Green circles represent RNA-binding/regulatory proteins, blue circles represent chromatin and DNA-binding proteins, pink circles highlight TFs and white circles label proteins with unknown functions. b Left panel: Venn diagram representing the overlap of proteins enriched in close proximity to the wild-type (Ubx^WT) and mutant (Ubx^N51A) versions of Ubx protein in the mesoderm, which showed that 33 proteins interacted with Ubx preferentially on the chromatin (purple), 27 in the nucleus (dark purple) and 46 in the nucleoplasm (brown). Right panel: Fold enrichment of gene ontology terms of proteins representing the different overlap classes (chromatin, nucleus, nucleoplasm). c Co-immunoprecipitation of Ubx close-proximity partners from nuclear extract of control (w¹¹¹⁸) or GFP-Ubx embryos, which carry a CRISPR/Cas9 engineered version of the Ubx gene, GFP-Ubx, at the endogenous locus². For co-immunoprecipitation, endogenous expression levels of all proteins were used. The input fraction is present as control (lane 1–2). Tin and CtBP were co-immunoprecipitated with GFP-Ubx (IP^GFP-lane 4), which was not the case using purified extracts from w¹¹¹⁸ embryos (IP^GFP-lane 3). As positive control, the known Ubx interactor M1BP was used, which was immunoprecipitated with GFP-Ubx, while Tubulin (Tub), histone H3 (H3) and Polycomb (Pc) were not pulled down with Ubx (lane 4). Protein size is indicated relative to ladder position. d–k Immunostaining of stage 11 embryos (3–6 h AEL) for Ubx (red) and the BioID-identified mesodermal close-proximity partners (green) CtBP (d, h), Cg (e, i), Tin (f, j) and Zld (g, k). To mark mesodermal cells, stainings were performed in the twi-INTACT background, which uses the tissue-specific biotinylation of the nuclear membrane protein RanGAP, and allows the detection of mesodermal nuclei by streptavidin staining (magenta). Bottom panel represents high-magnification images of mesodermal nuclei. GO term p-value are calculated with Fisher test and FDR correction. Images are representative of all embryos analysed over 2 sets of pooled embryos from independent collections. Scale bar = 50 µm; zoom scale bar = 10 µm. See also Supplementary Figs. 5 and 6 and Supplementary Data 36–39. Source data are provided as a Source Data file.