Fig. 5: Direct and functional interaction between Ubx and Tin.

a ChIP-seq of Ubx² and ChIP-on-chip profiles of Tin⁴⁵ at the dpp genomic locus in mesodermal cells. Isoforms of the dpp gene are shown (blue) and the known dpp visceral enhancer (light blue). The box highlights Ubx and Tin binding to the dpp674 enhancer. b–g Immunostaining of stage 11 tin³⁴⁶/TM6-Dfd>lacZ (b), Ubx¹/TM6-Dfd>lacZ (c), Ubx¹,tin³⁴⁶/TM6-Dfd>lacZ (d), tin³⁴⁶/tin³⁴⁶ (e), Ubx¹/Ubx¹ (f) and Ubx¹,tin³⁴⁶/tin³⁴⁶,Ubx¹ (g) embryos for dpp mRNA (green) and β-Galactosidase protein (red). Images are representative of all embryos analysed (n = 15) per genotype over 2 sets of pooled embryos from independent collection. h Quantification of relative signal intensity of dpp mRNA levels shows significant expression changes between tin³⁴⁶/TM6-Dfd>lacZ and Ubx¹,tin³⁴⁶/TM6-Dfd>lacZ heterozygous mutants (n = 15 independent embryos). i S2R+ cells were co-transfected with a dpp674-containing plasmid driving luciferase expression, myc-Ubx and V5-Tin encoding plasmids (100 ng). Increasing amounts of Ubx^WT or Ubx^N51A (left) or Tin expressing plasmids (right) was used. Transfection efficiency was normalized with Renilla activity originating from co-transfected pRT-TK plasmid. Results are indicated relative to basal activity of the dpp674-luciferase plasmid. Graphics represent mean +/− sem of three (n = 3) independent experiments performed in triplicates. j Schematic of Ubx fragments used for GST in vitro pull-down assays. Hexapeptide (HX) motif is highlighted in blue, the Homeodomain (HD) in green and the UbdA motif in dark-red. k Pull-down assay using the indicated GST-fused Ubx derivatives and in vitro purified Tin. Input is loaded as indicated. l, m Quantification of interactions relative to GST-Ubx^FL (full length) signal is indicated in (l) for V5-Tin (n = 3) and in (m) for myc-Exd (n = 2 independent experiments) as mean ± SD. n Pull-down assay using GST-fused Ubx derivatives and in vitro His-purified GFP (negative control), myc-Exd (positive control) and V5-Tin proteins. Input is loaded as indicated. Protein size is indicated relative to ladder position. Pull-down assays showed direct interaction of Ubx^WT as well as Ubx^N51A with Exd and Tin but not with GFP (lane 3–4). Ubx-Tin interaction is at least 10 times stronger than Ubx-Exd interaction as exemplified by intensity of signal in pull-downs compared with input (comparison of lane 3–4 with 1). Statistical tests were performed with one-way ANOVA (**p < 0.01, *p < 0.05). Scale bar = 50 µm. See also Supplementary Fig. 7. Source data are provided as a Source Data file.