Figure 5.
Sox100B Directly Regulates the Activity of the Sox21a Intronic Enhancer
(A) Representative images showing that 24 h Sox100B overexpression is sufficient to induce ectopic Sox21a1st intron dsRed in L3 stage larval wing imaginal disc.
(B) Representative confocal images showing that 24 h Sox100B overexpression is sufficient to induce ectopic Sox21a protein expression in larval wing imaginal disc.
(C–E) (C) Schematic diagram representing the experimental design to identify putative Sox100B binding sites in the first intron of Sox21a using Drosophila S2 cells. A series of reporter constructs using different fragments of the full-length first intron (fragments 1–4) identified a 106 bp region that is necessary and sufficient to mediate Sox100B-induced dsRed reporter expression. Then site-directed mutagenesis of three candidate sites (sites 1–3) in the mapped region were constructed and tested, revealing that both site 2 and site 3 are necessary for reporter induction. Representative images of S2 carrying the different fragments are shown in (D). Quantification of the dsRed signal, normalized to GFP expression, is shown in (E) for the full-length fragments carrying individual mutations of the predicted Sox binding sites. In (E), n represents the number of transfected cells (GFP-positive cells) analyzed; p values were calculated using unpaired two-tailed Student's t test.
(F) A proposed scheme depicting the role of Sox100B-Sox21a transcriptional cascade in the process of EB to EC differentiation. Sox100B is required for proper EB differentiation progression by regulating Sox21a and potentially other differentiation factors.