Fig. 3. SOX4 and SOX11 bind to and activate E4 via competition with SOX5.

a, Chromatin immunoprecipitation (ChIP) from Neuro-2a cells expressing V5-tagged SOX4 and SOX11. Captured DNA was analyzed by PCR using primers specific for E1-4. SOX4 and SOX11 bound and recruited Pol II to E4 but not E1-3. b, Analysis of SOXC transactivation using empty (pGL4) or E1-4 containing luciferase vectors. The activity of E4, but not E1-3, was significantly increased by Sox4 (≥ 4 fold) or Sox11 (≥ 13 fold), but not Sox12 (≤ 1.5 fold). c-d, Analysis of functional elements within E4 using luciferase vectors containing deletion fragments of E4 (E4F1-4) (c). E4F2, but not the other fragments, was significantly activated by co-transfection of Sox4 (≥ 5 fold) or Sox11 (≥ 9 fold), but not Sox12 (≤1.5 fold) (d). e, Analysis of putative SOX binding sites (SB1-3) using mouse (Mo) E4F2 luciferase vectors mutagenized by substitution (blue lowercase nucleotides) with zebrafish (Ze) sequence. Targeted mutation of SB2 (MoE4F2-m2) significantly diminished the trans-activating ability of SOX4 or SOX11. f, Electrophoretic mobility shift assay (EMSA) with biotin-labeled SB2 DNA. SOX4-V5 and SOX11(1-276)-V5 shifted wildtype (arrowhead) but not mutated SB2 DNA. SOXC-SB2 complexes were super-shifted (open arrowhead) by an anti-V5 antibody. g, A schematic model of E4 regulation by SOX5 and SOXC. h-i, Analysis of competition between SOX5 and SOXCs using the E4-containing luciferase vector. Decreasing concentrations of co-transfected Sox5 led to a dose-dependent increase in E4 activation in response to Sox4 or Sox11 (h), whereas decreasing concentrations of co-transfected Sox4 or Sox11 led to a dose-dependent increase in Sox5 repression of E4 (i). One-tailed Student’s t-test; **P<0.01, ***P<0.001. n = 4 per condition. Error bars represent s.e.m.