c-Myb and Cebp1 synergistically regulate lyz transcription. (A) Schematic diagram of lyz promoter region. The transcription initiation site is designated as 0. Seven putative c-Myb (marked by star) and 2 Cebp1 (marked by triangle) consensus sites are identified using promo 3.0 online software and their positions relative to the transcription initiation site are shown. (B-E) Transient reporter assay demonstrates that c-Myb and Cebp1 synergistically regulate lyz transcription. Embryos were injected at 1-cell stage with pTol-GFP reporter constructs driven by an intact 2.4-kb lyz promoter (lyz:GFP), c-Myb sites mutated lyz promoter pTol-lyz(mMBS):GFP, Cebp1 sites mutated lyz promoter pTol-lyz(mCBS):GFP or both sites mutated pTol-lyz(mMBS+mCBS):GFP, and lyz promoter activity were measured at 2 dpf by scoring the number of GFP⁺ cells. Scale bars represent 200 μm. (F) Quantification of GFP⁺ cells in 2 dpf pTol-lyz:GFP, pTol-lyz(mCBS):GFP, pTol-lyz(mMBS):GFP, and pTol-lyz(mCBS+mMBS):GFP-injected embryos (ANOVA, Dunnett T3, n ≥ 160). (G-H) Luciferase reporter assay. Bars showed the relative luciferase activity of transfection with lyz(mMBS):Luc, lyzCBS(mCBS):Luc or both sites mutated lyz(mMBS+mCBS):Luc compared with unmutated lyz:Luc control (G). Synergistic effect on transactivation of lyz promoter by c-Myb and Cebp1 (H). Bars showed the relative luciferase activity with overexpression of cebp1, c-myb, or both genes compared with control (H). The statistical significance was calculated using 1-way ANOVA followed by Dunnett T3 correction. The asterisk indicates a statistical difference (*P < .05, **P < .01, ***P < .001, ****P < .0001, triplicated). (I-N) WISH shows that lyz expression is further decreased in 36-hpf c-myb^hkz3/+;cebp1^smu1/smu1 double (cebp1 homozygous and cmyb heterozygous) mutants (N) compared with cebp1^smu1/smu1 single mutants (M). (O-P) Quantifications of lyz⁺ cells in 36-hpf WT (c-myb^+/+;cebp1^+/+), single-heterozygous (c-myb^hkz3/+ and cebp1^smu1/+), double-heterozygous (c-myb^hkz3/+;cebp1^smu1/+) mutant embryos, cebp1^smu1/smu1 single mutants and c-myb^hkz3/+;cebp1^smu1/smu1 double mutants shown in the statistical figure (O) and table (P). The statistical significance was calculated using 1-way ANOVA followed by Bonferroni correction. The asterisk indicates a statistical difference (**P < .01, ***P < .001, ****P < .0001, n ≥ 16). (Q) Coimmunoprecipitation experiment shows a physical interaction between c-Myb and Cebp1. Embryos overexpressed with Myc-tagged c-myb, and GFP-tagged cebp1 by heat-shock induction were immunoprecipitated with anti-GFP antibody. The immunoprecipitates were subjected to western blotting with anti-Myc and anti-GFP antibody.