Fig. 4

c-Myb and Runx1 synergistically regulate neutrophil-specific genes transcription. A, schematic diagram of the lyz, mpx, srgn, and npsn promoter region. The transcription starting site is designated as TSS. Putative c-Myb and Runx1 binding sites are marked by stars and triangles respectively using JASPAR online software. B, ChIP shows that MYC-tagged c-Myb and MYC-tagged Runx1 bind to the promoter region of the lyz, mpx, npsn, and srgn promoters. Lysates from the embryos injected with the Myc-c-myb (left panel) and Myc-runx1 plasmids (right panel) were precipitated with anti-MYC antibodies. The precipitates were then subjected to semiquantitative PCR analysis compared with anti-IgG control. Input DNA control was on the left of each panel. C, Upper panel shows representative procedures of GFP reporter assay. lyz(-2.4k):GFP, mpx(-8k):GFP, npsn(-2k):GFP and srgn(-5k):GFP coinjected with or without hs:c-myb, hs:runx1 plasmid. Two groups were classified by GFP fluorescence intensity and GFP+ cells number. Lower panel shows embryo percentage of each group (each n ≥ 20). Gray represents weaker GFP expression and less GFP+ cells. Dark gray represents stronger GFP expression and more GFP+ cells. D, in vitro coimmunoprecipitation experiment detected the interaction between c-Myb and Runx1. Myc-tagged c-myb and Flag-tagged runx1 were transfected into 293T cells as indicated and cell lysates were immunoprecipitated with anti-FLAG antibody. The immunoprecipitants were examined by western blot using anti-MYC and anti-FLAG antibodies. Input represents 10% of total cell lysates used for immunoprecipitation. E, deletions impinging on the c-Myb DBD (top panel) and Runx1 RHD (low panel) decrease the interaction between c-Myb and Runx1. MYC-c-Myb was immunoprecipitated, and western blots were probed with antibodies to MYC or FLAG. F, summary of c-Myb and Runx1 mapping experiments. AD, activation domain; DBD, DNA-binding domain; ID, inhibitory domain; NRD, negative-regulatory domain; RHD, RUNT-homology domain; TAD, transactivation domain.