Fig. 1. Hoxa2 binds to the Six2 promoter in vivo. (A,B) Western blot using anti-HA (A) and anti-Hoxa2 polyclonal antibody 43 (B) on whole extracts of human 293 cells transfected with empty vector (control), pCDNA3-Hoxa2-HA (Hoxa2-HA) or pCDNA3-Hoxb2-HA (Hoxb2-HA). Arrows indicate the expected position of Hoxb2-HA. (C) Side view of the facial region of an E11.0 mouse embryo, showing the areas isolated for ChIP (red, maxillary component of first arch and frontonasal mass; blue, second arch). (D) Schematic of the Six2 genomic locus around the transcriptional start site (+1), with red boxes indicating the relative position of the two Hoxa2 binding sites identified in vitro (Kutejova et al., 2005) and gray arrows indicating the position of the primers used for PCR amplification. (E) PCR amplification of the immunoprecipitated chromatin from E11.5 second branchial arch (2nd) or from frontonasal mass and first branchial arch (1st) using anti-Hoxa2 polyclonal antibodies 43 and 44 or normal rabbit IgG. (F) Same experiment as in E, using polyclonal anti-polymerase II antibodies to control for the quality of first arch chromatin: first arch chromatin is enriched for the Six2 proximal promoter fragment, as expected for a gene actively transcribed in this area (Oliver et al., 1995). (G) PCR amplification with Six2 or IP10 (Cxcl10; control) primers of E10.5 second branchial arch (2nd) chromatin, immunoprecipitated using anti-Hoxa2 antibody 43, or normal rabbit IgG. The number of PCR cycles is indicated in the bottom-right corner. EB, elution buffer. ChIP was performed on three independent pools of samples, and PCRs were performed in duplicate on each pool. Results shown are from a representative set.