Fig. 2.

Recruitment of retinoic acid receptors to the mouse Pitx2 promoter in vivo. (A) Schematic representation of the mouse Pitx2 upstream region containing two putative DR5 retinoic acid response elements (RARE) located 3.6 and 4.3 kb upstream of transcription start site. Arrows indicate the location of primer pairs used to amplify the RARE-containing regions and a non-specific negative control region (NSR) located 595 bp downstream for chromatin immunoprecipitation (ChIP) assay. (B) Nucleotide sequence alignment of conserved DR5a and DR5b RAREs in mouse, rat, and human Pitx2 genes. ChIP assay was performed using (C) E8.5 mouse embryos and (D) E12.5 eyes. The cross-linked protein-DNA complexes were immunoprecipitated with anti-RAR-α, RAR-β, and RAR-γ antibodies. Mouse IgG was used as a negative control. Input material and immunoprecipitates were analyzed by PCR with specific primers flanking the RARE and non-specific primers located several kb away for the mouse Pitx2, RARβ, and Raldh1 promoters. S: RARE specific primers, NS: Non-specific primers.