Fig. 1. Comparison of PcG associations with Hoxb8 genomic surrounding at 12.5 dpc between anterior and posterior tissues. (A) Embryonic tissues used in this study. Wild-type embryos at 12.5 dpc were dissected as illustrated and anterior (A) and posterior (P) tissues (paraxial mesoderm plus neuroectoderm) were subjected to the ChIP analyses. (B) Comparative analysis of Rnf2 association to regions 2, 14 and D1 between anterior and posterior tissues. The chromatin fraction purified from A or P tissue was subjected to the immunoprecipitation with anti-Rnf2 antibody. Amounts of genomic DNA immunoprecipitated by anti-Rnf2 (A+ or P+) were quantified by comparing with serially diluted genomic DNA isolated from the original chromatin fractions designated as `Input' (see Fig. S2A in the supplementary material) and equivalent amounts of immunoprecipitated DNA to that of `Input' DNA loaded into lane 1 were subjected to PCR reactions. Usually 10 to 20 ng of genomic DNA was used. Immunoprecipitated DNA was also serially diluted. Mock-immunoprecipitated DNA (A- and P-) derived from the same volume of the chromatin fraction as used for anti-Rnf2 immunoprecipitation were subjected to the PCR. The adam34 locus was used as a negative control. (C) Comparative analysis of Phc1 association to regions 2 and 14 between anterior and posterior tissues. (D) Comparative analyses for association of Cbx2 and Ring1 to regions 2 and 14. Amounts of genomic DNA (A+ and P+) immunoprecipitated by anti-Cbx2 and anti-Ring1 antibodies subjected to PCR were equivalent to that of `Input' DNA loaded into lane 1. Mock-immunoprecipitated DNA (A- and P-) derived from the same volume of the chromatin fraction as used for anti-Cbx2 and -Ring1 immunoprecipitation were subjected to the PCR. (E) Rnf110 association to regions 2 and 14. All experiments were conducted as described using anti-Rnf110 antibody. (F) Schematic comparisons of Rnf2, Ring1, Phc1, Cbx2 and Rnf110 association to the Hoxb8 genomic surrounding between anterior and posterior tissues. Genomic organization around Hoxb8 gene is shown at the top. Exonic regions are indicated by black boxes and the exon numbers are numerically shown in the boxes. Positions of known cis-acting regulatory elements are represented by overlying bold bars indicated as BH1100, KA and DE (Charité et al., 1995; Vogels et al., 1993; Oosterveen et al., 2003). Putative promotor regions are indicated by folded arrows. The genomic regions examined by PCR using specific primer pairs listed in Table 1 are shown by bars and numerically indicated. The relative quantity of each genomic region in immunoprecipitated genomic DNA from anterior and posterior tissues was estimated by referring to `Input' DNA isolated from the initial lysates and enrichment values against the `Input', and are represented by the black and gray bars, respectively. Genomic regions left unexamined are covered by boxes crossed with a diagonal line.