DEV02405 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Solubilization and fractionation of the chromatin and PcG complexes after chemical crosslinking of 12.5 dpc embryonic tissue. (A) Presence of the chromatin and PcG proteins in the soluble fraction of crosslinked embryonic tissues after extensive sonication. Equivalent volume of soluble and insoluble materials against the original lysate was loaded to each lane and subjected to immunoblot analyses for Rnf2, Ring1, Phc1, Cbx2 and Histone H3 and ethidium bromide staining for genomic DNA. Most of the genomic DNA and PcG proteins are solubilized under this condition. (B) Preparation of chromatin fraction by CsCl isopycnic centrifugation. The chromatin fractions were purified from the soluble fraction (also called whole-cell lysate in the text) by CsCl isopycnic centrifugation to purify as described by Orlando et al. (Orlando et al., 1997). Aliquots from each fraction were examined for the presence of PcG proteins, chromosomal DNA and Hoxb8 gene. Distribution of Rnf2, Ring1, Phc1 and Cbx2 was examined by immunoblotting. Hspa5 was used as a marker protein for endoplasmic reticulum and nuclear envelope. Chromosomal DNA in each fraction was visualized by ethidium bromide staining. Distribution of DNA fragments encompassing the Hoxb8 gene was examined by PCR. Chromatin predominantly distributed to fractions 5 to 7 as reported previously and these fractions were used for ChIP analyses (Suzuki et al., 2002).

• Supplemental Figure 2 -

  Fig. S2. Quantification of genomic DNA immunoprecipitated by anti-Rnf2 antibody from the chromatin fractions of anterior and posterior tissues of 12.5 dpc embryos. (A) Immunoprecipitated genomic DNA from the chromatin fractions purified from anterior or posterior tissue by anti-Rnf2 antibody. Genomic DNA immunoprecipitated by anti-Rnf2 (A+ or P+) and mock-immunoprecipitated DNA (A– and P–) derived from the same volume of the chromatin fraction as used for anti-Rnf2 immunoprecipitation are visualized by ethidium bromide staining. Their amounts are quantified by comparing with serially diluted genomic DNA isolated from the original chromatin fractions of the anterior tissue. Quantity of the genomic DNA loaded in each lane is shown. (B) Quantitative adjustment of immunoprecipitated DNA by anti-Rnf2 antibody from anterior and posterior tissues by comparing the inclusion of DNA fragments encompassing tbx2 gene, which is also bound by Rnf2. 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’. Almost equivalent amounts of immunoprecipitated (A+ and P+) genomic DNA to that of ‘Input’ DNA loaded in lane 1, which was approximately 20 ng, were subjected to PCR reaction for the tbx2 gene. Mock-immunoprecipitated DNA (A– and P–) derived from the same volume of the chromatin fraction as used for anti-Rnf2 immunoprecipitation were used as negative control.

• Supplemental Figure 3 -

  Fig. S3. Comparison of Rnf2 binding to regions 3, 5, 6, 9 and 10 between anterior and posterior tissues of 12.5 dpc embryos. Results are shown as described in the legend for Figure 2B. For regions 3 and 6, results from three different experiments were shown.