Dynamic Acetylation of All Lysine 4–Methylated Histone H3 in the Mouse Nucleus: Analysis at c-fos and c-jun
Figure 2
Effect of TSA Treatment on Acetylation of Nucleosomes Associated with c-fos and c-jun Genes
(A) Schematic diagram representing relative positions of regions of c-fos and c-jun amplified by primer pairs used in the PCR step of ChIP assays. Exons are indicated by boxes, open for untranslated regions and filled for coding regions. Polyadenylation sites (pA) are indicated.
(B and C) Cross-linked chromatin fragments were prepared from quiescent C3H 10T½ cells treated with TSA (10 ng/ml; 15 min to 4 h). “C” indicates control (unstimulated). Specific DNAs were immunoprecipitated with anti-acetyl-H3 antibodies. Recovered DNAs from antibody-bound fractions (AcH3 IP) as well as total input DNA (Input) from released chromatin used for ChIP were analysed for the presence of c-fos (B) and c-jun (C) gene sequences. Controls for PCR included a DNA minus (−DNA) reaction and 5- to 20-ng loadings of input DNA to ensure all amplifications were within the linear range. PCR reactions were carried out in triplicate and gels quantified by phosphorimaging. Representative gels are shown.
(D) Data are expressed graphically as average Bound/Input (± standard deviation). Note that to correct for the dilution factor applied to anti-acetyl-H3 immunoprecipitated DNA prior to PCR analyses, values obtained from quantification of PCR gels were multiplied by three.