FIG. 5.

The E1A Δ26-35 mutant causes the demethylation but not the acetylation of H3 or the binding of E2Fs at the promoters of cyclin A and Cdc6 in quiescent cells. (A) Cross-linked chromatin (left) was prepared from quiescent wild-type E1A-inducible cells without Dox and then immunoprecipitated in parallel with NR IgG or anti-H3-methyl-Lys-9 antibodies. Precipitated DNA fragments were analyzed by PCR as described in the legend for Fig. 3. Cross-linked chromatin (right) prepared from quiescent wild-type E1A-inducible cells treated with Dox for 12 h was immunoprecipitated in parallel with NR IgG, anti-H3-methyl-Lys-9, anti-H3-acetyl-Lys-9, or anti-H3-acetyl-Lys-14 antibodies. Precipitated DNA fragments were analyzed by PCR as described in the legend for Fig. 3. (B) E1A Δ26-35 mutant-inducible cells were rendered quiescent and then treated with or without Dox for 3-h intervals, ranging from 0 to 12 h. ChIP assays were then performed with the use of anti-H3-methyl-Lys-9, anti-H3-acetyl-Lys-9, anti-MyoD, or NR IgG antibodies. (C) Quiescent E1A.928-inducible cells were treated with Dox for 12 h and then subjected to ChIP analysis using NR IgG and antibodies specific for H3K9 di-methylation, H3K9 acetylation, or H3K14 acetylation. Precipitated DNA fragments were analyzed by PCR as described in the legend for Fig. 2.