FIG. 8.

Overall chromatin organization is similar in cells expressing H4-WT and H4-4A histone H4. (A) ChIP was performed as described in Materials and Methods. Fifteen microliters of each PCR product was analyzed on 1% agarose gels. Lanes: 1, 1-kb DNA ladder; 2, PCR of genomic DNA; 3 to 6, YJB7650 (H4-4A); 7 to 10, YJB7651 (H4-WT);11 to 14, YJB7902 (H4-4A/WT); 3, 7, and 11, PCRs of total immunoprecipitate; 4, 8, and 12, no-antibody control; 5, 9, and 13, immunoprecipitation with anti-acetyl-histone H3; 6, 10, and 14, immunoprecipitation with anti-HA antibody. (B) Protein ChIP. Formaldehyde cross-linked lysates were isolated from the H4-4A/WT strain containing SIR3-HA (YJB8651). Active chromatin was immunoprecipitated with the anti-acetyl-histone H3 antibody; silent chromatin was precipitated with an anti-HA antibody against Sir3-HA. Nuclear extracts (lysates) and immunoprecipitates were separated by SDS-22% PAGE, and Western analysis of histone H4 proteins was performed with anti-H4-17-33 antibody. To detect the histones associated with silent chromatin, the blot, indicated by an asterisk, was exposed approximately 10 times longer than for active chromatin.