In vivo association of Swi4-3XHA, Mbp1-3Xmyc and Swi6 with the promoter regions of genes known to be regulated by these transcription factors. Immunoblots (top panels a-c) of yeast extracts using anti-HA antibody 16B12 (a), anti-Myc antibody 9E10 (b) and affinity-purified anti-Swi6 polyclonal antibody (c). a, lane 1, extract from an untagged SWI4 strain; lane 2, extract from a strain carrying SWI4 with three copies of the hemagglutinin epitope at the very C-terminus. b, lane 1, extract from an untagged MBP1 strain; lane 2, extract from a strain carrying MBP1 with three copies of the Myc epitope at the very C-terminus. c, lane 1, extract from a swi6D strain; lane2, extract from a wild type SWI6 strain. The approximate positions of migration of RPN 800 molecular weight markers (Amersham) are indicated. PCR was performed using template DNA from immunoprecipitated chromatin. Immunoprecipitations were carried out with anti-HA antibody 12CA5 against Swi4-3XHA (a), anti-Myc antibody 9E10 against Mbp1-3Xmyc (b) and affinity-purified anti-Swi6 polyclonal antibody (c). PCR products were resolved on polyacrylamide gels. (Lane 1) 100bp DNA ladder (Gibco-BRL); the 200bp band is indicated by an asterisk (*). (Lane 2) PCR products using immunoprecipitated template DNA derived from a SWI4 strain (a), a MBP1 strain (b) and a swi6D strain (c). (Lane 3) PCR products using immunoprecipitated template DNA derived from a SWI4-3XHA strain (a), an MBP1-3XMYC strain (b) and a SWI6 strain (c). a, PCR products using primers for the promoter regions of CLN1 (panel 1), CLN2 (panel 2), HO (panel 3), ACT1 (panel 4) and HHT1 (panel 5). b, PCR products using primers for the promoter region of CDC21 (panel 1), CLB6 (panel 2), ACT1 (panel 3) and HHT1 (panel 4). c, PCR products using primers for the promoter regions of CDC21 (panel 1), CLB6 (panel 2), CLN1 (panel 3) and CLN2 (panel 4).

In vivo association of Mbp1-3Xmyc with the promoter regions of genes predicted to be MBF targets by ChIP and microarray analysis. PCR was performed using template DNA immunoprecipitated from extracts that had been sonicated to shear DNA to an average size of 0.5-2 kb and derived from formaldehyde cross-linked cells. Immunoprecipitations were anti-Myc antibody 9E10 against Mbp1-3Xmyc. PCR products were resolved on 8% polyacrylamide gels. (Lane 1) 100bp DNA ladder (Gibco-BRL). (Lane 2) PCR products using immunoprecipitated template DNA derived from, a MBP1 strain. (Lane 3) PCR products using immunoprecipitated template DNA derived from a MBP1-3XMYC strain. (Lane 4) PCR products using template of sheared DNA of whole cell yeast extract (input). PCR products using primers for the promoter regions of predicted MBF target genes: CDC7 (panel 1), DIN7 (panel 2), PRE1 (panel 3), RAD18 (panel 4) and SUN4 (panel 5) and for the promoter region of SUC2, a gene that is not a putative target (neg., panel 6). METHODS. QIAGEN Taq polymerase and the corresponding buffer system was used. Thermalcycling conditions included an initial denaturation step at 95 C for 3 min., then 28 cycles of 95°C for 45s, 50°C for 30s and 72°C for 1min. followed by a final extension phase at 72°C for 10 min. PCR products were separated on 8% polyacrylamide and visualized with 0.1mg/ml ethidium bromide.