FIG. 4.

ChIP analysis of Fkh1, SBF, Chl1, and Mcm1 binding to RE during the cell cycle. (A) Fkh1 binds RE in the G₂/M phase of the cell cycle. ChIP is shown for primers amplifying RE sequences, the CLB2 promoter, and the unrelated ARG5,6 coding region in a strain bearing the hemagglutinin (HA)-tagged Fkh1 at the natural FKH1 locus (CFY480). ChIP was performed on cells in exponential phase (Log), arrested with α-factor (G₁), arrested with α-factor and released in the presence of HU (S), or arrested with nocodazole (G₂). Sequence enrichment was determined by PCR prior to (NIP) and after immunoprecipitation (IP). (B) Fkh1-3xHA binding to RE and to the SUN4 and the PHO3 promoters in synchronized cells released from α-factor arrest. The binding efficiency is expressed as the ratio between IP and non-IP values obtained from real-time PCR quantitation. (C) ChIP in a strain bearing four copies of region A instead of RE (KS358). (D) The Swi4/Swi6 complex binds RE in the G₁/S phase of the cell cycle. The PCL1 promoter sequence was used as a positive control. (E) Chl1 does not bind RE. ChIP assay with primers amplifying RE sequences and the ARG5,6 ORF on strain ECY266 containing an integrated CHL1-9xMYC gene at the natural CHL1 locus. (F) Mcm1 binds RE all along the cell cycle. ChIP experiment with primers amplifying the RE region and the ARG5,6 coding region in a strain bearing Mcm1 tagged with MYC at the MCM1 locus (MJF190). All these experiments have been reproduced three times, except for that shown in panel B, which is supported by our data obtained with arrested cells.