Cell cycle-dependent regulation of Saccharomyces cerevisiae donor preference during mating-type switching by SBF (Swi4/Swi6) and Fkh1

Abstract

Saccharomyces mating-type switching occurs through a double-strand break-initiated gene conversion event at MAT, using one of two donors located distantly on the same chromosome, HMLalpha and HMRa. MATa cells preferentially choose HMLalpha, a decision that depends on the recombination enhancer (RE) that controls recombination along the left arm of chromosome III. We previously showed that an fhk1Delta mutation reduces HMLalpha usage in MATa cells, but not to the level seen when RE is deleted. We now report that donor preference also depends on binding of the Swi4/Swi6 (SBF) transcription factors to an evolutionarily conserved SCB site within RE. As at other SCB-containing promoters, SBF binds to RE in the G(1) phase. Surprisingly, Fkh1 binds to RE only in G(2), which contrasts with its cell cycle-independent binding to its other target promoters. SBF and Fkh1 define two independent RE activation pathways, as deletion of both Fkh1 and SCB results in nearly complete loss of HML usage in MATa cells. These transcription factors create an epigenetic modification of RE in a fashion that apparently does not involve transcription. In addition, the putative helicase Chl1, previously involved in donor preference, functions in the SBF pathway.

FIG. 1.

RE regulation and donor preference during mating-type switching in Saccharomyces cerevisiae. (A) Donor preference in MATα cells. A schematic representation of the mating-type locus (MAT), the two silent donor loci (HML and HMR), and the RE is shown. In MATα cells, the Matα2-Mcm1 complex recruits the general repressor Tup1 to RE, inactivating RE by formation of highly organized nucleosomes, resulting in the preferred use of HMR. (B) Donor preference in MATa cells. Mcm1 binds to the RE and removes the positioned nucleosomes, allowing binding of trans-acting factors, including Fkh1 and SBF, leading to activation of RE and consequently to the favored usage of HML.

FIG. 2.

Deletion and mutational analysis of region C shows that an SCB is necessary for complete RE activity. (A) Sequence comparison of region C among the Saccharomyces sensu stricto species. The MATα2-Mcm1 operator is shown. Sequences enclosed by the box correspond to the SCB necessary for RE activity (reverse complement of the consensus). Only perfectly conserved residues between the five species are indicated in the consensus sequence. (B) (Top) HML usage in strains bearing different mutations in region C. Some of these mutations have been introduced in the 270-bp synthetic RE which lacks region E (lower lane). Others were introduced in the 753-bp minimal RE (upper lane). (Middle) Sequence of region C. The Matα2-Mcm1 operator is underlined, and the SCB is shown in the box. (Bottom) HML usage in strains bearing the 753-bp minimal RE with several deletions of region C sequences.

FIG. 3.

Donor preference in swi4 and swi6 mutants. (A) Southern blot of genomic DNA extracted from wild-type (ECY384) and swi4 (ECY411) strains after inducting MAT switching. Genomic DNA was cut with the BamHI and HindIII restriction enzymes, and the Southern blot was probed with Yα sequences. (B) Papillation test measuring recombination between leu2 heteroalleles. In wild-type MATa cells, papillation is strongly reduced in a swi6 mutant (ECY304). Fluctuation tests confirmed these observations. (C) Southern blot of genomic DNA from wild-type (ECY507), RE^SCB (ECY507), swi6 (ECY508), and RE^SCB swi6 (ECY509) strains analyzed under the same conditions as in panel A.

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.

FIG. 5.

Donor preference is affected by an arrest in G₁/S and in G₂/M in MATa cells but not in cells with RE deleted. (A) Time course experiment of MAT switching in MATa CDC7 (XW652) and MATa cdc7-as3 (GI560) cells arrested or not by the ATP analogue NMPP1. Genomic DNA cut with the restriction enzymes StyI and BamHI and probed with a MAT distal fragment reveals the parental fragment MATa, the fragment resulting from the HO cut, and the MATα and MATα-BamHI products. (B) Expression of HO under a Gal promoter specific for the G₁ phase does not affect donor preference in MATa cells. Genomic DNA of the WT strain used in panel A (XW652) and of a G₁-Gal-HO strain (ECY273) here digested with HindIII and BamHI and probed with Yα sequences shows the two products, MATα and MATα-BamHI. (C) The same experiment as in panel A but carried out in strains with RE deleted, thus mimicking MATα cells (WT, XW676; cdc7-as3, ECY252). (D) MATa switching in strains with RE deleted or not (XW676 and XW652, respectively). HO is induced in exponential phase (Log) or when cells are arrested in the presence of nocodazole (Noc). Genomic DNA was cut with StyI and BamHI and probed with a MAT distal fragment.