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

Mating-type switching in Saccharomyces cerevisiae is initiated by the expression of the HO endonuclease, which creates a double-strand break (DSB) at a specific site within the MAT locus. The DSB is repaired by a gene conversion event using one of two donor loci, HMLα and HMRa, located near the left and right subtelomeric regions, respectively (20, 21). These donors are maintained in a heterochromatic stage; they are not transcribed and are refractory to HO endonuclease cleavage (39, 47, 64). Although MAT is able to use both HML and HMR to repair the break, there is a strong mating-type-dependent preference in the choice between the two donors. MATa cells preferentially recombine with HML, whereas MATα selects HMR (33, 61, 67, 68). HML usage is mainly regulated, while HMR is used by default (67). Donor preference is independent of whether the donor carries a or α information; moreover, donor selection does not depend on any sequences that uniquely define HML or HMR or any sequences flanking or distal to HML or HMR (61, 67). Preferential selection of HML in MATa cells depends on an approximately 700-bp cis-acting element, the recombination enhancer (RE), which is located 17 kb centromere proximal to HML (66). Deletion of RE causes reversed donor preference in MATa cells: HML usage is reduced to 10% compared to 90% in the wild type (WT) (66). However, RE deletion does not affect donor preference in MATα cells, showing that RE is simply turned off in these cells and is not responsible for the inhibition of HML usage. In fact, RE regulates the entire left arm of chromosome III for recombination. In MATa, when HML is moved to other locations of the left arm of chromosome III it is still the preferred donor (66). In addition, RE activity is not limited to mating-type switching. The rate of spontaneous recombination between two different leu2 alleles, one replacing HML and the other located near MAT, is ≥10 times higher in MATa versus MATα strains (67). This difference is lost when RE is deleted (66). Recently we showed that the two arms of chromosome III can be defined as two independent domains and that RE controls the accessibility between these domains (10), probably by increasing the movement and/or conformation of the left arm in the nucleus (4). Thus, when MAT is moved onto the left arm, it becomes the preferred donor in both MATa and MATα cells. RE is well conserved in the Saccharomyces sensu stricto species (30; Saccharomyces genome database [www.yeastgenome.org]). Comparison of RE sequences from S. cerevisiae, Saccharomyces carlsbergensis, and Saccharomyces bayanus (53, 65), and also from Saccharomyces mikatae and Saccharomyces kudriazvezii, defines five highly conserved regions A, B, C, D, and E. Deletion studies show that each of these regions with the exception of B is necessary for complete RE activity (53, 68). Regions A, D, and E contain Fkh1 binding sites, and this transcription factor has been shown to play an essential role in HML activation in MATa cells (53). Fkh1 was first described as a component of the SFF complex, involved in the regulation of transcription of the CLB2 cluster of genes at the transition between the G2 phase and mitosis. RE is regulated very similarly to a-specific genes (65). Binding of the Matα2-Mcm1 repressor complex to the region C inactivates RE in MATα cells, and binding of Mcm1 activates RE in MATa cells (54, 55, 65, 68). As at a-specific genes, binding of the Matα2-Mcm1 complex to RE in MATα cells is linked to the formation of a highly organized array of nucleosomes, covering the entire 2.5-kb intergenic region containing RE (63) (Fig. ​(Fig.1A).1A). Formation of this chromatin structure is dependent on Tup1 but not Ssn6 (63, 65). This structure is absent in MATa cells when Mcm1 binds RE in the absence of Matα2 (Fig. ​(Fig.1B).1B). Mcm1 function is limited to this opening, which allows trans-acting factors like Fkh1 to bind RE (14, 53). Recently, the DNA repair protein yKu80 has been shown to be involved in the activation of RE (48). 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 ... Because fkh1Δ does not reduce MATa donor preference as much as deletion of RE, we looked for additional regulatory elements within RE. We report that an SCB (Swi4/Swi6 cell cycle box), present in region C, is important for RE activity. SCB is usually found in promoters of genes induced at the transition from G1 to S phase. Activation of these genes depends on the SBF complex (Swi4/Swi6 cell cycle box binding factors) (3). SBF is necessary for complete RE activity, but its role in donor preference apparently does not involve its G1/S transcription activity. We found that SBF binds RE in G1/S, while Fkh1 binds in the G2 phase. SBF and Fkh1 activate RE through two independent pathways. Our genetic analysis also shows that the putative ATP-dependent DNA helicase Chl1 (18, 19, 24), previously shown to be involved in donor preference (62), acts through the pathway that involves SBF. We propose that the activation of the left arm of chromosome III for recombination requires the establishment of signals in both G1 and G2 to create a specific organization of chromosome III in the nucleus that leads to the preferential choice of donors present on the left arm.