New insights into donor directionality of mating-type switching in Schizosaccharomyces pombe

Abstract

Mating-type switching in Schizosaccharomyces pombe entails programmed gene conversion events regulated by DNA replication, heterochromatin, and the HP1-like chromodomain protein Swi6. The whole mechanism remains to be fully understood. Using a gene deletion library, we screened ~ 3400 mutants for defects in the donor selection step where a heterochromatic locus, mat2-P or mat3-M, is chosen to convert the expressed mat1 locus. By measuring the biases in mat1 content that result from faulty directionality, we identified in total 20 factors required for donor selection. Unexpectedly, these included the histone H3 lysine 4 (H3K4) methyltransferase complex subunits Set1, Swd1, Swd2, Swd3, Spf1 and Ash2, the BRE1-like ubiquitin ligase Brl2 and the Elongator complex subunit Elp6. The mutant defects were investigated in strains with reversed donor loci (mat2-M mat3-P) or when the SRE2 and SRE3 recombination enhancers, adjacent to the donors, were deleted or transposed. Mutants in Set1C, Brl2 or Elp6 altered balanced donor usage away from mat2 and the SRE2 enhancer, towards mat3 and the SRE3 enhancer. The defects in these mutants were qualitatively similar to heterochromatin mutants lacking Swi6, the NAD+-dependent histone deacetylase Sir2, or the Clr4, Raf1 or Rik1 subunits of the histone H3 lysine 9 (H3K9) methyltransferase complex, albeit not as extreme. Other mutants showed clonal biases in switching. This was the case for mutants in the NAD+-independent deacetylase complex subunits Clr1, Clr2 and Clr3, the casein kinase CK2 subunit Ckb1, the ubiquitin ligase component Pof3, and the CENP-B homologue Cbp1, as well as for double mutants lacking Swi6 and Brl2, Pof3, or Cbp1. Thus, we propose that Set1C cooperates with Swi6 and heterochromatin to direct donor choice to mat2-P in M cells, perhaps by inhibiting the SRE3 recombination enhancer, and that in the absence of Swi6 other factors are still capable of imposing biases to donor choice.

Integrity of a heterochromatic domain ensured by its boundary elements

“…In this procedure, spores, but not vegetative cells, are stained dark by iodine vapors ( 31 ). Because heterochromatin is required for efficient mating-type switching and thus for homothallic mating and sporulation ( 22 , 32 , 33 ), the Δ K mutant epitype with established heterochromatin forms brown colonies similar to wild-type ( h 90 ) strains, while the Δ K mutant epitype lacking heterochromatin forms light mottled colonies like clr4 Δ strains ( 29 , 30 ). We found that in a Δ K mutant lacking both boundaries the dark epitype could not be readily propagated ( Fig.…”

Mating-type switching by homology-directed recombinational repair: a matter of choice

“…Three genetic screens have produced S. pombe mutants with donor choice defects (Egel et al 1984 ; Thon et al 2005 ; Maki et al 2018 ). Donor choice, or directionality of mating-type switching, refers to a selection process, whereby the mat2-P cassette is preferentially chosen in M cells to convert the expressed mat1 locus, while the mat3-M cassette is preferentially chosen in P cells, resulting in equal proportions of the two cell types (Fig.…”

“…Another strategy identified the multi-subunit Clr4-containing complex (CLRC) responsible for the methylation of histone H3K9 as necessary for proper donor choice, by searching for mutations that reduce heterologous switching in the wild-type h 90 strain but increase heterologous switching in the h 09 strain, where the mat2 and mat3 cassette contents are swapped (Thon et al 2005 ). More recently, we used a sensitive high-throughput fluorescence microscopy approach to find mutants with aberrant ratios of P and M cells, among which directionality mutants were identified (Maki et al 2018 ). Table 1 presents the mutants obtained in this screen that have defects in histone-modifying enzymes.…”

A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata

“…In all cases studied so far, it implies a genomic DNA rearrangement of the mating-type locus ( MAT , encoding the key regulators of sexual identity) and species have evolved very different molecular pathways for the same aim. In the fission yeast Schizosaccharomyces pombe , an imprint at mat1 (it is unknown whether the imprint is an epigenetic mark or a single nick) is introduced, that leads to a DSB during DNA replication [ 7 , 8 ]. Repair occurs with one of the two silent copies of mat1 , called mat2 and mat3 .…”