A novel type of silencing factor, Clr2, is necessary for transcriptional silencing at various chromosomal locations in the fission yeast Schizosaccharomyces pombe

Abstract

The mating-type region of the fission yeast Schizosaccharomyces pombe comprises three loci: mat1, mat2-P and mat3-M. mat1 is expressed and determines the mating type of the cell. mat2-P and mat3-M are two storage cassettes located in a 17 kb heterochromatic region with features identical to those of mammalian heterochromatin. Mutations in the swi6+, clr1+, clr2+, clr3+, clr4+ and clr6+ genes were obtained in screens for factors necessary for silencing the mat2-P-mat3-M region. swi6+ encodes a chromodomain protein, clr3+ and clr6+ histone deacetylases, and clr4+ a histone methyltransferase. Here, we describe the cloning and characterization of clr2+. The clr2+ gene encodes a 62 kDa protein with no obvious sequence homologs. Deletion of clr2+ not only affects transcriptional repression in the mating-type region, but also centromeric silencing and silencing of a PolII-transcribed gene inserted in the rDNA repeats. Using chromatin immunoprecipitation, we show that Clr2 is necessary for histone hypoacetylation in the mating-type region, suggesting that Clr2 acts upstream of histone deacetylases to promote transcriptional silencing.

Figure 1

Complementation of the sporulation defect in a clr2-E22 mutant strain with cloned DNA. A black arrow points at a product of haploid meiosis and a white arrow points at a zygotic ascus in (A). Strain KE78 (clr2-E22) was transformed with vector DNA, pDW232 (A) or the clr2⁺-expressing plasmid pPB11 (B).

Figure 2

Clr2 is necessary for silencing in the mating-type region, centromere and the rDNA. Cells were serially diluted in steps of ten and spotted onto the indicated media. (A) The strains FY597 (wild type), Hu582 (clr2Δ), PG3088 (clr2-E22) and PG3089 (clr4Δ) have the ura4⁺ reporter gene inserted in the mating-type region [mat3-M(EcoRV)::ura4⁺]. (B) The strains FY498 (wild type), PJ42 (clr2Δ) PG3090 (clr2-E22) and PG3091 (clr4Δ) have the ura4⁺ reporter gene inserted in the repeats of centromere 1 [imr1R(NcoI)::ura4⁺]. (C) The strains FY412 (wild type), PJ32 (clr2Δ) PG3092 (clr2-E22) and PG3093 (clr4Δ) have the ura4⁺ reporter gene inserted into the central core of centromere 2 [cen2 (SphI)::ura4⁺]. (D) The strains Hu393 (wild type), PJ34 (clr2Δ), PG3094 (clr2-E22) and PG3095 (clr4Δ) have the ura4⁺ reporter gene inserted into the rDNA (rDNA::ura4⁺).

Figure 3

Antibodies against Clr2 recognize Clr2 from a total S.pombe protein extract when the protein is overexpressed. Protein extracts from S.pombe were (A) stained with Coomassie or (B) blotted and incubated with the Clr2p antibody. M, protein size markers; lane 1, PB254 (clr2Δ); lane 2, PB141 (clr2⁺) transformed with vector pREP82X; lane 3, PB141 (clr2⁺) transformed with plasmid pPB40 with the clr2⁺ gene under the control of the weakest nmt1 promoter; and lane 4, PB141 (clr2⁺) transformed with plasmid pPB63 where the clr2⁺ gene is under the control of the strongest nmt1 promoter.

Figure 4

Histone acetylation levels in the mating-type region are elevated in the absence of Clr2. Radiolabeled PCR products amplifying a portion of the ura4 gene placed in the mating-type region (ura4⁺) or at its normal chromosomal location (ura4-DS/E) were obtained using total chromatin (IN) or immunoprecipitated chromatin. −K is a negative control where no antibody was added in the immunoprecipitation step. Antibodies recognizing specifically H3AcK14 (H3K14), H4AcK8 (H4K8) or H4AcK12 (H4K12) were used in the immunoprecipitations. Those were conducted in duplicate or triplicate as shown. The mean values of normalized ratios (ura4⁺/ura4-DS/E) are reported below the lanes for each antibody used. The strains used were FY597 (wild type) and Hu582 (clr2Δ).