Ume6 is required for the MATa/MATalpha cellular identity and transcriptional silencing in Kluyveromyces lactis

Abstract

To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of the unscheduled meiotic gene expression 6 (UME6) gene in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLalpha and HMRa. Chromatin immunoprecipitation (ChIP) suggested that Ume6 acted directly by binding the cis-regulatory silencers of these loci. Unexpectedly, a MATa ume6 strain was mating proficient, whereas a MATalpha ume6 strain was sterile. This observation was explained by the fact that ume6 derepressed HMLalpha2 only weakly, but derepressed HMRa1 strongly. Consistently, two a/alpha-repressed genes (MTS1 and STE4) were repressed in the MATalpha ume6 strain, but were expressed in the MATa ume6 strain. Surprisingly, ume6 partially suppressed the mating defect of a MATa sir2 strain. MTS1 and STE4 were repressed in the MATa sir2 ume6 double-mutant strain, indicating that the suppression acted downstream of the a1/alpha2-repressor. We show that both STE12 and the MATa2/HMRa2 genes were overexpressed in the MATa sir2 ume6 strain. Consistent with the idea that this deregulation suppressed the mating defect, ectopic overexpression of Ste12 and a2 in a MATa sir2 strain resulted in efficient mating. In addition, Ume6 served as a block to polyploidy, since ume6/ume6 diploids mated as pseudo a-strains. Finally, Ume6 was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors.

Figure 1.—

Similarity between S. cerevisiae and K. lactis Ume6. (A) Schematic of the mating-type loci in K. lactis, including their chromosomal position (top) and the ORFs in the HMLα and HMRa loci with the transcriptional orientation depicted by arrows (middle). The boxes with L (left) and R (right) show the repetitive sequences that flank the mating-type loci, and the stars represent the position of the silencers. The arrowheads below the boxes represent the positions of the primers used in the qPCR experiments in Figure 2. (Bottom) Ume6- and Reb1-binding sites and the C-box in the HMLα and HMRa silencers. (B) Pairwise alignment of ScUme6p and KlUme6p displaying 41% similarity and 26% identity. Threonine 99/106 is boxed; the cysteines of the C-terminal Zn²⁺-finger domain and the residues of the amphiphatic helix are in boldface type.

Figure 2.—

Ume6 is required for silencing of the cryptic mating-type loci. (A) Quantitative RT–PCR analysis using primers specific for the a1 and a2 genes and RNA from MATα (SAY538), MATα ume6 (SAY541), MATa (SAY572), and MATa ume6 (SAY549) strains. (B) Same as in A, but using primers specific for the α1 and α2 genes. The amount of cDNA in each sample was first normalized to the ACT1 gene. The expression levels of a1 and a2 in SAY572 was set to 1.0 in A, and the expression levels of α1 and α2 in SAY538 was set to 1.0 in B. Values shown are the average of two independent experiments with the maximum and minimum values indicated. (C) ChIP was performed on strains SAY537 (UME6) and SAY721 (UME6-13XMYC) using an anti-Myc antiserum (9E10). Following ChIP, DNA associated with the HMRa and HMLα silencers and the MATα1/HMLα1, MATα2/HMLα2, MATα3/HMLα3, and ACT1 ORFs was analyzed by qPCR using locus-specific primers (as indicated by the colors). Mock immunoprecipitations, in which the 9E10 antiserum was omitted, are included. The y-axis represents the amount of precipitated DNA relative to a standard curve obtained from dilutions of the input DNA. Values shown are the standard error of the mean resulting from three independent experiments.

Figure 3.—

Mating phenotypes of ume6 strains. (A) Mating tests of MATa (CK213) and MATa ume6 (SAY549) (left) and of MATα (SAY538) and MATα ume6 (SAY541) (right) strains to MATα (WM52) and MATa (CK213) tester strains. Diploids were selected on synthetic dextrose plates containing uracil (SD + Ura; left) or on SD + Ura, Trp, and Met; right. (B) Mating tests of MATα hmra (SAY726), MATα ume6 (SAY541), and MATα hmra ume6 (SAY732) strains to a MATa tester strain (CK213). Diploids were selected on SD + Ura, Trp, and Met. (C) Mating tests of MATa strains; WT (SAY572), sir2 (SAY102), sir2 hmlαΔp (SAY189), and sir2 ume6 (SAY586) to MATα tester mater (WM52). Diploids were selected on SD + Ura. Below each panel are the results of quantitative matings in which the mating efficiency of the wild-type MATa or MATα strain was set to 1.0.

Figure 4.—

The ume6 mutation influences expression of haploid-specific genes. (A) RNA-blot analysis using strains MATα (SAY538), MATa (SAY572), MATα ume6 (SAY541), MATa ume6 (SAY549), MATa sir2 ume6 (SAY586), and MATa sir2 (SAY102). The ³²P-labeled probes used to detect transcripts were MTS1, STE4, or ACT1 specific, as indicated. (B) Quantification of the blot shown in A plus an independent experiment, indicating the minimum and maximum values as error bars. Shown is the ratio between the MTS1/ACT1 and STE4/ACT1 transcripts, with the ratio in SAY102 defined as 1.0. (C) Mating tests of MATa (CK213), MATa ume6 (SAY549), MATa ste4 ume6 (SAY1081), and MATa ste4 (SAY1084) strains to the MATα tester strain (WM52). Diploids were selected on SD + Ura.

Figure 5.—

Ume6 is required for maintaining the a/α cell identity. (A) Mating tests of the MATa/MATα diploid strains UME6/ume6 (SAY538X549) and ume6/ume6 (SAY541X549) to the MATα tester strain (WM52). Triploids were selected on SD + Ura. (B) FACS analysis of a haploid MATα strain (WM52), a wild-type diploid strain (SAY538XSAY572), and mating mixtures obtained from mixing WM52 with the UME6/ume6 heterozygous diploid (SAY538X549) or the ume6/ume6 homozygous diploid (SAY541X549). (C) RNA-blot analysis of total RNA prepared from the strains indicated above the lanes (same as in Figure 4A). The probes used in each panel are shown on the left side of each blot. Below the blots are quantifications of the relative expression levels expressed as the DIG1, a2, and STE12/ACT1 ratio. The average of two independent experiments is shown with the minimum and maximum values indicated as error bars. (D) Mating tests of a MATa sir2 strain (SAY569) containing empty plasmids (pCXJ18 + pCXJ20), a plasmid overexpressing the a2 gene from the ADH1 promoter (pCXJ18 + pADH-a2), a plasmid overexpressing the STE12 gene from the GAL1-10 promoter (pGAL-STE12 + pCXJ20), or plasmids overexpressing both STE12 and a2 (pGAL-STE12 + pADH-a2). The MATα tester strain was WM52, and diploids were selected on SD.

Figure 6.—

Ume6 represses meiotic genes during vegetative growth independently of Rpd3/Sin3. (A) RNA-blot analysis of total RNA prepared from MATα ume6 (SAY541), MATα (SAY119), and MATa ume6 (SAY549) strains as indicated above the lanes. The gene-specific probes used are indicated on the right, and the control hybridizations with an ACT1 probe are shown on the left. (B) RNA-blot analysis using RNA from strains MATa (SAY572), MATα (SAY119), MATa ume6 (SAY549), MATα ume6 (SAY541), MATa rpd3 (SAY276), MATα rpd3 (SAY789), MATa sin3 (SAY839), and MATα sin3 (SAY838). (C) ChIP was performed as described in Figure 2C by using locus-specific primers for the SPO69 regulatory region. Values on the y-axis are the average of two independent experiments displaying the maximum and minimum values as error bars. (D) Mating test of the MATa strains WT (CK213), rpd3 (SAY276), rpd3 sir2 (SAY945), sin3 (SAY839), and sin3 sir2 (SAY947) to the MATα tester strain (WM52) (left, top and bottom). Diploids were selected on SD + Ura. Mating tests of MATα strains WT (SAY119), rpd3 (SAY789), and sin3 (SAY838) to the MATa tester strain (CK213) (right, top and bottom). Diploids were selected on SD + Ura, Leu, Trp, and Met.