The Cdk1 and Ime2 protein kinases trigger exit from meiotic prophase in Saccharomyces cerevisiae by inhibiting the Sum1 transcriptional repressor

Abstract

The induction of middle meiotic promoters is a key regulatory event in the life cycle of Saccharomyces cerevisiae that controls exit from prophase, meiosis, and spore formation. The Sum1 repressor and Ndt80 activator proteins control middle promoters by binding to overlapping DNA elements. NDT80 is controlled by a tightly regulated middle meiotic promoter through a positive autoregulatory loop and is repressed in vegetative cells by Sum1. It has previously been shown that the meiosis-specific kinase Ime2 promotes the removal of Sum1 from DNA. Here, we show that Sum1 is also regulated by the cyclin-dependent kinase, Cdk1. While sum1 phosphosite mutants that are insensitive to Cdk1 or Ime2 complete meiosis and form spores, a mutant that is insensitive to both Ime2 and Cdk1 (sum1-ci) blocks meiotic development in prophase with an ndt80Delta-like phenotype. Ectopic expression of NDT80 or mutation of a Sum1-binding element in the NDT80 promoter bypasses the sum1-ci block. Hst1 is a NAD(+)-dependent histone deacetylase that is linked to Sum1 by the Rfm1 tethering factor. Deletion of HST1 or RFM1 also bypasses the sum1-ci block. These results demonstrate that Sum1 functions as a key meiotic brake through the NDT80 promoter and that Cdk1 and Ime2 trigger exit from meiotic prophase by inhibiting the Sum1 transcriptional repression complex.

FIG. 1.

Inhibition of Cdk1 prevents expression of Smk1 in sum1-i cells. cdk1-as1 SMK1-HA SUM1 (WT) or cdk1-as1 SMK1-HA sum1-i (sum1-i) cells were transferred to sporulation medium containing either 5 μM 1-NM-PP1 (+) to inhibit Cdk1-as1 or vehicle alone (−). Samples were withdrawn at 0, 5, 6.5, 8, and 9.5 h postinduction, and protein extracts were analyzed by immunoblotting using an HA antibody (Smk1) or a PSTAIR antiserum, which detects the constitutively expressed Cdk1 and Pho85 proteins as a loading control (Con).

FIG. 2.

NDT80 and middle genes are not induced in a sum1-ci mutant. Cells of the indicated genotype were transferred to sporulation medium; collected at 0, 5, 6.5, 8, 9.5, 11, and 24 h postinduction; and analyzed for the completion of meiosis (lower panel). Protein extracts prepared from the same cells were analyzed by immunoblotting (upper) using antibodies for the indicated proteins. Con, control.

FIG. 3.

sum1-c is defective in the NDT80-independent pathway for removing Sum1 repression. The indicated diploid strains were transferred to sporulation medium; collected at 0, 5, 6.5, 8, and 9.5 h; and analyzed by immunoblotting using an HA antibody (Smk1) or a PSTAIR antiserum as a loading control (Con).

FIG. 4.

sum1-ci and ndt80Δ phenotypes are similar. (A) ZIP1-GFP (WT), ndt80Δ ZIP-GFP (ndt80Δ), and sum1-ci ZIP1-GFP (sum1-ci) cells were transferred to sporulation medium, collected at various times, and fixed, and relative fluorescence emission was monitored in a fluorometer (n = 4). (B) Live cells were analyzed by visible differential interference contrast (DIC) microscopy or by fluorescence microscopy for Zip1-GFP (GFP) at 6 and 24 h, as indicated. The brightly staining complex (arrows) was observed at a similar frequency in all fluorescence-positive cells.

FIG. 5.

ß-Estradiol-inducible NDT80 bypasses the sum1-ci meiotic block. SUM1 GAL4-ER pGAL-NDT80 (WT) and sum1-ci GAL4-ER pGAL-NDT80 (sum1-ci) cells were transferred to sporulation medium, and 1 μM ß-estradiol (+) or vehicle alone (−) was added at 5 h to induce expression of NDT80. The completion of meiosis (MII) was monitored in three independent cultures at 24 h after cells were transferred to sporulation medium (error bars are too small to be visible). Equivalent numbers of cells were collected on a nitrocellulose filter and assayed for the presence of dityrosine, a component of the outer layer of the spore wall, by fluorescence.

FIG. 6.

NDT80 promoter mutations bypass the sum1-ci block. (A) ndt80Δ or sum1-ci diploids harboring an NDT80 plasmid controlled by the wild-type promoter (WT), the promoter carrying a deletion of the MSE1 Sum1/Ndt80 binding site (M1Δ), or the plasmid lacking NDT80 (ø) were transferred to sporulation medium, and meiosis (MI) was scored at 24 h postinduction (n = 4). (B) sum1-ci diploids containing the indicated number of NDT80, NDT80::TRP1, or NDT80-M1Δ::TRP1 genes were analyzed as described for panel A (n = 3). (C) Strains a, d, and f from panel B were collected at 72 h postinduction, stained with DAPI, and analyzed using phase-contrast or fluorescence photomicrography. The arrows in the middle photomicrographs point to cells that have completed meiosis but have failed to form spores.

FIG. 7.

hst1Δ and rfm1Δ bypass the sum1-ci block to middle-phase gene expression and meiosis. (A) Cells of the indicated genotype were harvested at 0, 3, 5, 6.5, 8, 9.5, and 24 h. RNA was prepared, and levels of the indicated mRNAs were analyzed by Northern blot hybridization. EtBr, the ethidium bromide-stained gel before transfer. (B) The completion of MII was quantified by fluorescence microscopy. (C) Equivalent numbers of cells of the indicated genotypes were collected on a nitrocellulose filter, and incorporation of dityrosine into spore walls was analyzed using the fluorescence assay. (D) Cells of the indicated genotype were harvested at 24 h postinduction, and the completion of MII was quantified by fluorescence microscopy.