The Ime2 protein kinase enhances the disassociation of the Sum1 repressor from middle meiotic promoters

Abstract

Meiotic development in Saccharomyces cerevisiae (sporulation) is controlled by the sequential transcription of temporally distinct sets of meiosis-specific genes. The induction of middle genes controls exit from meiotic prophase, the completion of the nuclear divisions, and spore formation. Middle promoters are controlled through DNA elements termed middle sporulation elements (MSEs) that are bound by the Sum1 repressor during vegetative growth and by the Ndt80 activator during meiosis. It has been proposed that the induction of middle promoters is controlled by competition between Ndt80 and Sum1 for MSE occupancy. Here, we show that the Sum1 repressor can be removed from middle promoters in meiotic cells independent of Ndt80 expression. This process requires the phosphorylation of Sum1 by the meiosis-specific cyclin-dependent kinase-like kinase Ime2. The deletion of HST1, which encodes a Sir2 paralog that interacts with Sum1, bypasses the requirement for this phosphorylation. These findings suggest that in the presence of Ndt80, Sum1 may be displaced from MSEs through a competition-based mechanism but that in the absence of Ndt80, Sum1 is removed from chromatin in a separate pathway requiring the phosphorylation of Sum1 by Ime2 and the inhibition of Hst1.

FIG. 1.

Sum1 repression of the SMK1 promoter is removed in an NDT80-independent fashion. (A) SMK1-HA or SMK1-HA ndt80Δ cells were transferred into sporulation medium, and samples were withdrawn at the indicated times. Total cellular extracts were analyzed with an HA antibody to monitor Smk1 expression (Smk1−) and with a PSTAIRE antibody that detects a closely spaced doublet consisting of Cdc28 and Pho85 as a loading control (Con−). The exposure time for the ndt80Δ samples was approximately four times longer than that for the wild-type samples. (B) NDT80-HA SUM1-MYC vegetative (V) cells and meiotic (M) cells (6.5 h postinduction) were analyzed by ChIP with HA or Myc antibodies. Values shown are increases in a PCR product containing the MSE in the SMK1 promoter relative to the DIT1 open reading frame PCR product (nonspecific control). Each bar represents the average of results from three separate ChIP experiments, each analyzed in triplicate. (C) ChIP assays were carried out with Sum1 antisera, and the results were analyzed as described in the legend to panel B.

FIG. 2.

Sum1 is present during middle meiosis. (A) Cells of the indicated genotypes were transferred into sporulation medium, and samples were withdrawn at the indicated times. Total cellular extracts were analyzed by immunoblotting with the indicated antibodies as described in the legend to Fig. 1. The arrows indicate the points where 50% of the cells had completed the nuclear divisions. Con, control.

FIG. 3.

The meiosis-specific Ime2 kinase phosphorylates Sum1 at residue T306 in vivo. (A) Strains expressing HH-tagged forms of Sum1 were incubated in sporulation medium for the indicated times, and Sum1-HH was purified and analyzed by immunoblotting using phosphospecific T306 antisera (T306P) or an HA antibody to control for total Sum1-HH levels. (B) SUM1-HH diploids were transferred into sporulation medium, and samples were withdrawn at the indicated times and analyzed as described in the legend to panel A. The arrow indicates the point at which 50% of the cells had completed meiosis. The amount of purified Sum1-HH that was recovered from meiotic cells was smaller than that recovered from mitotic cells in all of these experiments. In the blots in both panels A and B, a larger fraction of the material purified from the meiotic cells than of that from the mitotic cells was loaded in order to equalize the amounts of Sum1 analyzed.

FIG. 4.

Smk1-HA expression is delayed in a sum1(T306A) mutant in the presence of Ndt80. (A) Logarithmic cells of the indicated genotypes were transferred into sporulation medium, and samples were withdrawn at the indicated times. Total cellular extracts were analyzed by immunoblotting with the indicated antibodies as described in the legend to Fig. 1. Wild-type cells reached 50% meiosis at 6.4 h and sum1(T306A) cells reached 50% meiosis at 6.7 h in this experiment. Con, control. (B) Three independent isolates of coisogenic wild-type and sum1(T306A) mutants were sporulated, and the fraction of cells that had completed MI was quantified.

FIG. 5.

Smk1 is not expressed and Sum1-T306A is not removed from the SMK1 promoter in ndt80Δ cells. (A) Cells of the indicated genotypes were transferred into sporulation medium, and samples were withdrawn at the indicated times. Total cellular extracts were analyzed by immunoblotting with the indicated antibodies as described in the legend to Fig. 1. (B) Cells of the indicated genotypes were analyzed by ChIP during vegetative growth (V) and during meiosis (M) (6.5 h postinduction). Values shown are increases (% input) of a PCR product containing the MSE in the SMK1 promoter relative to an intragenic DIT1 (negative control) PCR product. Each bar represents the average of results from three separate ChIP experiments, each analyzed in triplicate.

FIG. 6.

Deletion of HST1 restores Smk1-HA expression and the removal of Sum1-T306A in ndt80Δ cells. (A) Cells of the indicated genotypes were transferred into sporulation medium, and samples were withdrawn at the indicated times. Total cellular extracts were analyzed by immunoblotting with the indicated antibodies as described in the legend to Fig. 1. Con, control. (B) Cells of the indicated genotypes were analyzed by ChIP during vegetative growth (V) and during meiosis (M) (6.5 h postinduction) as described in the legend to Fig. 5.

FIG. 7.

Multiple Sum1-repressible middle-gene mRNAs accumulate in an Ndt80-independent process that is regulated by Ime2 and Hst1. Strains of the indicated genotypes were collected at the indicated times after being transferred into sporulation medium, and total RNA was prepared and analyzed by Northern blot hybridization with the indicated probes. The rRNA sample from the ethidium bromide (EtBr)-stained electrophoretic gel prior to Northern transfer is shown as a loading control. The wild-type (WT) RNA samples were included in this analysis to demonstrate the contribution of Ndt80 to the accumulation of the indicated mRNAs.

FIG. 8.

Sum1 is removed from multiple promoters in an NDT80-independent pathway that is regulated by Ime2 and Hst1. Sum1 ChIP samples were analyzed by PCR with primers specific for the indicated promoters as described in the legend to Fig. 5B. The same samples were used for the analyses presented in Fig. 5B and 6B. Each bar represents the average of results from three separate ChIP experiments, each analyzed in triplicate. V, vegetative cells (6.5 h postinduction).

FIG. 9.

Multistep model for the Ndt80-independent removal of the Sum1 repressor from chromatin in meiotic cells. Sum1 recruits the Hst1 deactylase via the Rfm1 bridging protein to middle-promoter chromatin in vegetative cells (state 1). The phosphorylation of Sum1 by the meiosis-specific Ime2 protein kinase is shown to negatively regulate the associated Rfm1-Hst1 complex to generate state 2. While this regulation may occur by directly promoting the removal of Rfm1-Hst1 as indicated, this possibility has not been tested. The influence of Hst1 implies that a histone acetyltransferase (HAT) acts after Hst1 downregulation and that the increase in acetylation of an unidentified chromatin component generates a state that is permissive for Sum1 removal (state 3). The deletion of HST1 or RFM1 is insufficient to derepress many middle genes in mitotic cells, and this finding implies that the removal of Sum1 requires an additional regulatory factor (indicated by X) that leads to Sum1 removal (state 4). The changes leading to state 4 regulate the NDT80 promoter. The removal of Sum1 repression at the NDT80 promoter increases the Ndt80 protein concentration in the cell, leading to Ndt80 occupancy and transcriptional induction (state 5). Ndt80 can also promote one or more of the preceding steps as indicated (for example, by direct competition) to enhance the feed-forward and switch-like attributes of the system.