The G1/S cyclin Cig2p during meiosis in fission yeast

Abstract

Cyclin-dependent kinases (CDKs) are important for both mitotic and meiotic cell cycles. In fission yeast, the major CDK, Cdc2p is involved in premeiotic DNA replication and in meiosis II. One of its partners, the mitotic cyclin Cdc13p is known to be required for meiosis, whereas there are no studies on the G1/S cyclin Cig2p. In this article, we have studied the regulation of the Cdc2p/Cdc13p and Cdc2p/Cig2p complexes during synchronous meiosis. We observed that Cdc2p/Cig2p kinase is activated in an unexpected biphasic manner, first at onset of premeiotic S phase and again during meiotic nuclear divisions. The role of Cig2p during meiosis was investigated using cig2-deleted strains that exhibit delays in onset of both S phase and meiotic divisions as well as an inefficient completion of MII. Furthermore, analysis of cig2 transcripts revealed a meiosis-specific regulation of cig2 expression during MI/MII dependent upon the Mei4p transcription factor leading to a different transcription start site at this stage of meiosis.

Figure 1

Cyclin-B protein levels and Cdc2p-associated kinase activities during pat1-induced meiosis. (A) pat1^ts/pat1^ts diploid cells (strain PN2158) were grown to late log-phase, washed, transferred to −N media for 16 h at 25°C and shifted to 34°C at time 0 h. Samples were taken every 0.5 h until completion of MII. The number of nuclei were counted under the microscope after DAPI staining and is given as a percentage of total cells. (□), Cells with 1 nucleus; (⋄). cells that completed meiosis I (2 nuclei); and (○), cells that completed meiosis II (3–4 nuclei). (B) Cells progressing into meiosis were analyzed by FACS. (C) Boiled samples were analyzed by Western blotting with anti-Cdc13p (SP4), anti-Cig2p (5E3–4), anti-PY15, and anti-Cdc2p (Y63–2) antibodies. (D) Cdc2p/cyclin B complexes from native extracts have been immunoprecipitated using antibodies directed against Cdc13p (polyclonal SP4) and Cig2p (polyclonal Moc8) and analyzed for their histone H1 kinase activities. [³²P]phosphate incorporation in histone H1 was measured on PhosphorImager after SDS-PAGE. The maximum of Cdc2p kinase activity (100%) was determined for Cdc13p and Cig2p and served as a reference for the other experiments (Figures 2, 4, and 5).

Figure 2

Cig2p absence delays meiotic progression. pat1^ts/pat1^ts cig2Δ/cig2Δ cells (strain PN2991) were induced to undergo meiosis as in Figure 1. Cells were analyzed by DAPI staining (A) and FACS (B). Protein levels were analyzed by Western blotting (C) and Cdc2p/Cdc13p kinase activity was measured (D) as in Figure 1.

Figure 3

Cig1p has a minor role during meiosis. Meiosis was induced in pat1^ts (A), pat1^{ts cig2Δ} (B), and pat1^ts cig2Δ cig1Δ (C) cells (strains PN1675, PN2298, and HM1391, respectively) as in Figure 1. Cells were analyzed by DAPI staining (left panels) and FACS (right panels).

Figure 4

The second Cig2p peak is not present in cells blocked before meiosis I. Meiosis was induced in pat1^ts mei4Δ cells (strain PN2448) as in Figure 1 and was followed during 8 h. (A) DNA content was measured by FACS analysis. Western blots (B) and kinase assays (C) were performed as in Figure 1.

Figure 5

The second Cig2p peak is detected in meiosis II arrested cells. pat1^ts/pat1^ts mes1Δ/mes1Δ cells (strain PN2468) were induced to undergo meiosis as in Figure 1. (A) The number of nuclei was counted indicating a block at a two-nuclei stage. (B) DNA content was analyzed by FACS. Western blots (C) and kinase assays (D) were performed as in Figure 1.

Figure 6

In the absence of cig2, meiosis generates dyads and diploid cells. Strains deleted (PN1926 h+, PN1942 h−, PN3796 h90) or not (PN1 h−, PN4 h+, PN2 h90) for cig2 were crossed (h+ × h−) or spread (h90) on YEPD plates and incubated at 25°C for 2 d. (A) The asci were observed under the microscope to determine the percentage of dyads and then dissected using the Singer-MSM micromanipulator and analyzed by FACS to determine the ploidy of spores coming from dyads and tetrads (ND: non determined). (B) Another group of asci was used to perform random spore analysis. Asci were digested with helicase, plated on YES and YEP and incubated at 25°C to follow the formation of diploid colonies. The percentage of dyads (A) and diploid colonies (B) reported are an average from three different experiments. The SDs are also reported.

Figure 7

cig2 presents a smaller transcript later in meiosis, which is dependent on mei4. RNA was extracted from cells taken during pat1^ts/pat1^ts (strain PN2158, left) and pat1^ts mei4Δ (strain PN2448, right) progression into meiosis and analyzed by Northern blotting using cdc13, cig2, cig1, and his3 (loading control) probes. Each probes are DNA fragments located in the ORF of the genes.

Figure 8

The cig2 transcript is modified in its 5′ untranslated region in MI. RNA from pat1^ts/pat1^ts (strain PN2158) meiotic time course was analyzed by Northern blotting using DNA probes from the 5′ untranslated (top) and 3′ untranslated (bottom) regions of cig2.

Figure 9

Cig2 has two different transcription start sites. (A) Diagram of the cig2 gene with probes used in Figure 8 indicated. (B) Detailed diagram of the 5′ untranslated region and position of primers A–F used to perform the primer extension experiment. A putative Mei4p-binding site (GTAAACA) is also indicated. (C) Primer extension experiment was performed by hybridizing primers A–F with RNA extracted from cells taken at 2 or 4 h of a pat1-induced meiosis (strain PN2158). The results obtained for primers A and F are shown. To determine the size of the extended products labeled φx174 DNA/HinfI markers (M) were loaded (first lane). The polyacrylamide gel was exposed to a BioMax film.