Gametogenesis in Yeast Is Regulated by a Transcriptional Cascade Dependent on Ndt80  Shelley Chu, Ira Herskowitz  Molecular Cell  Volume 1, Issue 5, Pages 685-696 (April 1998) DOI: 10.1016/S1097-2765(00)80068-4

Figure 1 Transcription Pattern of Sporulation Genes in Wild-Type and Ndt80-Deficient Cells (A) Ndt80 is necessary for transcriptional induction of middle sporulation genes. Wild-type (YSC328) and ndt80 mutant (YSC330) cells were transferred to sporulation medium at t = 0. RNA was harvested at hourly intervals and used to make duplicate Northern blots, one probed with SPS1 and TCM1 (upper), the other with SMK1 and TCM1 (lower). TCM1 codes for the ribosomal L3 protein and served as a loading control. (B) Transcription of NDT80, CLB1, and SPC42 relative to representative early and mid–late genes. RNA samples obtained from the same time course as Figure 1 were used to make duplicate Northern blots. The first blot was hybridized with probes for DMC1 (an early sporulation gene), NDT80, CLB1, and, and TCM1 (Figure 2, upper). The second blot was probed with SPC42, DIT1, and TCM1 (Figure 2, lower). Molecular Cell 1998 1, 685-696DOI: (10.1016/S1097-2765(00)80068-4)

Figure 2 Ndt80 Is Necessary for Transcriptional Induction of CLB1, CLB3, CLB4, CLB5, and CLB6 during Meiosis The same blots of Figure 1B were hybridized with probes for CLB1, CLB2, CLB5, and TCM1 (upper), or CLB3, CLB4, CLB6, and TCM1 (lower). Progression through meiotic division was monitored at each interval by fixing and staining cells with the DNA-specific dye DAPI. From t = 0 to t = 5 hours, 99% of the cells were mononucleate (single DAPI-staining body). By t = 6, 58% of the cells were mononucleate, 15% were binucleate (indicative of cells that have completed meiosis I), and 27% were tetranucleate (indicative of cells that have completed meiosis I and II). The percentages of mono-, bi-, and tetranucleate cells for subsequent intervals were the following: t = 7 (24%, 16%, 60%); t = 8.5 (20%, 7%, 73%); t = 9 (16%, 8%, 74%). Molecular Cell 1998 1, 685-696DOI: (10.1016/S1097-2765(00)80068-4)

Figure 4 Regulation of NDT80 Transcription (A) NDT80 transcription is dependent on Ime1. Wild-type (YSC7) and ime1 mutant (YSC794) cells were transferred to sporulation medium at t = 0. RNA was harvested at the indicated intervals. The Northern blot was probed with NDT80 and TCM1. (B) Ndt80 can activate its own synthesis. Exponentially growing strains were induced with galactose and examined by phase (i, iii, v, and vii) or immunofluorescence (ii, iv, vi, and viii) microscopy. A functional GFP-tagged Ndt80 protein under control of the NDT80 promoter (pNDT80–NDT80–GFP) was expressed in strains carrying pGAL–NDT80 (YSC921, i and ii) or pGAL (YSC918, v and vi). A pGAL–NDT80–GFP construct was also expressed in either background of pGAL–NDT80 (YSC922, iii and iv) and pGAL (YSC919, vii and viii). Glucose-grown cells exhibited no immunofluorescence (data not shown). Molecular Cell 1998 1, 685-696DOI: (10.1016/S1097-2765(00)80068-4)

Figure 3 Ectopic Expression of Ndt80 Induces Middle Gene Expression in Vegetative Cells RNA was harvested from vegetative cells (YSC531, YSC552, and YSC553) in the absence or presence of ectopic Ndt80-HA expression. Northern blots were hybridized with probes for CLB1–CLB6 and TCMI. (A) Comparison of CLB1 and CLB2 expression in strains containing pGAL–NDT80–HA grown with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) galactose treatment. Asynchronous a/α (YSC531; lanes 1, 2, 5, and 6) or α-factor-treated a haploid (YSC553; lanes 3 and 4) cells were used. (B) Comparison of CLB3 and CLB4 expression in an asynchronous population of cells from strains containing pGAL (YSC552) and pGAL–NDT80–HA (YSC553) grown in the presence of galactose. Similar induction was observed when these blots were probed with CLB1, CLB5, and CLB6 (data not shown). (C) Comparison of CLB5, CLB6, and SPS1 expression in an asynchronous population of cells from strain YSC531 processed as described in (A). Molecular Cell 1998 1, 685-696DOI: (10.1016/S1097-2765(00)80068-4)

Figure 5 Ndt80 Recognizes the SPS4 MSE (A) Extracts from cells expressing Ndt80-HA have a DNA-binding activity specific for the SPS4 MSE. Extracts made from a galactose-induced pGAL-NDT80-HA strain (YSC562, lanes 2–15) were incubated with a [32P] MSE probe. The probe alone was run in lane 1. The reactions contained no competitor (lane 2), 29 bp wild-type competitor (A, lanes 3, 4 and 9), or mutant competitors GCCAAC (B, lanes 5–6), GCCAGTAACAC (C, lanes 7–8), GCCACGCTGAC (D, lanes 10–11), or TCATGTAAGAC (E, lanes 12–13). A change from the consensus is indicated by an underline. 10- or 100-fold excess competitor was added to the reactions as indicated. Arrow, protein-DNA complex specific to GAL-NDT80-HA extracts. Asterisk, nonspecific complex. (B) Mbp–Ndt80 isolated from E.coli binds the MSE. Gel mobility shift assays were performed using Mbp (lane 2) or Mbp–Ndt80 fusion protein (lanes 3–13) purified from E. coli and incubated with a [32P] MSE probe. The probe alone was run in lane 1. The reactions included no competitor (lanes 3 and 13), wild-type competitor (A, lanes 4–6), or mutant competitor oligonucleotides GCCAGTAACAC (C, lanes 7–9) or TCATGTAAGAC (E, lanes 10–13). A change from the consensus is indicated by an underline. 10-, 100-, or 200-fold excess competitor was added to the reactions as indicated. Molecular Cell 1998 1, 685-696DOI: (10.1016/S1097-2765(00)80068-4)

Figure 6 The Meiotic Recombination Checkpoint Gene RAD17 Controls Transcription of the Ndt80-Regulated Gene CLB1 rad17 (YSC927), dmc1 (YSC907), and dmc1 rad17 (YSC928) strains were transferred to sporulation medium at t = 0. RNA was harvested at hourly intervals. The Northern blot was probed with NDT80, CLB1, and TCM1. Molecular Cell 1998 1, 685-696DOI: (10.1016/S1097-2765(00)80068-4)

Figure 7 Ndt80 Is a Central Regulator of the Sporulation Transcriptional Cascade Both nutritional and mating type signals initiate entrance into sporulation by activating the transcription factor Ime1 (reviewed byMalone 1990). Ime1 together with its DNA-binding partner, Ume6, turns on expression of the early genes involved in chromosome synapsis and recombination (Kupiec et al. 1997). NDT80 transcription is dependent on Ime1 and occurs after early gene expression. Ndt80 can activate its own transcription and that of the middle sporulation genes. Transcription of the middle genes, which function in both meiotic nuclear division, e.g., CLBs, and spore formation, e.g., SPS1 (Friesen et al. 1994), is regulated by the meiotic recombination checkpoint machinery. The activities responsible for subsequent transcription of the mid–late and the late genes, whose products are required for spore wall maturation (Mitchell 1994), remain to be identified. The Ndt80-dependent, coordinate induction of genes involved in meiotic division and gamete morphogenesis results in the production of an ascus containing a tetrad of four haploid spores. Molecular Cell 1998 1, 685-696DOI: (10.1016/S1097-2765(00)80068-4)