Regulation of the premiddle and middle phases of expression of the NDT80 gene during sporulation of Saccharomyces cerevisiae

Abstract

The NDT80 gene of Saccharomyces cerevisiae, which encodes a global activator of transcription of middle sporulation-specific genes, is first expressed after the activation of early meiotic genes but prior to activation of middle sporulation-specific genes. Both upstream repression sequence 1 (URS1) and mid-sporulation element (MSE) sites are present in the promoter region of the NDT80 gene; these elements have been shown previously to contribute to the regulation of expression of early and middle sporulation-specific genes, respectively, by mediating repression in growing cells and activation at specific times during sporulation. In this study, we have shown that the overlapping windows of URS1- and MSE-mediated repression and activation are responsible for the distinctive premiddle expression pattern of the NDT80 gene. Our data suggest that a Sum1-associated repression complex bound at the NDT80 MSE sites prevents Ime1 tethered at the NDT80 URS1 sites from activating transcription of the NDT80 gene at the time that Ime1-dependent activation of early URS1-regulated meiotic genes is occurring. We propose that a decrease in the efficiency of Sum1-mediated repression as cells progress through the early events of the sporulation program allows the previously inactive Ime1 tethered at the URS1(NDT80) sites to promote a low level of expression of the NDT80 gene. This initial phase of URS1-dependent NDT80 expression is followed by Ndt80-dependent upregulation of its own expression, which requires the MSE(NDT80) sites and occurs concomitantly with Ndt80-dependent activation of a set of middle MSE-regulated sporulation-specific genes. Mutation of IME2 prevents expression of NDT80 in sporulating cells. We show in this study that NDT80 is expressed and that middle genes are activated in cells of an Deltaime2/Deltaime2 Deltasum1/Deltasum1 strain in sporulation medium. This suggests that Ime2 activates expression of NDT80 by eliminating Sum1-mediated repression.

FIG. 1.

Sequence of the 5′-flanking region of the NDT80 gene. The sequence upstream of the initiator ATG codon of the NDT80 gene is given. All numbering in the text is with respect to the A of this ATG codon being nucleotide +1. This ATG codon and the stop codon of the upstream gene, EPT1, are boxed. Putative TATA boxes of the NDT80 gene are underlined. Arrows are over sequences that correspond to the consensus for a URS1 or an MSE and indicate the orientation of the element. See the text for the coding of the sites as URS1-1, URS1-2, MSE-1, and MSE-2. The NDT80 sequence inserted into pLGΔ312(Bgl) to create the CYC1-(URS1-1-MSE-1)^NDT80-lacZ reporter gene (see Fig. 3) is underlined with a dashed line.

FIG. 2.

Model for the temporal regulation of expression of the NDT80 gene. See text for details. The horizontal line represents the NDT80 promoter, with the URS1 and MSE sites depicted as shaded boxes with arrowheads (see Fig. 1). For simplicity, regulatory molecules are shown only at the URS1-1 and MSE-1 sites. Curved lines ending in an arrowhead indicate activation of transcription; curved lines ending with a bar indicate repression of transcription. URS1- or MSE-bound proteins, or complexes, that mediate repression are represented in gray; proteins, or complexes, that mediate activation are represented by shapes outlined with shadows. The reductions in size of the Sum1 symbol (C) and the Ime1-Ume6 symbols (D) denote a reduction in activity. (A) The NDT80 promoter in mitotic cells. (B to D) The NDT80 promoter in sporulating cells at the time that early meiotic genes, including IME2, are being expressed (B), after early genes, but before middle sporulation-specific genes have been activated (C), or at the time of middle sporulation-specific gene expression (D).

FIG. 3.

Derepression of a CYC1-(URS1-1-MSE-1)^NDT80-lacZ reporter gene in mitotic cells requires mutation of both UME6 and SUM1. Duplicate patches of cells of a ume6 sum1 strain (first column), a sum1 strain (second column), a ume6 strain (third column), and a wild-type strain (fourth column) contained the following plasmids: (A) pLGΔ312(Bgl), which contains the CYC1-lacZ reporter gene described by Hepworth et al. (16); (B) pAV138-2, which contains two tandem copies of the URS1 site of the HOP1 gene inserted between the CYC1 UAS sites and TATA box in pLGΔ312(Bgl) (48); (C) pCYC1-SPS4-lacZ, which contains four tandem copies of a 29-bp fragment that spans the MSE site of the SPS4 gene inserted between the CYC1 UAS sites and TATA box in pLGΔ312(Bgl) (16, 17); (D) pCYC1-(URS1-1-MSE-1)^NDT80-lacZ, which contains a 91-bp fragment that extends from the URS1-1 site to the MSE-1 site in the promoter region of the NDT80 gene (see Fig. 1), inserted between the CYC1 UAS sites and TATA box in pLGΔ312(Bgl) (see Materials and Methods); (E) pJX43, which contains a fragment spanning the MSE of the SMK1 promoter inserted into the URS1 site of a HOP1-lacZ reporter gene (37). The patches of cells were overlaid with X-Gal-containing agar (see Materials and Methods).

FIG. 4.

The URS1 sites are responsible for initial expression of the NDT80 gene. The Northern filters represented under the RNA heading contained RNA extracted from wild-type cells (lanes 1 to 8) and Δndt80/Δndt80 cells (lanes 9 to 16) harvested during vegetative growth (0 h) or at the indicated times, as noted above the top panel (in hours), after transfer of cells to sporulation medium. Cells used for the experiment of each panel harbored the following plasmid-borne ndt80 minigenes: (A) (−505^WT)ndt80; (B) (−505^ΔU1ΔU2)ndt80; (C) (−505^ΔU1)ndt80; and (D) (−505^ΔU2)ndt80. A schematic diagram of the promoter region of each of these minigenes is given under the promoter heading. Abbreviations: U1, URS1-1; M1, MSE-1; U2, URS1-2; M2, MSE-2. Δ denotes that the specified element has been deleted. The Northern filters were hybridized with a radioactively labeled NDT80-specific probe (top portion of each panel) and a control probe (bottom portion of each panel) prepared with pC4 (see Materials and Methods). The closed and open arrowheads denote the full-length chromosome-derived NDT80 transcripts and the truncated ndt80 minigene-derived transcripts, respectively. The ratio column presents a normalized ratio for the expression of each plasmid-borne (−505^mutant)ndt80 minigene relative to the chromosomal NDT80 gene in the same cells, determined as follows. The WT column gives the intensity of the hybridization signal for transcripts derived from each plasmid-borne (−505^mutant)ndt80 minigene at 6 h of sporulation (lane 5) relative to the intensity of the hybridization signal for transcripts derived from the chromosomal NDT80 gene in the same cells at 6 h of sporulation, normalized to the same ratio obtained for transcripts derived from cells containing the (−505^WT)ndt80 plasmid-borne minigene (in panel A). The ndt80 ratio column gives the intensity of the hybridization signal for transcripts derived from each (−505^mutant)ndt80 minigene in Δndt80/Δndt80 cells at 6 h after transfer to sporulation medium (lane 13), normalized to the loading control, relative to the amount of chromosome-derived NDT80 transcripts in wild-type cells at 6 h of sporulation (lane 5), normalized to the control probe, and then normalized to the same ratio obtained for transcripts derived from the (−505^WT)ndt80 minigene (in panel A). Relative intensities of hybridization signals were determined by quantitation of phosphorimages obtained with a Molecular Dynamics STORM 860 PhosphorImager. The images were analyzed with ImageQuant (Molecular Dynamics, Inc.) and IPLab Gel (Signal Analytics Corporation) software. The images of autoradiograms presented in this figure are scans obtained with Adobe Photoshop 5.0 LE software and assembled with the use of Adobe Illustrator 10 and PowerPoint 98.

FIG. 5.

SUM1 prevents premature expression of the NDT80 gene. Northern filters were prepared with RNA from Δndt80/Δndt80 cells (lanes 1 to 8), Δndt80/Δndt80 Δsum1/Δsum1 cells (lanes 9 to 16), and Δsum1/Δsum1 cells (lanes 17 to 24) that contained the plasmid-borne (−505^WT)ndt80 minigene. Cells were harvested during vegetative growth (0 h) or at the indicated time (in hours), as noted above panel A, after transfer of cells to sporulation medium. The filter was hybridized sequentially with the gene-specific probes denoted on the left. The closed and open arrowheads on the right of panel A denote the full-length chromosome-derived NDT80 transcripts and the truncated ndt80 minigene-derived transcripts, respectively.

FIG. 6.

The MSE sites prevent premature expression of the NDT80 gene and mediate upregulation of its expression midway through sporulation. (A to F) The Northern filters represented under the RNA column heading contained RNA extracted from wild-type cells (lanes 1 to 8) and Δndt80/Δndt80 cells (lanes 9 to 16) harvested during vegetative growth (0 h) or at the indicated times, as noted above the top panel (in hours), after transfer of cells to sporulation medium. (G) The Northern filter contained RNA extracted from Δndt80/Δndt80 cells (lanes 1 to 8) and Δndt80/Δndt80 Δsum1/Δsum1 cells (lanes 9 to 16). Cells used for the experiment of each panel harbored the following plasmid-borne ndt80 minigenes: (A) (−505^WT)ndt80; (B) (−505^ΔM1ΔM2)ndt80; (C and G) (−505^ΔM1)ndt80; (D) (−505^ΔM2)ndt80; (E) (−505^ΔU1ΔM1ΔM2)ndt80; (F) (−505^ΔM1ΔU2ΔM2)ndt80. The closed and open arrowheads on the right of panel A denote the full-length chromosome-derived NDT80 transcripts and the truncated ndt80 minigene-derived transcripts, respectively. For experimental details and explanations of nomenclature, see the legend to Fig. 4. The filter represented in panel A is the same as that shown in Fig. 4A.

FIG. 7.

IME2 activates middle sporulation-specific gene expression by alleviating Sum1-mediated repression of the NDT80 gene. A Northern filter was prepared with RNA from ime2/ime2 cells (lanes 1 to 8), ime2/ime2 Δsum1/Δsum1 cells (lanes 9 to 16), and Δsum1/Δsum1 cells (lanes 17 to 24) harvested during vegetative growth (lanes 1, 9, and 17) or at the indicated time after transfer of cells to sporulation medium, as noted above panel A. The filter was hybridized sequentially with the gene-specific probes denoted on the right.

FIG. 8.

Ndt80 that is ectopically expressed in ime2/ime2 cells in sporulation medium is active. A Northern filter was prepared with RNA extracted from wild-type cells (lanes 1 to 9) and ime2/ime2 cells (lanes 10 to 18) during vegetative growth (lanes 1 and 9) or at the indicated time after transfer of cells to sporulation medium, as noted above panel A. Both the wild-type cells and the ime2/ime2 cells harbored a plasmid that contained a HOP1-NDT80 fusion gene, in which the NDT80 coding region was fused to the promoter region (see Materials and Methods). The filter was hybridized sequentially with the gene-specific probes denoted on the right.