Transcriptional regulation of the SMK1 mitogen-activated protein kinase gene during meiotic development in Saccharomyces cerevisiae

Abstract

Meiotic development (sporulation) in Saccharomyces cerevisiae is characterized by an ordered pattern of gene expression, with sporulation-specific genes classified as early, middle, mid-late, or late depending on when they are expressed. SMK1 encodes a mitogen-activated protein kinase required for spore morphogenesis that is expressed as a middle sporulation-specific gene. Here, we identify the cis-acting DNA elements that regulate SMK1 transcription and characterize the phenotypes of mutants with altered expression patterns. The SMK1 promoter contains an upstream activating sequence (UASS) that specifically interacts with the transcriptional activator Abf1p. The Abf1p-binding sites from the early HOP1 and the middle SMK1 promoters are functionally interchangeable, demonstrating that these elements do not play a direct role in their differential transcriptional timing. Timing of SMK1 expression is determined by another cis-acting DNA sequence termed MSE (for middle sporulation element). The MSE is required not only for activation of SMK1 transcription during middle sporulation but also for its repression during vegetative growth and early meiosis. In addition, the SMK1 MSE can repress vegetative expression in the context of the HOP1 promoter and convert HOP1 from an early to a middle gene. SMK1 function is not contingent on its tight transcriptional regulation as a middle sporulation-specific gene. However, promoter mutants with different quantitative defects in SMK1 transcript levels during middle sporulation show distinct sporulation phenotypes.

FIG. 1

Analysis of the SMK1 promoter using SMK1-lacZ expression plasmids. (A) Comparison of relative SMK1 mRNA levels and SMK1-lacZ enzyme activities during sporulation. β-Galactosidase activity levels were measured at 2-h intervals postinduction by using strain LNY150 transformed with pMDP83. Activity is expressed as units of β-galactosidase per milligram of total protein. SMK1 mRNA levels were quantitated by Northern blot hybridization (20). (B) Deletion analysis of the SMK1 promoter. The indicated deletions were generated from the −219 promoter construct pMDP83 (see Table 2). β-Galactosidase activities were determined in vegetative cells (V) and 10 h postinduction (S) and are averages from at least two separate determinations. In each case the deletion point indicates the number of base pairs between the initiator ATG and the C that is the most 3′ base of the KpnI restriction endonuclease site of the parental plasmid. (C) Nucleotide sequence of the SMK1 promoter. Deletion endpoints diagrammed in panel B are indicated (*), and the initiator ATG of SMK1 is boldfaced. Consensus elements referred to in the text and the KpnI restriction enzyme site used in the construction of pMDP83 are underlined.

FIG. 2

Comparison of UAS^S and UAS^H sites. (A) EMSA of Abf1p binding. Partially purified Abf1p was serially diluted fivefold and used in binding reactions to radiolabeled 13-bp oligonucleotide duplexes containing the HOP1 Abf1p-binding site (lanes 1 to 5), the wild-type SMK1 Abf1p-binding site (lanes 6 to 10), and the mutant SMK1 Abf1p sites containing the −115A (lanes 11 to 15) and −118A (lanes 16 to 20) substitutions. (B) Analysis of HOP1 and SMK1 promoters containing heterologous Abf1p-binding sites. The HOP1 promoter (upper panel) lacking its Abf1p-binding element (open circles), reconstituted with the HOP1 binding site (open squares), or reconstituted with the SMK1 Abf1 site (closed squares) was tested by using the HOP1-lacZ plasmid pAV130, pCC83, or pJX33, respectively. The SMK1 promoter (lower panel) lacking a functional Abf1p-binding site (−118A mutant; open circles), containing its normal Abf1p-binding site (open squares), or reconstituted with the Abf1p-binding site from HOP1 (closed squares) was tested for expression of β-galactosidase by using the SMK1-lacZ plasmid pMDP126-118A, pMDP126, or pMDP113, respectively, in yeast strain LNY150 as described in Materials and Methods. The experiment was performed independently three times with similar results.

FIG. 3

Mutational analysis of UAS^S. (A) Effects of UAS^S mutations on expression of SMK1 and binding to Abf1p. Strains with the indicated mutations in the −124 promoter SMK1-lacZ plasmid pMDP126 were assayed for β-galactosidase activity in vegetative (V) and sporulated cultures at 10 h postinduction (S). β-Galactosidase activities are averages from at least two separate comparisons to the wild-type plasmid (pMDP126). Both vegetative and sporulation values are shown as percentages of the wild-type sporulation value (79 ± 8 U/mg of total protein). Relative complex formation between oligonucleotide duplexes containing the indicated mutation and partially purified Abf1p (Binding) was quantitated from a single titration curve performed as shown in Fig. 2. The mutations are referred to by the numbers at the left. Sequence requirements for Abf1p binding (Abf1 con) are shown above for comparison. The HOP1-Abf1 mutation contains a 6-bp substitution to generate the HOP1 Abf1p-binding site, which reads 5′-ATCACTTCACACG-3′. (B) Hybridization analysis of UAS^S promoter mutants. Indicated promoter mutations in the context of the wild-type SMK1 gene were integrated at the ura3 locus, and RNA was prepared from vegetative (V) or sporulated (S) cultures 10 h postinduction. Hybridization analysis was performed on total RNA by Northern blot analysis using an SMK1-specific probe. The same filter was subsequently hybridized with the middle sporulation SPO12-specific probe as a normalization control. The SMK1-specific hybridization signal in sporulating samples (normalized for SPO12 hybridization) was reduced to 51, 48, 23, and 17% of the wild-type signal in the −116A, −115A, −115A −106A, −117A, and −118A mutant strains, respectively.

FIG. 4

Expression of β-galactosidase by mse^S and pseudo-urs1^S SMK1 promoter plasmids during sporulation. Yeast strain LNY150 transformed with a plasmid expressing β-galactosidase under the control of the wild-type (A), pseudo-urs1^S (B), mse^S (C), or pseudo-urs1^S mse^S (D) −139 promoter (pMDP89, pMDP119, pMDP174, or pMDP176, respectively) was synchronously sporulated, and β-galactosidase levels (expressed as units per milligram of total protein) were determined at 2-h intervals. The experiment was performed at least twice for each promoter with similar results.

FIG. 5

Expression of SMK1 mRNA by mse^S and urs1^S promoter mutants during sporulation. Diploid yeast containing a single integrated copy of wild-type SMK1 coding information under the control of the indicated mutant promoter was transferred to sporulation medium, and total RNA was prepared from cells harvested at the indicated times and assayed by Northern blot hybridization. The indicated smk1 promoter mutants were generated using the following integrating plasmids, from left to right: wild-type (pMDP199), pseudo-urs1^S (pMDP183), mse^S (pMDP185), and pseudo-urs1^S mse^S (pMDP187) (see Table 2). The same filter was probed with sequences specific for the SPO12 middle gene, the HOP1 early gene, and the pC4/2 constitutive expression control.

FIG. 6

Reconstitution of the HOP1 promoter with MSE^S and URS1^S. Yeast cells of strain LNY273 transformed with HOP1 promoter-lacZ plasmids containing the indicated cis-regulatory elements were synchronously sporulated, and β-galactosidase levels were measured. pAV124 contains a 5-bp substitution that destroys the naturally occurring URS1^H and introduces an XhoI site (open circles); pCC51 contains URS1^H (closed circles); pJX42 contains pseudo-URS^S (open triangles); and pJX43 contains MSE^S (closed squares) in the XhoI site of pAV124. The control is pMDP89 (open squares), which is the SMK1 promoter-lacZ plasmid. Data are averages from three determinations.