Multiple and Distinct Activation and Repression Sequences Mediate the Regulated Transcription of IME1, a Transcriptional Activator of Meiosis-Specific Genes in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae the choice between meiosis-sporulation and alternative developmental pathways such as the mitotic cell cycle, pseudohyphae growth, or G1 arrest depends on the expression and activity of a master regulator, Ime1p. This is deduced from the observation that cells deleted for IME1 are sporulation deficient and arrest in meiosis at G1 prior to any meiotic event, i.e., transcription of meiosis-specific genes, premeiotic DNA replication, meiotic recombination, and nuclear divisions (15, 49). IME1 encodes a transcriptional activator (23, 48) that is recruited to the promoters of early meiosis-specific genes by interacting with a sequence-specific DNA binding protein, Ume6p (41). Initiation of meiosis depends on two signals: starvation for nutrients and the presence of MATa1 and MATα2 gene products (17). The nutrient signal is required at several levels: for the transcription of IME1 (15), for the translation of IME1 mRNA (47), for the association of Ime1p with its meiotic target, Ume6p (41), and for entry into the first meiotic division (21). The MAT signal is also required in more than one step: for the transcription of IME1 and for efficient meiosis (15, 47). The purpose of this study has been to identify the elements in IME1 that are required for its regulated transcription and to determine the role of the RAS-cyclic AMP-dependent protein kinase (cAPK) pathway in the activity of the regulated upstream activating sequence (UAS) elements. Therefore, we shall summarize below the known information concerning the transcription of IME1. Transcripts of IME1 are not detected in the presence of glucose, and a low basal level is present in vegetative acetate media (15). Upon nitrogen depletion the level of IME1 mRNA increases in MATa/MATα diploids, reaching a peak at about 6 to 8 h and then declining (15, 49). The transcription of IME1 is not induced in cells that do not carry both the MATa1 and MATα2 alleles (MAT-insufficient cells) (15, 49). Very little is known about the organization of the IME1 locus. The sequence of IME1 identifies three putative TATA boxes: TATATTA at −353, TATTTAA at −330, and TATAAAT at −158. Deletions of these TATA boxes revealed that the functional TATA is at −330 (1). Accordingly, the main transcription initiation site of IME1 RNA was mapped to −229 (1, 47). The complete genomic sequence of S. cerevisiae reveals that upstream of IME1 there is an extremely large region, 4,122 bp long, that is devoid of open reading frames, tRNA, or rRNA. This suggests the possibility that a large region may be involved in the transcriptional regulation of IME1. Indeed, previous reports have pointed to this phenomenon (5, 12). Covitz and Mitchell reported that the region between −2243 and −1743 upstream of IME1 ATG carries a negative element that prevents the expression of IME1 in MATa/MATa cells (5). Furthermore, a 21-bp element (RRE) located at base pair −2024 to −2044 binds Rme1p (5), a zinc finger protein that represses the transcription of IME1 in MAT-insufficient cells (15, 16, 37). The regulated region may extend even further, since multiple copies of IME1 sequences from −3166 to −3762 promote sporulation in both the presence of nutrients and in MATa/MATa diploids (12). Except for Rme1p, the transcriptional activators and repressors that directly affect the transcription of IME1 are unknown. Nonetheless, several genes that affect the transcription of IME1 have been identified. IME4 encodes a positive regulator that is absolutely required for the transcription of IME1 (44). The transcription of IME4 is induced only in MATa/MATα diploids that are shifted to nitrogen-depleted medium (44), suggesting that Ime4p transmits both MAT and nitrogen signals. IME4 does not encode a DNA binding protein, and its mode of action is not known. The third gene that mediates MAT regulation to IME1 is RES1; a dominant mutation, RES1-1, promotes sporulation of MAT-insufficient diploids (14). RES1 has yet to be cloned, but epistasis tests suggest that it acts in a pathway distinct from either Ime4p or Rme1p (14, 44). The nitrogen depletion signal seems to be transmitted to IME1 via the RAS-cAPK pathway: mutations that cause lower activity of cAPK, such as cdc25, ras2, and cyr1, lead to the expression of IME1 and to meiosis in the presence of nitrogen (references 26, 27, and 49 and references therein). On the other hand, mutations that cause constitutive activity of cAPK, such as RAS2-val19 and bcy1, are sporulation deficient and are suppressed by overexpression of IME1 (27). Mutations in several genes lower the level of IME1 RNA; these include the serine-threonine protein kinase MCK1 (34) and the DNA binding protein RIM1 (50) and its proteolytic cleavage regulators RIM8, RIM9, and RIM13 (22, 51). In this paper we report a systematic analysis of the 5′ untranslated region of IME1 and identify the elements that are required for its regulated transcription. We show that IME1 is regulated by an unusually large region that is composed of alternate negative and positive elements. Our analysis reveals the presence of distinct elements responding to MAT, carbon, and nitrogen regulation. We demonstrate that the RAS-cAPK pathway transmits a glucose signal to one of the regulated UAS elements, IREu. Moreover, gel-shift and expression assays show that Msn2p and Msn4p (Msn2/4p) function as the transcription factors mediating the UAS activity of IREu in the presence of acetate as the sole carbon source.