Transcriptional regulation of meiosis in budding yeast

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It is not clear if the activity of Ime1 as a transcriptional activator is subject to regulation. Using the SK1 strain and a LexA-Ime1 fusion, it was shown that transcriptional activation of the reporter gene lexAop-lacZ depends on nitrogen depletion and the presence of a protein kinase, Rim11 (Smith et al., 1993). Genetic analysis showed that in this system Rim11 was required to relieve a repression activity of Ime1 that was modulated by the C-terminal domain of Ime1. This conclusion was based on the following results: i. A lexA-Ime1 fusion truncated for the C-terminal 66 amino acids activated transcription of lexAop-lacZ in rim11∆ cells (Smith et al., 1993). ii. LexA-Ime1L321F mutant protein is impaired in both association with Rim11 and transcriptional activation (Malathi et al., 1997). On the other hand, using the S288C strain and a Gal4(bd)-Ime1 fusion, it was shown that transcriptional activation of the reporter gene gal1-lacZ is independent of growth conditions or Rim11 (Mandel et al., 1994; Rubin-Bejerano et al., 1996). In both strains Rim11 is required for the transcription of EMG and sporulation (Mandel et al., 1994; Mitchell and Bowdish, 1992). The reasons for the disparity between these reports are not known, but several possibilities can be suggested. i. The use of different strain backgrounds, S288C and SK1. ii. The binding of the Gal4(bd) and lexA to the DNA requires its dimerization. The gal4(bd)- IME1 gene includes the Gal4 dimerization signal, whereas the lexA-IME1 gene might lack the lexA dimerization sequence (Golemis and Brent, 1992). Under meiotic conditions Ime1 can oligomerize, and this activity depends on Rim11 (Rubin-Bejerano et al., 1996). Therefore, it is possible that the ability of the lexA-Ime1 protein to activate transcription only under meiotic conditions and only in the RIM11 strain is due to the ability of the protein to dimerize and bind the DNA only under these conditions. Deletion and mutation analysis reveals that Ime1 is composed of at least two domains essential for meiosis: a transcriptional activation domain (ad) (amino acids 165-228), and an interaction domain (id) (amino acids 270-360) (Mandel et al., 1994; Smith et al., 1993). The N-terminal 160 amino acids are not essential for meiosis (Mandel et al., 1994). However, an Ime1 protein truncated for this domain gives rise to lower levels of asci in comparison to cells expressing this truncated protein fused to the Gal4(bd), suggesting that the later might either provide a nuclear localization signal or increase the stability of the protein (Mandel et al., 1994). Two hybrid assays reveal that Ime1 interacts with Ume6, and that this interaction is through amino acids 270-360 of Ime1 [Ime1(id)] and amino acids 1-232 of Ume6 [Ume6(id)] (Colomina et al., 1999; Rubin-Bejerano et al., 1996; Xiao and Mitchell, 2000). The validity of this interaction is evident from the use of different strain backgrounds and different two-hybrid systems, namely, the Gal4(bd)-Ime1(id) with Ume6(id)-Gal4(ad) (Rubin-Bejerano et al., 1996; Xiao and Mitchell, 2000), and the tetR-Ime1(id) with Ume6(id)-VP16 (Colomina et al., 1999). 23
However, direct demonstration of physical association between Ime1 and Ume6 is still missing. The use of the two-hybrid assay enables the demonstration that this interaction is negatively regulated by both glucose and nitrogen. The interaction is absent in vegetative growth conditions with glucose as the sole carbon source, and excessive interaction takes place under meiotic conditions (SPM media) (Colomina et al., 1999; Rubin-Bejerano et al., 1996). Shifting glucose grown stationary cells to acetate growth media leads to partial interaction (Rubin-Bejerano et al., 1996), suggesting that the interaction of Ime1 with Ume6 depends on the absence of both glucose and nitrogen. Furthermore, this interaction is absolutely dependent on Rim11 (Colomina et al., 1999; Rubin-Bejerano et al., 1996), and partially dependent on Rim15 (Vidan and Mitchell, 1997). Two models were suggested for the role of Ime1 Ume6 and Rim11 in the transcription of EMG. I. Bowdish et al proposed that Ume6 is converted from a repressor to an activator following phosphorylation by Rim11, and that Ime1, which associates with Rim11 (Bowdish et al., 1994; Rubin-Bejerano et al., 1996), is required to recruit Rim11 to Ume6 (Bowdish et al., 1995). ii. Rubin-Bejerano et al proposed that Ume6 recruits Ime1 to the URS1 element present in EMG, and that this recruitment promotes the Ime1 transcriptional activation domain to induce the transcription of EMG. According to the later, Rim11 is required for the association between Ime1 and Ume6 (Rubin-Bejerano et al., 1996). The following results support the second model: i. Ime1 functions as a transcriptional activator (Mandel et al., 1994; Smith et al., 1993). ii. Fusion of an heterologous transcriptional activation domain, that of Gal4, to Ime1(id), leads to the expression of the EMG, IME2, and sporulation (Mandel et al., 1994). Moreover, this Ime1(id)-Gal4(ad) fusion protein promotes meiosis of ime1∆ diploid cells only when galactose is added to the sporulation media (Mandel et al., 1994). Since galactose is required to relieve repression of Gal80 on the transcriptional activation of Gal4(ad) (Johnston and Carlson, 1992), it was suggested that the normal function of Ime1 is to activate transcription (Mandel et al., 1994). iii. A Gal4(ad)- Ume6(159-836) fusion protein that is expressed from the IME1 promoter bypasses the requirement for IME1 for the transcription of EMG and sporulation: it leads to expression of the EMG, HOP1, and to 40% sporulation (Rubin-Bejerano et al., 1996). iv. A Gal4(ad)-Ume6 fusion protein can activate the transcription of the EMG SPO13 in SD media if it carries mutations that prevent its association with Sin3 (Washburn and Esposito, 2001). In the absence of the Gal4(ad), these ume6 mutants do not promote expression of EMG (Washburn and Esposito, 2001). These results suggest that by itself Ume6 does not function as a transcriptional activator, and its conversion into a positive regulator depends on the recruitment of the transcriptional activation domain of Ime1 (Rubin-Bejerano et al., 1996; Washburn and Esposito, 2001). Currently, this is 24
Page 1 and 2: Transcriptional regulation of meios
Page 3 and 4: 3. Proteins required for the associ
Page 5 and 6: I. Introduction The budding yeast S
Page 7 and 8: et al., 2000). On the other hand, i
Page 9 and 10: the UCS4 element upstream of the CY
Page 11 and 12: egulator of Cyr1, CDC25 encodes the
Page 13 and 14: 1998). Furthermore, unlike IREu, IR
Page 15 and 16: In the absence of glucose Sok2 does
Page 17 and 18: autophosphorylation, including on a
Page 19 and 20: hours in SPM, followed by a decline
Page 21 and 22: 4. The SIN3, UME6, and RPD3 genes.
Page 23: 1993). Nevertheless, when Ime1 is t
Page 27 and 28: 3. Proteins required for the associ
Page 29 and 30: heterologous promoter does not supp
Page 31 and 32: expressed only in the absence of gl
Page 33 and 34: structure of hCDK2 reveals that cyc
Page 35 and 36: terminal non-essential domain (Komi
Page 37 and 38: is dependent on Ime2, but in cells
Page 39 and 40: VI. Transcriptional regulation of l
Page 41 and 42: The transcriptional activator that
Page 43 and 44: Functional interaction between Ime1
Page 45 and 46: Bowdish, K. S., and Mitchell, A. P.
Page 47 and 48: Foiani, M., Nadjar-Boger, E., Capon
Page 49 and 50: Jeffrey, P. D., Russo, A. A., Polya
Page 51 and 52: Lindgren, A., Bungard, D., Pierce,
Page 53 and 54: Pazin, M. J., and Kadonaga, J. T. (
Page 55 and 56: Shenhar, G. (2001). TRANSCRIPTIONAL
Page 57 and 58: Szent-Gyorgyi, C. (1995). A biparti
Page 59 and 60: Yoshida, M., Kawaguchi, H., Sakata,
Page 61 and 62: Fig. 4. The function of positive an
Page 63 and 64: Fig. 9. A schematic structure of IM
Page 65 and 66: activation by Ime2 is due to phosph
Page 67 and 68: MCK1 Protein kinase required for ef
Page 69: UME3 A cyclin C homolog that associ

Transcriptional regulation of meiosis in budding yeast

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