Academic journal article Genetics

The Snf1 Protein Kinase and Sit4 Protein Phosphatase Have Opposing Functions in Regulating TATA-Binding Protein Association with the Saccharomyces Cerevisiae INO1 Promoter

Academic journal article Genetics

The Snf1 Protein Kinase and Sit4 Protein Phosphatase Have Opposing Functions in Regulating TATA-Binding Protein Association with the Saccharomyces Cerevisiae INO1 Promoter

Article excerpt

ABSTRACT

To identify the mechanisms by which multiple signaling pathways coordinately affect gene expression, we investigated regulation of the S. cerevisiae INO1 gene. Full activation of INO1 transcription occurs in the absence of inositol and requires the Snfl protein kinase in addition to other signaling molecules and transcription factors. Here, we present evidence that the Sit4 protein phosphatase negatively regulates INO1 transcription. A mutation in 5IT4 was uncovered as a suppressor of the inositol auxotrophy of snf1Δ strains. We found that sit4 mutant strains exhibit an Spt^sup -^ phenotype, suggesting a more general role for Sit4 in transcription. In fact, like the gene-specific regulators of INO1 transcription, Opil, Ino2, and Ino4, both Snf1 and Sit4 regulate binding of TBP to the INO1 promoter, as determined by chromatin immunoprecipitation analysis. Experiments involving double-mutant strains indicate that the negative effect of Sit4 on INO1 transcription is unlikely to occur through dephosphorylation of histone H3 or Opil. Sit4 is a known component of the target of rapamycin (TOR) signaling pathway, and treatment of cells with rapamycin reduces INO1 activation. However, analysis of rapamycin-treated cells suggests that Sit4 represses INO1 transcription through multiple mechanisms, only one of which may involve inhibition of TOR signaling.

IN response to changes in their environment, cells frequently alter their patterns of gene expression. Often, these alterations are mediated by signaling pathways that transmit information to the nucleus and regulate the promoters of genes needed to adapt or respond to the new condition. The mechanisms by which the signaling pathways influence the transcriptional machinery have been dissected in only a few cases. From the analysis of Saccharomyces cerevisiae genes required for growth in the presence of galactose (BASH and LOHR 2001; LARSCHAN and WINSTON 2001; KAO et al 2004) or in the absence of phosphate (see references in SVAREN and HORZ 1997; AUESUKAREE et al 2004; MARTINEZCAMPA et al 2004; REINKE and HORZ 2004), for example, a general picture has emerged in which signaling molecules lead to the recruitment of gene-specific transcriptional regulatory proteins and chromatin-modifying proteins. In turn, these proteins recruit components of the RNA polymerase II (pol II) general transcription machinery, such as the TATA-binding protein (TBP). However, the precise mechanisms by which signal transduction pathways direct changes in transcription are diverse and appear to depend on the promoters being regulated and on the signaling molecules themselves (CosMA 2002).

The ability of yeast to sense the availability of inositol and to adjust the expression of genes required for lipid biosynthesis, in particular the INOl gene, has been studied as a model for the integration of signaling pathways that affect transcription. The INOl gene encodes inositol-1-phosphate synthase, which converts glucose-6-phosphate to inositol-1-phosphate for use in phospholipid synthesis. Transcription of the INOl gene is repressed under conditions of exogenous inositol (HENRY and PATTON-VOGT 1998). In the absence of inositol, the INOl promoter is derepressed through the actions of a number of gene-specific and general transcription factors and cofactors (HENRY and PATTON-VOGT 1998). The heterodimeric Ino2-Ino4 complex binds to upstream activating sequences in the INOl promoter and is required for activation of transcription (HENRY and PATTON-VOGT 1998). The negative regulator Opil appears to sense levels of the lipid precursor phosphatidic acid (PA) through a direct binding mechanism that involves Scs2, a protein that resides in the endoplasmic reticulum (ER) membrane (LOEWEN et al. 2004). In the presence of inositol, PA is converted to phosphatidylinositol, and Opil is released from the ER and translocates to the nucleus. The mechanisms by which Opil represses the INOl promoter and by which its absence permits INOl transcription are not well understood. …

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