Genome-wide Analysis Reveals Inositol, Not Choline, as the Major Effector of Ino2p-Ino4p and Unfolded Protein Response Target Gene Expression in Yeast

Abstract

In the yeast Saccharomyces cerevisiae, the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline. A genome-wide approach using cDNA microarray technology was used to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline. We report that the global response to inositol is completely distinct from the effect of choline. Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating. Moreover, the combination of inositol and choline increased the number of repressed genes compared with inositol alone and enhanced the repression levels of a subset of genes that responded to inositol. In all, 110 genes were repressed in the presence of inositol and choline. Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild-type cells. One set of genes contained the UAS INO sequence and were bound by Ino2p and Ino4p. Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p. Another nonoverlapping set of genes was coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but was not dependent on OPI1 and did not show further repression when choline was present together with inositol. These results suggest that inositol is the major effector of target gene expression, whereas choline plays a minor role.Phospholipids are the key structural elements of membranebounded organelles and play important roles in signaling and membrane trafficking pathways. Each membrane compartment is composed of a unique set of phospholipids whose biophysical properties contribute to the function of each organelle. Phospholipid metabolism is highly regulated by the cell, ensuring the biogenesis and growth of membranes by coordinating the relative rates of synthesis of individual phospholipids with numerous factors, such as the availability of exogenous supplies of phospholipid precursors, growth stage, and membrane trafficking (1, 2).

Multiple bHLH proteins regulate CIT2 expression in Saccharomyces cerevisiae

“…The inositol response in yeast is known to be mediated by the Ino2p-Ino4p bHLH dimer, which regulates the phospholipid biosynthetic genes (Carman and Henry, 1999;Greenberg and Lopes, 1996;Henry and Patton-Vogt, 1998;Jesch et al, 2005;Robinson and Lopes, 2000a;Santiago and Mamoun, 2003). Therefore, the current study suggests that CIT2 expression is coordinated with phospholipid biosynthesis.…”

“…This induction was not evident in the ρ + wild-type strain (Figure 2A). This inositol/choline-mediated induction was the reverse of that which occurs with the phospholipid biosynthetic genes via Ino2p and Ino4p (Jesch et al, 2005;Santiago and Mamoun, 2003). However, the ino2∆ and ino4∆ deletion mutants did not display inositol/choline-mediated induction but, interestingly, did reveal phosphate induction ( Figure 2B).…”

“…This is not entirely surprising, since these genome-wide studies did not use ρ 0 strains, which is where the effect of Ino2p and Ino4p is most prominent ( Figure 2B). The expression-profiling studies each identified a significant number of inositol/choline-induced genes (sets of 35 and 11 in the two studies) (Jesch et al, 2005;Santiago and Mamoun, 2003), but the sets did not overlap significantly. The lack of consistency has been attributed to differences in experimental design and media composition (Jesch et al, 2005).…”

“…Therefore, the current study suggests that CIT2 expression is coordinated with phospholipid biosynthesis. It is worth noting that the role of INO2 and INO4 in regulating CIT2 was not revealed by two expression-profiling studies that identified inositol-regulated or Ino2p : Ino4p-regulated genes (Jesch et al, 2005;Santiago and Mamoun, 2003) and a genome-wide study of transcription factor binding sites (ChIP-chip) (Harbison et al, 2004). This is not entirely surprising, since these genome-wide studies did not use ρ 0 strains, which is where the effect of Ino2p and Ino4p is most prominent ( Figure 2B).…”

“…It has been determined that, while inositol represses phospholipid biosynthetic gene expression and choline enhances this regulation, inositol and choline can affect gene expression separately (Jesch et al, 2005;Santiago and Mamoun, 2003). .…”