The consumption of nutrients is one of the main ways organisms interact with their local environments. Especially considering the importance of nutrition in human disease, it is vital to understand the nature of this interaction. All the work described in this thesis was motivated by this goal, through experiments done on various yeast species. I explored whether deficiency of the vitamin folate affected a potentially heritable form of gene regulation, histone methylation. Also, I conducted experiments to understand the factors mediating the transcriptional regulation involved in the synthesis of sulfur-containing amino acids. Finally, I sought to characterize a highly-conserved gene predicted be involved in methionine synthesis.
The vitamin folate is required for proper methionine homeostasis in all organisms. Methionine is the precursor to s-adenosyl-methionine (SAM), which is used in myriad cellular methylation reactions, including all histone methylation reactions. Thus, I conducted experiments that showed that folate and methionine deficiency led to reduced methylation of lysine 4 of histone H3 (H3K4). This was not due to a general growth defect, as cells grown at low temperature did not show any effect on H3K4 methylation. The effect on H3K79 methylation was less pronounced. It was possible to exacerbate the effect on H3K79 methylation through the use of a hypomorphic allele, indicating the enzyme responsible for H3K79 methylation, Dot1, was less sensitive to changes in SAM concentration than the enzymes responsible for H3K4 methylation, Set1. Folate deficiency also caused changes in gene transcription that mirrored changes seen during complete loss of H3K4 methylation. Finally, methionine deficiency was also seen to affect H3K4 methylation in the fission yeast Schizosaccharomyces pombe, though folate deficiency did not.
Transcription of the MET regulon, encoding the proteins involved in the synthesis of the sulfur-containing amino acids, methionine and cysteine, is repressed by the presence of either methionine or cysteine in the environment. This is accomplished by ubiquitination of the transcription factor Met4, carried out by the SCF(Met30) E3 ligase. Previous work indicates cysteine is the repressive agent, and methionine downregulates the MET regulon through its ability to be converted to cysteine. However, I found that a previously untested member of the MET regulon, STR3, was controlled by methionine availability, rather than cysteine. This fits the logic of the synthesis pathway, as Str3 is involved in synthesis of methionine from cysteine. Also, in order to understand how the activity of SCF(Met30) towards Met4 is controlled, I investigated whether there are post-translational modifications of Met30 that are differential between cells grown with and without sulfur-containing amino acids. Finally, I conducted a screen to find more components of the machinery controlling MET regulon repression. I found that loss of Cho2, which is involved in the methylation of phosphatidylethanolamine to produce phosphatidylcholine, leads to upregulation of the MET regulon, which is due to reduced cysteine synthesis in cho2∆ cells.
Methylenetetrahydrofolate reductase (MTHFR) produces the substrate methyltetrahydrofolate (MTHF), which is required for methionine synthesis. Genomic analysis indicates that many fungal species have two copies of MTHFR-encoding genes. In S. cerevisiae deletion of one of the MTHFR-encoding genes (MET13) causes methionine auxotrophy, whereas deletion of the other (MET12) does not. I found that the MET13 ortholog was also essential for methionine prototrophy in Saccharomyces bayanus, indicating that MET12 has not been able to support MTHF synthesis for a significant evolutionary period. To determine the critical differences between MET12 and MET13, I tested a series of chimeric proteins composed of parts of Met12 and Met13 for the ability to support methionine prototrophy, and found that much of Met12 could be substituted into Met13 to produce functional chimeras. However, two small critical regions of Met12 crippled MTHFR function when substituted into Met13, both of which were in the catalytic domain.
