Branched chain amino acid regulation of the ILV2 locus in Saccharomyces cerevisiae

Mutant regulatory loci of the branched pathway for the biosynthesis of isoleucine-valine and leucine were identified with the unusual phenotype of an amino acid dependent auxotrophy. Two mutant loci, bcs1 and bcs2, conferred branched chain amino acid sensitivity and showed independent segregation. Linkage studies defined bcs1 as a cis-acting regulatory site of ILV2 (SMR1). ILV2 upstream deletion analyses and high-copy transformation of the positive regulatory locus LEU3 ruled out the possibility of LEU3 protein binding palindromes mediating the branched chain amino acid dependent auxotrophy. In the presence of leucine and valine, the general amino acid control system (GCN4) was epistatic to bcs1 and bcs2, and under nonstarvation conditions GCN4 strains showed an increased acetolactate synthase activity over gcn4 strains. Thus in addition to general regulation of ILV2, GCN4 functions in basal level expression when the locus is subject to specific repression by pathway end product.     

Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI.

In Saccharomyces cerevisiae, the TPI gene product, triosephosphate isomerase, makes up about 2% of the soluble cellular protein. Using in vitro and in vivo footprinting techniques, we have identified four binding sites for three factors in the 5' noncoding region of TPI: a REB1-binding site located at positions -401 to -392, two GCR1-binding sites located at positions -381 to -366 and -341 to -326, and a RAP1-binding site located at positions -358 to -346. We tested the effects of mutations at each of these binding sites on the expression of a TPI::lacZ gene fusion which carried 853 bp of the TPI 5' noncoding region integrated at the URA3 locus. The REB1-binding site is dispensable when material 5' to it is deleted; however, if the sequence 5' to the REB1-binding site is from the TPI locus, expression is reduced fivefold when the site is mutated. Because REB1 blocks nucleosome formation, the most likely function of its binding site in the TPI controlling region is to prevent the formation of nucleosomes over the TPI upstream activation sequence. Mutations in the RAP1-binding site resulted in a 10-fold reduction in expression of the reporter gene. Mutating either GCR1-binding site alone had a modest effect on expression of the fusion. However, mutating both GCR1-binding sites resulted in a 68-fold reduction in the level of expression of the reporter gene. A LexA-GCR1 fusion protein containing the DNA-binding domain of LexA fused to the amino terminus of GCR1 was able to activate expression of a lex operator::GAL1::lacZ reporter gene 116-fold over background levels. From this experiment, we conclude that GCR1 is able to activate gene expression in the absence of REB1 or RAP1 bound at adjacent binding sites. On the basis of these results, we suggest that GCR1 binding is required for activation of TPI and other GCR1-dependent genes and that the primary role of other factors which bind adjacent to GCR1-binding sites is to facilitate of modulate GCR1 binding in vivo.     

Sinomenine increases the methylation level at specific GCG site in mPGES-1 promoter to facilitate its specific inhibitory effect on mPGES-1

Prostaglandin E₂ (PGE₂) in cancer and inflammatory diseases is a key mediator of disease progression. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to inhibit the expression of PGE₂ by depressing cyclooxygenase (COX) in inflammatory treatments. However, the inhibition to COXs may cause serious side effects. Thus, it is urgent to develop new anti-inflammatory drugs aiming new targets to inhibit PGE₂ production. Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the final step of PGE₂ biosynthesis. Therefore, the selective inhibition of mPGES-1 has become a promising strategy in the treatments of cancer and inflammatory diseases. Our previous studies confirmed that sinomenine (SIN) is a specific mPGES-1 inhibitor. However, the exact mechanism by which SIN inhibits mPGES-1 remains unknown. This study aimed to explain the regulation effect of SIN to mPGES-1 gene expression by its DNA methylation induction effect. We found that the demethylating agent 5-azacytidine (5-AzaC) reversed the inhibitory effect of SIN to mPGES-1. Besides, SIN selectively increased the methylation level of the promoter region in the mPGES-1 gene while the pretreatment of 5-AzaC suppressed this effect. The results also shows that pretreatment with SIN increased the methylation level of specific GCG sites in the promoter region of mPGES-1. This specific methylation site may become a new biomarker for predicting and diagnosing RA and cancer with high expression of mPGES-1. Also, our research provides new ideas and solutions for clinical diagnosis and treatment of diseases related to mPGES-1 and for targeted methylation strategy in drug development.     

The yeast ILV2 gene is under general amino acid control

The yeast ILV2 gene encodes acetolactate synthase, the first enzyme in the biosynthesis of isoleucine and valine. Its multiple regulation has precluded the clear demonstration of whether ILV2 is under general amino acid control. Nonderepressible gcn4 strains were used as recipients for transformation with a YCp plasmid carrying GCN4. Parental gcn4 cells and their isogenic GCN4 transformants were evaluated for ALS derepression following induced amino acid starvation. GCN4 cells showed 1.5- to 1.7-fold derepression but no derepression was observed in isogenic control gcn4 strains. A similar depression of ILV2 mRNA was also observed. Genetic evidence for general amino acid control was the gcn4 suppression of high level resistance to sulfometuron methyl by the SMRI-410 allele of ILV2.