Growth-regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes.

Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2-8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3'-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3'-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron-exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.

Gene dosage effects in yeast support broader roles for the LOG1, HAM1 and DUT1 genes in detoxification of nucleotide analogues.

Purine and pyrimidine analogues have important uses in chemotherapies against cancer, and a better understanding of the mechanisms that cause resistance to these drugs is therefore of importance in cancer treatment. In the yeast Saccharomyces cerevisiae, overexpression of the HAM1 gene encoding inosine triphosphate pyrophosphatase confers resistance to both the purine analogue 6-N-hydroxylaminopurine (HAP) and the pyrimidine analogue 5-fluorouracil (5-FU) (Carlsson et al., 2013, PLoS One 8, e52094). To find out more about the mechanisms of resistance to nucleotide analogues, and possible interdependencies between purine and pyrimidine analogue resistance mechanisms, we screened a plasmid library in yeast for genes that confer HAP resistance when overexpressed. We cloned four such genes: ADE4, DUT1, APT2, and ATR1. We further looked for genetic interactions between these genes and genes previously found to confer resistance to 5-FU. We found that HMS1, LOG1 (YJL055W), HAM1, and ATR1 confer resistance to both 5-FU and HAP, whereas ADE4, DUT1 and APT2 are specific for HAP resistance, and CPA1 and CPA2 specific for 5-FU resistance. Possible mechanisms for 5-FU and HAP detoxification are discussed based on the observed genetic interactions. Based on the effect of LOG1 against both 5-FU and HAP toxicity, we propose that the original function of the LOG (LONELY GUY) family of proteins likely was to degrade non-canonical nucleotides, and that their role in cytokinin production is a later development in some organisms.

The Ability of a Charophyte Alga Hexokinase to Restore Glucose Signaling and Glucose Repression of Gene Expression in a Glucose-Insensitive Arabidopsis Hexokinase Mutant Depends on Its Catalytic Activity.

Hexokinases is a family of proteins that is found in all eukaryotes. Hexokinases play key roles in the primary carbon metabolism, where they catalyze the phosphorylation of glucose and fructose, but they have also been shown to be involved in glucose signaling in both yeast and plants. We have characterized the Klebsormidium nitens KnHXK1 gene, the only hexokinase-encoding gene in this charophyte alga. The encoded protein, KnHXK1, is a type B plant hexokinase with an N-terminal membrane anchor localizing the protein to the mitochondrial membranes. We found that KnHXK1 expressed in Arabidopsis thaliana can restore the glucose sensing and glucose repression defects of the glucose-insensitive hexokinase mutant gin2-1. Interestingly, both functions require a catalytically active enzyme, since an inactive double mutant was unable to complement gin2-1. These findings differ from previous results on Arabidopsis AtHXK1 and its orthologs in rice, where catalytic and glucose sensing functions could be separated, but are consistent with recent results on the rice cytoplasmic hexokinase OsHXK7. A model with both catalytic and non-catalytic roles for hexokinases in glucose sensing and glucose repression is discussed.

Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination.

The moss Physcomitrella patens is unique among plants in that homologous recombination can be used to knock out genes, just like in yeast. Furthermore, transformed plasmids can be rescued from Physcomitrella back into Escherichia coli, similar to yeast. In the present study, we have tested if a third important tool from yeast molecular genetics, auxotrophic selection markers, can be used in Physcomitrella. Two auxotrophic moss strains were made by knocking out the PpHIS3 gene encoding imidazoleglycerol-phosphate dehydratase, and the PpTRP1 gene encoding phosphoribosylanthranilate isomerase, disrupting the biosynthesis of histidine and tryptophan, respectively. The resulting PpHIS3Δ and PpTRP1Δ knockout strains were unable to grow on medium lacking histidine or tryptophan. The PpHIS3Δ strain was used to test selection of transformants by complementation of an auxotrophic marker. We found that the PpHIS3Δ strain could be complemented by transformation with a plasmid expressing the PpHIS3 gene from the CaMV 35S promoter, allowing the strain to grow on medium lacking histidine. Both linearized plasmids and circular supercoiled plasmids could complement the auxotrophic marker, and plasmids from both types of transformants could be rescued back into E. coli. Plasmids rescued from circular transformants were identical to the original plasmid, whereas plasmids rescued from linearized transformants had deletions generated by recombination between micro-homologies in the plasmids. Our results show that cloning by complementation of an auxotrophic marker works in Physcomitrella, which opens the door for using auxotrophic selection markers in moss molecular genetics. This will facilitate the adaptation of shuttle plasmid dependent methods from yeast molecular genetics for use in Physcomitrella.

The histone demethylase activity of Rph1 is not essential for its role in the transcriptional response to nutrient signaling.

Rph1 and Gis1 are two related yeast zinc finger proteins that function as downstream effectors in the Ras/PKA, TOR and Sch9 nutrient signaling pathways. Both proteins also contain JmjC histone demethylase domains, but only Rph1 is known to be an active enzyme, demethylating lysine 36 of histone H3. We have studied to what extent the demethylase activity of Rph1 contributes to its role in nutrient signaling by performing gene expression microarray experiments on a yeast strain containing a catalytically inactive allele of RPH1. We find that the enzymatic activity of Rph1 is not essential for its role in growth phase dependent gene regulation. However, the ability of Rph1 to both activate and repress transcription is partially impaired in the active site mutant, indicating that the demethylase activity may enhance its function in vivo. Consistent with this, we find that the Rph1 mutation and a deletion of the histone H3 methylase Set2 affect the same target genes in opposite directions. Genes that are differentially expressed in the Rph1 mutant are also enriched for binding of Rpd3, a downstream effector in silencing, to their promoters. The expression of some subtelomeric genes and genes involved in sporulation and meiosis are also affected by the mutation, suggesting a role for Rph1-dependent demethylation in regulating these genes. A small set of genes are more strongly affected by the active site mutation, indicating a more pronounced role for the demethylase activity in their regulation by Rph1.

A Ham1p-dependent mechanism and modulation of the pyrimidine biosynthetic pathway can both confer resistance to 5-fluorouracil in yeast.

5-Fluorouracil (5-FU) is an anticancer drug and pyrimidine analogue. A problem in 5-FU therapy is acquired resistance to the drug. To find out more about the mechanisms of resistance, we screened a plasmid library in yeast for genes that confer 5-FU resistance when overexpressed. We cloned five genes: CPA1, CPA2, HMS1, HAM1 and YJL055W. CPA1 and CPA2 encode a carbamoyl phosphate synthase involved in arginine biosynthesis and HMS1 a helix-loop-helix transcription factor. Our results suggest that CPA1, CPA2, and HMS1 confer 5-FU resistance by stimulating pyrimidine biosynthesis. Thus, they are unable to confer 5-FU resistance in a ura2 mutant, and inhibit the uptake and incorporation into RNA of both uracil and 5-FU. In contrast, HAM1 and YJL055W confer 5-FU resistance in a ura2 mutant, and selectively inhibit incorporation into RNA of 5-FU but not uracil. HAM1 is the strongest resistance gene, but it partially depends on YJL055W for its function. This suggests that HAM1 and YJL055W function together in mediating resistance to 5-FU. Ham1p encodes an inosine triphosphate pyrophosphatase that has been implicated in resistance to purine analogues. Our results suggest that Ham1p could have a broader specificity that includes 5-FUTP and other pyrimidine analogoue triphosphates.

The jmjN and jmjC domains of the yeast zinc finger protein Gis1 interact with 19 proteins involved in transcription, sumoylation and DNA repair.

The jumonji domain is a highly conserved bipartite domain made up of two subdomains, jmjN and jmjC, which is found in many eukaryotic transcription factors. The jmjC domain was recently shown to possess the histone demethylase activity. Here we show that the jmjN and jmjC domains of the yeast zinc finger protein Gis1 interact in a two-hybrid system with 19 yeast proteins that include the RecQ helicase Sgs1, the silencing factors Esc1 and Sir4, the URI-type prefoldin Bud27 and the PIAS type SUMO ligase Nfi1/Siz2. Extensive interaction cross dependencies further suggest that the proteins form a larger complex. Consistent with this, 16 of the proteins also interact with a Bud27 two-hybrid bait, and three of them co-precipitate with TAP-tagged Gis1. The Gis1 jumonji domain can repress transcription when recruited to a promoter as a lexA fusion. This effect is dependent on both the jmjN and jmjC subdomains, as were all 19 two-hybrid interactions, indicating that the two subdomains form a single functional unit. The human Sgs1 homolog WRN also interacts with the Gis1 jumonji domain. Finally, we note that several jumonji domain interactors are related to proteins that are found in mammalian PML nuclear bodies.

Functional and physical interactions within the middle domain of the yeast mediator.

Med21 (Srb7) is a small essential subunit of the middle domain of the Mediator, which is conserved in all eukaryotes. It is thought to play an important role in both transcriptional activation and repression. In the yeast Saccharomyces cerevisiae, Med21 is known to interact both with the Mediator subunit Med6 and the global co-repressor Tup1. We have made a temperature-sensitive med21-ts mutant, which we used in a high copy number suppressor screen. We found ten yeast genes that can suppress the med21-ts mutation in high copy number. The three strongest suppressors were MED7 and MED10 (NUT2), which encode other Mediator subunits, and ASH1, which encodes a repressor of the HO gene. 2-Hybrid experiments confirmed multiple interactions between Med21, Med10, Med7 and Med4, and also revealed a Med21 self-interaction. The interactions of Med21 with Med7 and Med10 were verified by co-immunoprecipitation of tagged proteins produced in insect cells and E. coli, where both interactions were found to depend strongly on the amino acid residues 2-8 of Med21. These interactions, and the interactions of Med21 with Med6 and Tup1, suggest that Med21 may serve as a molecular switchboard that integrates different signals before they reach the core polymerase.

The structural and functional role of Med5 in the yeast Mediator tail module.

Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5delta and med15delta deletions. The med5delta and med16delta deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.

Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-microm plasmid.

The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro, and its function is conserved in all eukaryotes. Mediator interacts with both transcriptional activators and RNA polymerase II, but little is known about the mechanisms by which it operates at the molecular level. Here, we show that the cyclin-dependent kinase Srb10 interacts with, and phosphorylates, the Med2 subunit of Mediator both in vivo and in vitro. A point mutation of the single phosphorylation site in Med2 results in a strongly reduced expression of the REP1, REP2, FLP1, and RAF1 genes, which are all located on the endogenous 2-microm plasmid. Combined with previous studies on the effects of SRB10/SRB11 deletions, our data suggest that posttranslational modifications of Mediator subunits are important for regulation of gene expression.