Jasper Rine

Epigenetic inheritance of transcriptional states in S. cerevisiae

SIR1, one of several genes required for repression of yeast silent mating type loci, has a unique role in repression of the HML alpha locus. Single-cell assays revealed that cells with mutant alleles of SIR1, including presumptive null alleles, existed as populations of genetically identical cells whose members were in one of two different regulatory states. A minority of cells had a repressed HML alpha locus whereas the majority had a derepressed HML alpha locus. The two states were mitotically stable, although rare changes in state were observed during mitotic growth, possibly reflecting heritable changes to the HML alpha locus at or before replication. Analysis of changes in state suggests that SIR1 protein has a role in the establishment but not the maintenance of repression of silent mating type genes, whereas SIR2, SIR3, and SIR4 are required for maintenance.

Upc2p and Ecm22p, Dual Regulators of Sterol Biosynthesis in Saccharomyces cerevisiae

ABSTRACT Sterol levels affect the expression of many genes in yeast and humans. We found that the paralogous transcription factors Upc2p and Ecm22p of yeast were sterol regulatory element (SRE) binding proteins (SREBPs) responsible for regulating transcription of the sterol biosynthetic genes ERG2 and ERG3. We defined a 7-bp SRE common to these and other genes, including many genes involved in sterol biosynthesis. Upc2p and Ecm22p activatedERG2 expression by binding directly to this element in the ERG2 promoter. Upc2p and Ecm22p may thereby coordinately regulate genes involved in sterol homeostasis in yeast. Ecm22p and Upc2p are members of the fungus-specific Zn[2]-Cys[6] binuclear cluster family of transcription factors and share no homology to the analogous proteins, SREBPs, that are responsible for transcriptional regulation by sterols in humans. These results suggest that Saccharomyces cerevisiae and human cells regulate sterol synthesis by different mechanisms.

The Awesome Power of Yeast Evolutionary Genetics: New Genome Sequences and Strain Resources for the Saccharomyces sensu stricto Genus

High-quality, well-annotated genome sequences and standardized laboratory strains fuel experimental and evolutionary research. We present improved genome sequences of three species of Saccharomyces sensu stricto yeasts: S. bayanus var. uvarum (CBS 7001), S. kudriavzevii (IFO 1802T and ZP 591), and S. mikatae (IFO 1815T), and describe their comparison to the genomes of S. cerevisiae and S. paradoxus. The new sequences, derived by assembling millions of short DNA sequence reads together with previously published Sanger shotgun reads, have vastly greater long-range continuity and far fewer gaps than the previously available genome sequences. New gene predictions defined a set of 5261 protein-coding orthologs across the five most commonly studied Saccharomyces yeasts, enabling a re-examination of the tempo and mode of yeast gene evolution and improved inferences of species-specific gains and losses. To facilitate experimental investigations, we generated genetically marked, stable haploid strains for all three of these Saccharomyces species. These nearly complete genome sequences and the collection of genetically marked strains provide a valuable toolset for comparative studies of gene function, metabolism, and evolution, and render Saccharomyces sensu stricto the most experimentally tractable model genus. These resources are freely available and accessible through www.SaccharomycesSensuStricto.org.

Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins

Significance Chromatin immunoprecipitation (ChIP) is a gold standard technique for genomic protein localization. We have discovered an artifact in ChIP that leads to reproducible but biologically meaningless enrichment of proteins at highly expressed genes, caused by high levels of polymerase II and polymerase III transcription. These findings call into question reports of unexpected localization of transcription factors, repressors, and cytosolic proteins to highly expressed genes. We suggest caution when interpreting ChIP enrichment at highly expressed genes and suggest a heterologous protein control in ChIP experiments to discern biologically meaningful from artifactual enrichment. Chromatin immunoprecipitation (ChIP) is the gold-standard technique for localizing nuclear proteins in the genome. We used ChIP, in combination with deep sequencing (Seq), to study the genome-wide distribution of the Silent information regulator (Sir) complex in Saccharomyces cerevisiae. We analyzed ChIP-Seq peaks of the Sir2, Sir3, and Sir4 silencing proteins and discovered 238 unexpected euchromatic loci that exhibited enrichment of all three. Surprisingly, published ChIP-Seq datasets for the Ste12 transcription factor and the centromeric Cse4 protein indicated that these proteins were also enriched in the same euchromatic regions with the high Sir protein levels. The 238 loci, termed ”hyper-ChIPable“, were in highly expressed regions with strong polymerase II and polymerase III enrichment signals, and the correlation between transcription level and ChIP enrichment was not limited to these 238 loci but extended genome-wide. The apparent enrichment of various proteins at hyper-ChIPable loci was not a consequence of artifacts associated with deep sequencing methods, as confirmed by ChIP-quantitative PCR. The localization of unrelated proteins, including the entire silencing complex, to the most highly transcribed genes was highly suggestive of a technical issue with the immunoprecipitations. ChIP-Seq on chromatin immunoprecipitated with a nuclear-localized GFP reproduced the above enrichment in an expression-dependent manner: induction of the GAL genes resulted in an increased ChIP signal of the GFP protein at these loci, with presumably no biological relevance. Whereas ChIP is a broadly valuable technique, some published conclusions based upon ChIP procedures may merit reevaluation in light of these findings.

A role for yeast oxysterol-binding protein homologs in endocytosis and in the maintenance of intracellular sterol-lipid distribution.

The seven yeast OSH genes (OSH1-OSH7) encode a family of orthologs of the mammalian oxysterol-binding protein (OSBP). The OSH genes share at least one essential overlapping function, potentially linked to the regulation of secretory trafficking and membrane lipid composition. To investigate the essential roles of the OSH genes, we constructed conditional OSH mutants and analyzed their cellular defects. Elimination of all OSH function altered intracellular sterol-lipid distribution, caused vacuolar fragmentation, and resulted in an accumulation of lipid droplets in the cytoplasm and within vacuolar fragments. Gradual depletion of Osh proteins also caused cell budding defects and abnormal cell wall deposition. In OSH mutant cells endocytosis was severely impaired, but protein transport to the vacuole and the plasma membrane was largely unaffected. Other mutants affecting sterol-lipid function and distribution, namely erg2Δ and arv1Δ, shared similar defects. These findings suggested that OSH genes, through effects on intracellular sterol distribution, establish a plasma membrane lipid composition that promotes endocytosis.

The biology of HMG-CoA reductase: the pros of contra-regulation

Hydroxymethylglutaryl-CoA reductase (HMG-R) is a key enzyme in the mevalonate pathway, from which thousands of molecules are derived including cholesterol and prenyl moieties. The regulation of HMG-R is complex and includes feedback control, cross-regulation by independent bio-chemical processes and contra-regulation of separate isozymes. From studies in yeast, these separate modes of regulation can be considered in an integrated fashion.

One hundred and one new simple sequence repeat-based markers for the canine genome.

One hundred and one new dinucleotide repeat polymorphisms specific for the canine genome have been identified and characterized. Screening of both primary libraries and marker selected libraries enriched for simple sequence repeats led to the isolation of large numbers of genomic clones that contained (CA)n repeats. Over 200 of these clones were sequenced, and PCR primers that bracket the repeat were developed for those that contained ten or more continuous (CA)n units. This effort led to the production of 101 polymorphic markers, which were assigned to one of four categories depending on their degree of polymorphism. Fiftyfour markers were found to be highly or very highly polymorphic as they had four or more alleles when tested on a panel of unrelated dogs. This group of markers will be useful for following inheritance of traits in crosses between dogs.

A missense mutation in the endothelin-B receptor gene is associated with Lethal White Foal Syndrome: An equine version of Hirschsprung Disease

Abstract. Lethal White Foal Syndrome is a disease associated with horse breeds that register white coat spotting patterns. Breedings between particular spotted horses, generally described as frame overo, produce some foals that, in contrast to their parents, are all white or nearly all white and die shortly after birth of severe intestinal blockage. These foals have aganglionosis characterized by a lack of submucosal and myenteric ganglia from the distal small intestine to the large intestine, similar to human Hirschsprung Disease. Some sporadic and familial cases of Hirschsprung Disease are due to mutations in the endothelin B receptor gene (EDNRB). In this study, we investigate the role of EDNRB in Lethal White Foal Syndrome. A cDNA for the wild-type horse endothelin-B receptor gene was cloned and sequenced. In three unrelated lethal white foals, the EDNRB gene contained a 2-bp nucleotide change leading to a missense mutation (I118K) in the first transmembrane domain of the receptor, a highly conserved region of this protein among different species. Seven additional unrelated lethal white foal samples were found to be homozygous for this mutation. No other homozygotes were identified in 138 samples analyzed, suggesting that homozygosity was restricted to lethal white foals. All (40/40) horses with the frame overo pattern (a distinct coat color pattern that is a subset of overo horses) that were tested were heterozygous for this allele, defining a heterozygous coat color phenotype for this mutation. Horses with tobiano markings included some carriers, indicating that tobiano is epistatic to frame overo. In addition, horses were identified that were carriers but had no recognized overo coat pattern phenotype, demonstrating the variable penetrance of the mutation. The test for this mutant allele can be utilized in all breeds where heterozygous animals may be unknowingly bred to each other including the Paint Horse, Pinto horse, Quarter Horse, Miniature Horse, and Thoroughbred.

Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae.

Two protein prenyltransferase enzymes, farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I), catalyze the covalent attachment of a farnesyl or geranylgeranyl lipid group to the cysteine of a CaaX sequence (cysteine [C], two aliphatic amino acids [aa], and any amino acid [X]. In vitro studies reported here confirm previous reports that CaaX proteins with a C-terminal serine are farnesylated by FTase and those with a C-terminal leucine are geranylgeranylated by GGTase-I. In addition, we found that FTase can farnesylate CaaX proteins with a C-terminal leucine and can transfer a geranylgeranyl group to some CaaX proteins. Genetic data indicate that FTase and GGTase-I have the same substrate preferences in vivo as in vitro and also show that each enzyme can prenylate some of the preferred substrates of the other enzyme in vivo. Specifically, the viability of yeast cells lacking FTase is due to prenylation of Ras proteins by GGTase-I. Although this GGTase-I dependent prenylation of Ras is sufficient for growth, it is not sufficient for mutationally activated Ras proteins to exert deleterious effects on growth. The dependence of the activated Ras phenotype on FTase can be bypassed by replacing the C-terminal serine with leucine. This altered form of Ras appears to be prenylated by both GGTase-I and FTase, since it produces an activated phenotype in a strain lacking either FTase or GGTase-I. Yeast cells can grow in the absence of GGTase-I as long as two essential substrates are overexpressed, but their growth is slow. Such strains are dependent on FTase for viability and are able to grow faster when FTase is overproduced, suggesting that FTase can prenylate the essential substrates of GGTase-I when they are overproduced.

Yeast cell-type regulation of DNA repair

The mating-type locus (MAT) in the yeast Saccharomyces cerevisiae provides information about whether cells are of the a or α mating type, and genes at this locus encode transcriptional regulators that determine the phenotypes associated with the different cell types. In a/α diploid cells, the a1/α2 repressor is formed, which inhibits haploid-specific gene expression and indirectly promotes meiosis. Mutations in SIR (silent information regulator) genes cause a loss of both heterochromatin and transcriptional silencing, resulting in the expression of cryptic a and α genes resident at the HML and HMR loci. As a result, sir mutant strains have the properties of a/α diploids.