Faculty Bibliography

Isw2 ATP-dependent chromatin-remodeling activity is targeted to early meiotic and MATa-specific gene promoters in Saccharomyces cerevisiae. Unexpectedly, preferential cross-linking of wild-type Isw2p was not detected at these loci. Instead, the catalytically inactive Isw2p-K215R mutant is enriched at Isw2 targets, suggesting that Isw2p-K215R, but not wild-type Isw2p, is a sensitive chromatin immunoprecipitation (ChIP) reagent for marking sites of Isw2 activity in vivo. Genome-wide ChIP analyses confirmed this conclusion and identified tRNA genes (tDNAs) as a new class of Isw2 targets. Loss of Isw2p disrupted the periodic pattern of Ty1 integration upstream of tDNAs, but did not affect transcription of tDNAs or the associated Ty1 retrotransposons. In addition to identifying new Isw2 targets, our localization studies have important implications for the mechanism of Isw2 association with chromatin in vivo. Target-specific enrichment of Isw2p-K215R, not wild-type Isw2p, suggests that Isw2 is recruited transiently to remodel chromatin structure at these sites. In contrast, we found no evidence for Isw2 function at sites preferentially enriched by wild-type Isw2p, leading to our proposal that wild-type Isw2p cross-linking reveals a scanning mode of the complex as it surveys the genome for its targets.

The yeast long terminal repeat (LTR) retrotransposon Ty1, like retroviruses, encodes a terminally redundant RNA, which is packaged into virus-like particles (VLPs) and is converted to a DNA copy by the process of reverse transcription. Mutations predicted to interfere with the priming events during reverse transcription and hence inhibit replication are known to dramatically decrease transposition of Ty1. However, additional cis-acting sequences responsible for Ty1 replication and RNA dimerization and packaging have remained elusive. Here we describe a modular mini-Ty1 element encoding the minimal sequence that can be retrotransposed by the Ty1 proteins, supplied in trans by a helper construct. Using a mutagenic screening strategy, we recovered transposition-deficient modular mini-Ty1-HIS3 elements with mutations in sequences required in cis for Ty1 replication and integration. Two distinct clusters of mutations mapped near the 5'-end of the Ty1 RNA. The clusters define a GAGGAGA sequence at the extreme 5'-end of the Ty1 transcript and a complementary downstream UCUCCUC sequence, 264 nt into the RNA. Disruption of the reverse complementarity of these two sequences decreased transposition and restoration of complementarity rescued transposition to wild-type levels. Ty1 cDNA was reduced in cells expressing RNAs with mutations in either of these short sequences, despite nearly normal levels of Ty1 RNA and VLPs. Our results suggest that the intramolecular interaction between the 5'-GAGGAGA and UCUCCUC sequences stabilizes an RNA structure required for efficient initiation of reverse transcription.

Ty1 elements are long terminal repeat (LTR) retrotransposons that reside within the genome of Saccharomyces cerevisiae. It has been known for many years that the 2'-5' phosphodiesterase Dbr1p, which debranches intron lariats, is required for efficient Ty1 transposition. A recent report suggested the intriguing possibility that Ty1 RNA forms a lariat as a transposition intermediate. We set out to further investigate the nature of the proposed Ty1 lariat branchpoint. However, using a wide range of techniques we were unable to find any evidence for the proposed lariat structure. Furthermore, we demonstrate that some of the techniques used in the initial study describing the lariat are capable of incorrectly reporting a lariat structure. Thus, the role of the Dbr1 protein in Ty1 retrotransposition remains elusive.

The silent information regulator protein (Sir2) and its homologs (collectively known as sirtuins) are NAD+-dependent deacetylase enzymes involved in chromosome stability, gene silencing and cell aging in eukaryotes and archaea. The discovery that sirtuin-dependent protein deacetylation is a NAD+-consuming reaction established a link with the energy generation systems of the cell. This link to metabolism was recently extended to the post-translational control of the activity of short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast. The crystal structure of the Sir protein complexed with a peptide of a protein substrate provided insights into how sirtuins interact with their protein substrates.

Study of mutant phenotypes is a fundamental method for understanding gene function. The construction of a near-complete collection of yeast knockouts (YKO) and the unique molecular barcodes (or TAGs) that identify each strain has enabled quantitative functional profiling of Saccharomyces cerevisiae. By using these TAGs and the SGA reporter, MFA1pr-HIS3, which facilitates conversion of heterozygous diploid YKO strains into haploid mutants, we have developed a set of highly efficient microarray-based techniques, collectively referred as dSLAM (diploid-based synthetic lethality analysis on microarrays), to probe genome-wide gene-chemical and gene-gene interactions. Direct comparison revealed that these techniques are more robust than existing methods in functional profiling of the yeast genome. Widespread application of these tools will elucidate a comprehensive yeast genetic network.

Post-translational modifications of the histone tails are correlated with distinct chromatin states that regulate access to DNA. Recent proteomic analyses have revealed several new modifications in the globular nucleosome core, many of which lie at the histone-DNA interface. We interpret these modifications in light of previously published data and propose a new and testable model for how cells implement the histone code by modulating nucleosome dynamics.

When telomerase is inactivated in Saccharomyces cerevisiae, telomeric DNA shortens with every cell division, and cells stop dividing after approximately 100 generations. Survivors that form in these senescent populations and resume growing have variably amplified arrays of subtelomeric Y' elements. We marked a chromosomal Y' element with the his3AI retrotransposition indicator gene and found that Y'HIS3 cDNA was incorporated into the genome at approximately 10- to 1,000-fold-higher frequencies in survivors compared to telomerase-positive strains. Y'HIS3 cDNA mobility was significantly reduced if assayed at 30 degrees C, a nonpermissive temperature for Ty1 retrotransposition, or in the absence of Tec1p, a transcription factor for Ty1. Microarray analysis revealed that Y' RNA is preferentially associated with Ty1 virus-like particles (VLPs). Genomic copies of Y'HIS3 cDNA typically have downstream oligo(A) tracts, followed by a complete Ty1 long terminal repeat and TYA1 or TYB1 sequences. These data are consistent with the use of Ty1 cDNA to prime reverse transcription of polyadenylated Y' RNA within Ty1 VLPs. Unmarked Y'-oligo(A)-Ty1 cDNA was also detected in survivors, reaching copy numbers of approximately 10(-2) per genome. We propose that Y'-oligo(A)-Ty1 cDNA recombines with Y' elements at eroding telomeres in survivors and may play a role in telomere maintenance in the absence of telomerase.

FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate beta-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate beta-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.