Faculty Bibliography

A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3-6% and 15-18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens.

Retrotransposons are RNA elements that reverse transcribe their RNA genomes and make a cDNA copy that is inserted back into a new genomic location by the element-encoded integrase protein. Ty1 is a long terminal repeat (LTR) retrotransposon in Saccharomyces cerevisiae that inserts into an approximately 700-bp integration window upstream of tRNA genes with a periodicity of approximately 80 bp. ATP-dependent chromatin remodeling by Isw2 upstream of tRNA genes leads to changes in chromatin structure and Ty1 integration site selection. We show that the N terminus of Bdp1p, a component of the RNA polymerase III transcription factor TFIIIB, is required for periodic integration of Ty1 into the integration window. Deletion of the Bdp1p N terminus and mutation of ISW2 result in similar disruption of nucleosome positioning upstream of some tRNA genes, and the N-terminal domain of Bdp1p is required for targeting of Isw2 complex to tRNA genes. This study provides the first example for recruitment of an ATP-dependent chromatin-remodeling factor by a general transcription factor in vivo.

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 papillomavirus E2 protein functions in viral transcriptional regulation, DNA replication, and episomal genome maintenance. Viral genomes are maintained in dividing cells by attachment to mitotic chromosomes by means of the E2 protein. To investigate the chromosomal tethering function of E2, plasmid stability assays were developed in Saccharomyces cerevisiae to determine whether the E2 protein could maintain plasmids containing the yeast autonomous replication sequence replication element but with the centromeric element replaced by E2-binding sites. E2 expression was not sufficient to maintain such plasmids, but plasmid stability could be rescued by expression of the mammalian protein Brd4. In the presence of both Brd4 and E2 proteins, plasmids with multiple E2-binding sites were stable without selection. S. cerevisiae encodes a homolog of Brd4 named Bdf1 that does not contain the C-terminal domain that interacts with the E2 protein. A fusion protein of Bdf1 and the Brd4 C-terminal "tail" could support E2-mediated plasmid maintenance in yeast. Using a panel of mutated E2 proteins, we determined that plasmid stability required the ability of E2 to bind DNA and to interact with Brd4 and mammalian mitotic chromosomes but did not require its replication initiation and transactivation functions. The S. cerevisiae-based plasmid maintenance assays described here are invaluable tools for dissecting mechanisms of episomal viral genome replication and screening for additional host protein factors involved in plasmid maintenance.

We predicted gene function using synthetic lethal genetic interactions between null alleles in Saccharomyces cerevisiae. Phenotypic and protein interaction data indicate that synthetic lethal gene pairs function in parallel or compensating pathways. Congruent gene pairs, defined as sharing synthetic lethal partners, are in single pathway branches. We predicted benomyl sensitivity and nuclear migration defects using congruence; these phenotypes were uncorrelated with direct synthetic lethality. We also predicted YLL049W as a new member of the dynein-dynactin pathway and provided new supporting experimental evidence. We performed synthetic lethal screens of the parallel mitotic exit network (MEN) and Cdc14 early anaphase release pathways required for late cell cycle. Synthetic lethal interactions bridged genes in these pathways, and high congruence linked genes within each pathway. Synthetic lethal interactions between MEN and all components of the Sin3/Rpd3 histone deacetylase revealed a novel function for Sin3/Rpd3 in promoting mitotic exit in parallel to MEN. These in silico methods can predict phenotypes and gene functions and are applicable to genomic synthetic lethality screens in yeast and analogous RNA interference screens in metazoans.

A report on the Third Annual International Conference on Transposition and Animal Biotechnology, Minneapolis, USA, 23-24 June 2005, and the FASEB Summer Research Conference 'Mammalian Mobile Elements', Tuscon, USA, 4-9 June, 2005.

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.