Faculty Bibliography

Genetic experiments have identified two structurally similar nucleosomal domains, SIN and LRS, required for transcriptional repression at genes regulated by the SWI/SNF chromatin remodeling complex or for heterochromatic gene silencing, respectively. Each of these domains consists of histone H3 and H4 L1 and L2 loops that form a DNA-binding surface at either superhelical location (SHL) +/-2.5 (LRS) or SHL +/-0.5 (SIN). Here we show that alterations in the LRS domain do not result in Sin(-) phenotypes, nor does disruption of the SIN domain lead to loss of ribosomal DNA heterochromatic gene silencing (Lrs(-) phenotype). Furthermore, whereas disruption of the SIN domain eliminates intramolecular folding of nucleosomal arrays in vitro, alterations in the LRS domain have no effect on chromatin folding in vitro. In contrast to these dissimilarities, we find that the SIN and LRS domains are both required for recruitment of Sir2p and Sir4p to telomeric and silent mating type loci, suggesting that both surfaces can contribute to heterochromatin formation. Our study shows that structurally similar nucleosomal surfaces provide distinct functionalities in vivo and in vitro.

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.

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.

The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these modifications.

Saccharomyces cerevisiae knockout collection TAG microarrays are an emergent platform for rapid, genome-wide functional characterization of yeast genes. TAG arrays report abundance of unique oligonucleotide 'TAG' sequences incorporated into each deletion mutation of the yeast knockout collection, allowing measurement of relative strain representation across experimental conditions for all knockout mutants simultaneously. One application of TAG arrays is to perform genome-wide synthetic lethality screens, known as synthetic lethality analyzed by microarray (SLAM). We designed a fully defined spike-in pool to resemble typical SLAM experiments and performed TAG microarray hybridizations. We describe a method for analyzing two-color array data to efficiently measure the differential knockout strain representation across two experimental conditions, and use the spike-in pool to show that the sensitivity and specificity of this method exceed typical current approaches.

The L1 retrotransposon is the most highly successful autonomous retrotransposon in mammals. This prolific genome parasite may on occasion benefit its host through genome rearrangements or adjustments of host gene expression. In examining possible effects of L1 elements on host gene expression, we investigated whether a full-length L1 element inserted in the antisense orientation into an intron of a cellular gene may actually split the gene's transcript into two smaller transcripts: (1) a transcript containing the upstream exons and terminating in the major antisense polyadenylation site (MAPS) of the L1, and (2) a transcript derived from the L1 antisense promoter (ASP) that includes the downstream exons of the gene. Bioinformatic analysis and experimental follow-up provide evidence for this L1 "gene-breaking" hypothesis. We identified three human genes apparently "broken" by L1 elements, as well as 12 more candidate genes. Most of the inserted L1 elements in our 15 candidate genes predate the human/chimp divergence. If indeed split, the transcripts of these genes may in at least one case encode potentially interacting proteins, and in another case may encode novel proteins. Gene-breaking represents a new mechanism through which L1 elements remodel mammalian genomes.

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.

LINE-1 (L1) retrotransposons are replicating repetitive elements that, by mass, are the most-abundant sequences in the human genome. Over one-third of mammalian genomes are the result, directly or indirectly, of L1 retrotransposition. L1 encodes two proteins: ORF1, an RNA-binding protein, and ORF2, an endonuclease/reverse transcriptase. Both proteins are required for L1 mobilization. Apart from the obvious function of self-replication, it is not clear what other roles, if any, L1 plays within its host. The sheer magnitude of L1 sequences in our genome has fueled speculation that over evolutionary time L1 insertions may structurally modify endogenous genes and regulate gene expression. Here we provide a review of L1 replication and its potential functional consequences.

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.

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.