Faculty Bibliography

The yeast synthetic lethal genetic interaction network contains rich information about underlying pathways and protein complexes as well as new genetic interactions yet to be discovered. We have developed a graph diffusion kernel as a unified framework for inferring complex/pathway membership analogous to "friends" and genetic interactions analogous to "enemies" from the genetic interaction network. When applied to the Saccharomyces cerevisiae synthetic lethal genetic interaction network, we can achieve a precision around 50% with 20% to 50% recall in the genome-wide prediction of new genetic interactions, supported by experimental validation. The kernels show significant improvement over previous best methods for predicting genetic interactions and protein co-complex membership from genetic interaction data.

The previously identified LRS (Loss of rDNA Silencing) domain of the nucleosome is critically important for silencing at both ribosomal DNA and telomeres. To understand the function of the LRS surface in silencing, we performed an EMS mutagenesis screen to identify suppressors of the H3 A75V LRS allele. We identified dominant and recessive mutations in histones H3, H4, and dominant mutations in the BAH (Bromo Adjacent Homology) domain of SIR3. We further characterized a surface of Sir3p critical for silencing via the LRS surface. We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles. Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4. Mutations in H3 and H4 genes that lose silencing tend to make the LRS surface more electronegative, whereas mutations that increase silencing make it less electronegative. Conversely, suppressors of LRS alleles in either SIR3, histone H3, or H4 also tend to make their respective surfaces less electronegative. Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain. Based on these findings, we propose an electrostatic model for how an extensive surface on the Sir3p BAH domain may regulate docking onto the LRS surface.

Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants that probes the contribution of each residue to nucleosome function was generated in Saccharomyces cerevisiae. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.

Integrated HIV-1 genomes are found within actively transcribed host genes in latently infected CD4(+) T cells. Readthrough transcription of the host gene might therefore suppress HIV-1 gene expression and promote the latent infection that allows viral persistence in patients on therapy. To address the effect of host gene readthrough, we used homologous recombination to insert HIV-1 genomes in either orientation into an identical position within an intron of an actively transcribed host gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT). Constructs were engineered to permit or block readthrough transcription of HPRT. Readthrough transcription inhibited HIV-1 gene expression for convergently orientated provirus but enhanced HIV-1 gene expression when HIV-1 was in the same orientation as the host gene. Orientation had a >10-fold effect on HIV-1 gene expression. Due to the nature of HIV-1 integration sites in vivo, this orientation-dependent regulation can influence the vast majority of infected cells and adds complexity to the maintenance of latency.

Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the histone variant Htz1p at Lys 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic interactions may help illuminate the mechanisms of intricate cellular processes.

In this issue of Developmental Cell, Soper et al. (2008) report that the mammalian MAELSTROM (MAEL) protein is critical for transposon silencing in the male germline. Loss of MAEL is associated with meiotic failure and DNA damage, suggesting that efficient transposable element restraining mechanisms must be in place for the preservation of germline integrity.

Deacetylation of histone H3 K56, regulated by the sirtuins Hst3p and Hst4p, is critical for maintenance of genomic stability. However, the physiological consequences of a lack of H3 K56 deacetylation are poorly understood. Here we show that cells lacking Hst3p and Hst4p, in which H3 K56 is constitutively hyperacetylated, exhibit hallmarks of spontaneous DNA damage, such as activation of the checkpoint kinase Rad53p and upregulation of DNA-damage inducible genes. Consistently, hst3 hst4 cells display synthetic lethality interactions with mutations that cripple genes involved in DNA replication and DNA double-strand break (DSB) repair. In most cases, synthetic lethality depends upon hyperacetylation of H3 K56 because it can be suppressed by mutation of K56 to arginine, which mimics the nonacetylated state. We also show that hst3 hst4 phenotypes can be suppressed by overexpression of the PCNA clamp loader large subunit, Rfc1p, and by inactivation of the alternative clamp loaders CTF18, RAD24, and ELG1. Loss of CTF4, encoding a replisome component involved in sister chromatid cohesion, also suppresses hst3 hst4 phenotypes. Genetic analysis suggests that CTF4 is a part of the K56 acetylation pathway that converges on and modulates replisome function. This pathway represents an important mechanism for maintenance of genomic stability and depends upon proper regulation of H3 K56 acetylation by Hst3p and Hst4p. Our data also suggest the existence of a precarious balance between Rfc1p and the other RFC complexes and that the nonreplicative forms of RFC are strongly deleterious to cells that have genomewide and constitutive H3 K56 hyperacetylation.

The efficacy of microarrays in examining gene expression, gene and genome structure, protein-DNA interactions, whole-genome similarities and differences, microRNA expression, methylation (and more) is no longer in question. It is a fast-developing, cutting edge technology that has grown up along with massive sequence databases and is likely to become part of everyday patient care. Many advances have recently expanded the power and utility of microarrays; among them is our development of a new array tiling technique that dramatically increases the scope of coverage of an oligonucleotide tiling array without substantially increasing its cost.