Faculty Bibliography

Transposable elements are DNA segments with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for genome manipulation. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field suggests that transposable elements will serve as indispensable tools. In particular, transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. In addition, transposons will gain importance in future cell-based clinical applications, including nonviral gene transfer into stem cells and the rapidly developing field of induced pluripotent stem cells. Here we provide an overview of transposon-based methods used in vertebrate model organisms with an emphasis on the mouse system and highlight the most important considerations concerning genetic applications of the transposon systems.

Histones are the basic protein components of nucleosomes. They are among the most conserved proteins and are subject to a plethora of post-translational modifications. Specific histone residues are important in establishing chromatin structure, regulating gene expression and silencing, and responding to DNA damage. Here we present HistoneHits, a database of phenotypes for systematic collections of histone mutants. This database combines assay results (phenotypes) with information about sequences, structures, post-translational modifications, and evolutionary conservation. The web interface presents the information through dynamic tables and figures. It calculates the availability of data for specific mutants and for nucleosome surfaces. The database currently includes 42 assays on 677 mutants multiply covering 405 of the 498 residues across yeast histones H3, H4, H2A, and H2B. We also provide an interface with an extensible controlled vocabulary for research groups to submit new data. Preliminary analyses confirm that mutations at highly conserved residues and modifiable residues are more likely to generate phenotypes. Buried residues and residues on the lateral surface tend to generate more phenotypes, while tail residues generate significantly fewer phenotypes than other residues. Yeast mutants are cross referenced with known human histone variants, identifying a position where a yeast mutant causes loss of ribosomal silencing and a human variant increases breast cancer susceptibility. All data sets are freely available for download.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys514 was crucial for enzymatic activity and the ability of yeast cells to grow on nonfermentable carbon sources. Furthermore, Sir2p deacetylated Pck1p both in vitro and in vivo. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production were dependent on TIP60, the human homolog of ESA1. Our findings demonstrate a regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme.

A major challenge in undergraduate life science curricula is the continual evaluation and development of courses that reflect the constantly shifting face of contemporary biological research. Synthetic biology offers an excellent framework within which students may participate in cutting-edge interdisciplinary research and is therefore an attractive addition to the undergraduate biology curriculum. This new discipline offers the promise of a deeper understanding of gene function, gene order, and chromosome structure through the de novo synthesis of genetic information, much as synthetic approaches informed organic chemistry. While considerable progress has been achieved in the synthesis of entire viral and prokaryotic genomes, fabrication of eukaryotic genomes requires synthesis on a scale that is orders of magnitude higher. These high-throughput but labor-intensive projects serve as an ideal way to introduce undergraduates to hands-on synthetic biology research. We are pursuing synthesis of Saccharomyces cerevisiae chromosomes in an undergraduate laboratory setting, the Build-a-Genome course, thereby exposing students to the engineering of biology on a genomewide scale while focusing on a limited region of the genome. A synthetic chromosome III sequence was designed, ordered from commercial suppliers in the form of oligonucleotides, and subsequently assembled by students into approximately 750-bp fragments. Once trained in assembly of such DNA "building blocks" by PCR, the students accomplish high-yield gene synthesis, becoming not only technically proficient but also constructively critical and capable of adapting their protocols as independent researchers. Regular "lab meeting" sessions help prepare them for future roles in laboratory science.

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.