Faculty Bibliography

In budding yeast, silent chromatin is defined at the region of telomeres, rDNA loci, and silent mating loci. Although the silent chromatin at different loci shows structural similarity, the underlying mechanism to establish, maintain, and inherit these structures may be fundamentally different. In this study, we found two arginine residues within histone H2B, which are specifically required to maintain either the telomeric or the rDNA silenct chromatin. Arginine 95 (R95) plays a specific role at telomeres, whereas arginine 102 (R102) is required to maintain the silent chromatin at rDNA and to ensure the integrity of rDNA loci by suppressing recombination between rDNA repeats. R95 mutants show enhanced rDNA silencing but a paradoxically low Sir2 protein abundance. Furthermore weakened silencing at telomeres in R95 mutants can be suppressed by a specific SIR3 allele, SIR3-D205N, which increases the affinity of Sir proteins to telomeres, suggesting H2B-R95 may directly mediate telomeric Sir protein-nucleosome interactions. Double mutations of R95 and R102 lead to desilencing of both rDNA and telomeres, indicating both arginines are necessary to ensure integrity of silent chromatin at these loci. Furthermore, mutations of R102 cause accumulation of extrachromosomal rDNA circles and reduce life span, suggesting that histone H2B contributes to longevity.

Characterizing structural variants in the human genome is of great importance, but a genome wide analysis to detect interspersed repeats has not been done. Thus, the degree to which mobile DNAs contribute to genetic diversity, heritable disease, and oncogenesis remains speculative. We perform transposon insertion profiling by microarray (TIP-chip) to map human L1(Ta) retrotransposons (LINE-1 s) genome-wide. This identified numerous novel human L1(Ta) insertional polymorphisms with highly variant allelic frequencies. We also explored TIP-chip's usefulness to identify candidate alleles associated with different phenotypes in clinical cohorts. Our data suggest that the occurrence of new insertions is twice as high as previously estimated, and that these repeats are under-recognized as sources of human genomic and phenotypic diversity. We have just begun to probe the universe of human L1(Ta) polymorphisms, and as TIP-chip is applied to other insertions such as Alu SINEs, it will expand the catalog of genomic variants even further.

The study of temperature-sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter, we describe two "shuffling" methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the "plasmid" version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the "chromosomal" version, The Ts alleles are integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have their strengths and weaknesses, which are discussed here.

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.