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James Broach

Gene expression in the yeast Saccharomyces cerevisiae

The research in my laboratory is directed toward understanding cellular regulation at the molecular level, using the yeast Saccharomyces cerevisiae as a model system. Specifically, we are investigating (l) signal transduction regulating initiation of cell growth, and (2) the mechanism underlying the control of cell type in yeast. We approach these problems using a combination of yeast genetics, biochemical analysis, cell biology and genomic techniques.

Yeast oncogene homologs

Two proteins in yeast, structurally and functionally homologous to the ras family of mammalian oncoproteins, mediate initiation of the cell cycle in response to nutrient availability and regulate the developmental switch between yeast and pseudohyphal morphology. The ras pathway acts in parallel with a second pathway mediated by protein kinases, called Tor, that are targets of the anti-fungal and immunosuppressive drug, rapamycin. We have examined how the balanced activity of these two pathways regulates yeast cell growth and development, with the expectation that such studies will shed light on the mechanism by which imbalance of signaling through these pathways causes tumorigenic transformation in larger cells.

This expectation is fortified by our prior observations that the human ras protein can functionally substitute for the cognate yeast protein and that the Tor pathway is composed of the same components in yeast as it is in humans. This indicates a conservation of function of signaling proteins and their regulation in all eukaryotic cells.

Through a combination of classical and molecular genetics, we have developed a detailed description of the Ras and Tor regulatory pathways in yeast (see Figure). Besides identifying additional members of these regulatory pathways and dissecting the interactions between the members of these pathways, our current efforts are directed towards developing global transcriptional approaches to define exhaustively the circuitry comprising these regulatory pathways. This novel application of DNA microarray technology should yield knowledge of the biological signals impinging on the pathways and the biological processes specifically regulated by the pathways. In addition, this approach should be directly applicable to dissection of signaling pathways in larger eukaryotes.

Control of cell type in yeast

Genes that determine the mating type of the cell are actively expressed at one site in the genome, but the same genes located at another site are completely inactive. This difference in expression is apparently determined by the chromatin structures of the different loci. We have recently determined that specific modifications of histones packaging the different genes are critical for differential expression, and we are currently examining the mechanism by which these modifications affect expression. This regulatory process is strikingly similar to chromosomal imprinting and X-chromosome inactivation observed in mammalian cells. Our analysis should yield clues as to the mechanistic underpinnings of these more complex systems.

In normal yeast cells, the inactive mating-type genes can be activated only by their physical transposition to an active region of the genome. This process is precisely regulated and involves specific interactions of sites on a chromosome located ~300,000 bp apart, controlled by an unprecedented mechanism of chromosome activation. We are further dissecting this process by molecular genetic techniques to gain an understanding of how very distant regions of the genome can associate in a highly ordered and programmed fashion.

Publications

Zawadzki KA, Morozov AV, Broach JR. (2009) Chromatin-dependent transcription factor accessibility rather than nucleosome remodeling predominates during global transcriptional restructuring in Saccharomyces cerevisiae. Mol Biol Cell. [Epub ahead of print]

Freckleton G, Lippman SI, Broach JR, Tavazoie S. (2009) Microarray profiling of phage-display selections for rapid mapping of transcription factor-DNA interactions. PLoS Genet. 5: e1000449. PubMed

Zaman S, Lippman SI, Schneper L, Slonim N, Broach JR. (2009) Glucose regulates transcription in yeast through a network of signaling pathways. Mol Syst Biol. 5: 245. PubMed

Airoldi EM, Huttenhower C, Gresham D, Lu C, Caudy AA, Dunham MJ, Broach JR, Botstein D, Troyanskaya OG. (2009) Predicting cellular growth from gene expression signatures. PLoS Comput Biol. 5: e1000257. PubMed

Zaman S, Lippman SI, Zhao X, Broach JR. (2008) How Saccharomyces responds to nutrients. Annu Rev Genet 42: 27-81. PubMed.

Tan MP, Broach JR, Floudas CA. (2007) Evaluation of normalization and pre-clustering issues in a novel clustering approach: global optimum search with enhanced positioning. J Bioinform Comput Biol 5: 895-913. PubMed

McClean NM, Broach JR, Ramanathan S. (2007) Cross-talk and decision making in MAPK pathways. Nat Genet 39: 409-414. PubMed

Urban J, Soulard A, Huber A, Lippman S, Mukhopadhyay D, Deloche O, Wanke V, Anrather D, Ammerer G, Riezman H, Broach JR, De Virgilio C, Hall MN, Loewith R. (2007) Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol Cell 26: 663-674. PubMed

Tan MP, Broach JR, Floudas CA (2007) A novel clusting approach and prediction of optimum number of clusters: Global optimization search with enhanced positioning. J Global Optim 39: 323-346.

Ramanathan S, Broach JR. (2007) Do cells think? Cell Mol Life Sci 64: 1801-1804. PubMed

Yorimitsu T, Zaman S, Broach JR, Klionsky DJ. (2007) Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae. Mol Biol Cell 18: 4180-4189. PubMed

Tan MP, Broach JR, Floudas CA. (2007) Evaluation of normalization and pre-clustering issues in a novel clustering approach: global optimum search with enhanced positioning. J Bioinform Comput Biol.5: 895-913. PubMed

Xu EY, Zawadzki KA, Broach JR. (2006) Single cell observations reveal intermediate transcriptional silencing states. Mol Cell 23: 219-229. PubMed

Houston PL, Broach JR. (2006) The dynamics of homologous pairing during mating type interconversion in budding yeast. PLoS Genet 2: e98.  PubMed

Ault AD, Broach JR. (2006) Creation of GPCR-based chemical sensors by directed evolution in yeast. Protein Eng Des Sel 19: 1-8.  PubMed

Broach JR. (2006) Cell Growth. In: Landmark Papers in Yeast Biology. Cold Spring Harbor Lab Press, Cold Spring Harbor, NY, pp. 127-140.

Ault AD, Broach JR. (2005) Creation of GPCR based chemical sensors by directed evolution in yeast. Protein Eng Des Sel 19: 1-8. PubMed

Niida A, Wang Z, Tomita K, Oishi S, Tamamura H, Otaka A, Navenot JM, Broach JR, Peiper SC, Fujii N. (2005) Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity. Bioorg Med Chem Lett 16: 134-137.  PubMed

Xu E, Bi X, Holland M, Gottschling DE, Broach JR. (2005) Mutations in the nucleosome core enhance transcriptional silencing. Mol Cell Biol 25: 1846-1859.  PubMed

Broach JR. (2004) Making the right choice--long-range chromosomal interactions in development. Cell 119: 583-589.  PubMed

Schneper L, Düvel K, Broach JR. (2004) Sense and sensibility: Nutritional response and signal integration in yeast. Curr Opin Microbiol 7: 624-630.  PubMed

Jorgensen P, Rupes I, Sharom JR, Schneper L, Broach JR, Tyers M. (2004) A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes Dev 18: 2491-2505.  PubMed

Santhanam A, Hartley A, Düvel K, Broach JR, Garrett S. (2004) PP2A phosphatase is required for stress and TOR kinase regulation of yeast stress response factor Msn2. Euk Cell 3: 1261-1271.  PubMed

Düvel K, Broach JR. (2004) The role of phosphatases in TOR signaling in yeast. Curr Top Microbiol Immunol 279: 19-38.  PubMed

Houston P, Simon P, and Broach JR (2004). The Saccharomyces cerevisiae recombination enhancer biases recombination during inter-chromosomal mating type switching but not in inter-chromosomal homologous recombination. Genetics 166: 1187-1197.  PubMed

Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K, Ito D, Peiper SC, Broach JR, Oishi S, Niida A, Fujii N, Imamura M (2004). Metastin and its variant forms suppress migration of pancreatic cancer cells. Biochem Biophys Res Commun 315: 85-92.  PubMed

Zhang WB, Wang ZX, Murray JL, Fujii N, Broach J, Peiper SC (2004). Functional expression of CXCR4 in S. cerevisiae: development of tools for mechanistic and pharmacologic studies. Ernst Schering Res Found Workshop 45: 125-152.  PubMed

Schneper L, Krauss A, Miyamoto R, Fang S and Broach JR (2004). The Ras/Protein kinase A pathway acts in parallel with the Mob2/Cbk1 pathway to effect cell growth and proper bipolar bud site selection. Euk Cell 3: 108-120.  PubMed

Wang Y, Pierce M, Schneper L, Guldul C, Zhang X, Tavazoie S, and Broach JR (2004). Ras and Gpa2 mediate the primary branch of a redundant glucose signal pathway in yeast. PLOS 2: 610-622.  PubMed

Fujii N, Oishi S, Hiramatsu K, Araki T, Ueda S, Tamamura H, Otaka A, Kusano S, Terakubo S, Nakashima H, Broach JR, Trent JO, Wang ZX, Peiper SC (2003). Molecular-size reduction of a potent CXCR4-chemokine antagonist using orthogonal combination of conformation- and sequence-based libraries. Angew Chem Int Ed Engl 42: 3251-3253.  PubMed

Lin X, Floudas C, Wang Y and Broach JR (2003). Theoretical and computational studies of glucose signaling pathways in yeast using global gene expression data. Biotechnol Bioeng 84: 864-886.  PubMed

Arias DA, Navenot JD, Zhang W, Broach JR and Peiper SC (2003). Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved T-X-P motif in transmembrane helix 2. J Biol Chem 278: 36513-36521.  PubMed 

Düvel K, Santhanam A, Garrett S and Broach JR (2003). Multiple roles of Tap42 in mediating rapamycin-induced transcriptional changes in yeast. Mol Cell 11: 1467-1489. PubMed

Yu Q, Qiu R, Foland TB, Griesen D, Galloway CS, Chiu Y, Broach JR and Bi X (2003). Rap1p and other transcriptional regulators can function in defining distinct domains of gene expression. Nucleic Acids Res 31: 1224-1233.  PubMed

Simon P, Houston P and Broach JR (2002). Directional bias during mating type switching in Saccharomyces is independent of chromosomal architecture. EMBO J 21: 2282-2291.  PubMed

Zhang WB, Navenot JM, Haribabu B, Tamamura H, Hiramatu K, Omagari A, Pei G, Manfredi JP, Fujii N, Broach JR, Peiper SC (2002). A point mutation that confers constitutive activity to chemokine receptor CXCR4 reveals T140 is an inverse agonist and AMD3100 and ALX40-4C are weak partial agonists. J Biol Chem 277: 24515-24521.  PubMed

Johnston SD, Enomoto S, Schneper L, McClellan MC, Twu F, Montgomery N, Haney S, Broach JR and Berman J (2001). Suppression of the RAS/cAMP signal transduction pathway by CAC3/MS11 is independent of Chromatin Assembly Factor-I and is mediated by NPR1. Mol Cell Biol 21: 1784-1794. PubMed

Nielsen KH, Gredsted L, Broach JR, and Willumsen BM (2001). Sensitivity of wild type and mutant Ras alleles to ras specific exchange factors: Identification of factor specific requirements. Oncogene 20: 2091-2100. PubMed

Haney S, Xu J, Lee S-Y, Broach JR, and Manfredi JP (2001). Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities. Mol Gen Genet 265: 1120-1128.  PubMed