Singer, Richard - Dalhousie University - Department of Biochemistry & Molecular Biology

研究领域

Chromatin and transcription FACT is an abundant and highly conserved general transcription factor for protein-coding genes. One of the functions of FACT is to facilitate transcription elongation on a chromatin template (DNA plus associated proteins), as found in the nucleus of eukaryotic cells (reviewed in Singer and Johnston, 2004). We initially identified the yeast version of FACT by purifying the Spt16/Cdc68 protein (it goes by both names) and its associated partner protein Pob3 (Brewster et al., 1998), having already characterized the gene for Spt16/Cdc68 through genetic and molecular approaches (Rowley et al., 1991). That study, and those of others, showed that FACT also has a negative effect on transcription, mediating repression at gene promoters caused by the structure of chromatin. Genetic investigations of FACT activity in vivo have shown that FACT can be subject to interference in ways related to protein turnover (Xu et al., 1993), and is regulated by protein folding/refolding, still poorly understood, that takes place during the transcription process (Xu et al., 1995). We have carried out structure/function analysis of yeast FACT (Evans et al., 1998; Brewster et al., 2001; O'Donnell et al., 2004), and are beginning to assign functions to the several domains of the FACT proteins (O'Donnell et al., 2009). Current research is focused on the several functional roles of FACT in transcription elongation (Stevens et al., 2011). Vesicular transport and the resumption of cell proliferation The Gcs1 protein (Ireland et al., 1994) has special significance (Drebot et al., 1987; Johnston and Singer, 2003) for the resumption of cell proliferation from the differentiated quiescent state (Werner-Washburne et al., 1993; Gray et al., 2004). Gcs1 mediates intracellular vesicular transport (Wang et al., 1996) for protein and membrane traffic, most likely as a GTPase-activating protein (GAP) for the Arf type of regulatory GTPases (Poon et al., 1996). Gcs1 is one of several structurally related yeast ArfGAPs, which seem to have overlapping functions. For example, Gcs1 and the Gcs1-like ArfGAP protein Glo3 (Ireland et al., 1994) can each facilitate transport from the Golgi to the endoplasmic reticulum (Poon et al., 1999; Lewis et al., 2004). Likewise, Gcs1 and a third GAP, Age2, can each mediate post-Golgi endosomal transport (Poon et al., 2001). Studies are ongoing to characterize the additional Gcs1-like proteins (Benjamin et al., 2011 JBC), understand the functional interactions of these GAPs (Yanagisawa et al., 2002; Wong et al., 2005; Robinson et al., 2006; Schindler et al., 2009), and learn how Gcs1 impinges so specifically on the resumption of cell proliferation. For this latter activity, Gcs1-mediated regulation of the Arf-related GTPase Arl1 has been found to be particularly important (Benjamin et al., 2011 MBC).

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Benjamin, J.J.R., Poon, P.P., Lewis, S.M., Auger, A., Wong, T.A., Singer, R.A. and Johnston, G.C., (2011) The yeast Arf GTPase-activating protein Age1 is regulated by phospholipase D for post-Golgi vesicular transport. J. Biol. Chem. 286:5187-5196 [PubMed] Benjamin, J.R.R., Poon, P.P., Drysdale, J.D., Wang, X., Singer, R.A. and Johnston, G.C., (2011) Dysregulated Arl1, a regulator of post-Golgi vesicle tethering, can inhibit endosomal transport and cell proliferation in yeast. Mol. Biol. Cell 22:2337-2347 [PubMed] Stevens, J.R., O'Donnell, A.F., Perry, T.F., Benjamin, J.J.R., Barnes, C.A., Johnston, G.C. and Singer, R.A., (2011) FACT, the Bur kinase pathway, and the histone co-repressor HirC have overlapping nucleosome-related roles in yeast transcription elongation. PLoS ONE 6:e25644 [PubMed] Lin, L.-J., Minard, L.V., Johnston, G.C., Singer, R.A. and Schultz, M.C., (2010) Asf1 can promote trimethylation of H3 K36 by Set2. Mol. Cell. Biol. 30: 1116-1129 [PubMed] Schindler, C., Rodriguez, F., Poon, P.P., Singer, R.A., Johnston, G.C. and Spang, A., (2009) The GAP domain and the SNARE, coatomer and cargo interaction region of the ArfGAP2/3 Glo3 are sufficient for Glo3 function. Traffic 10:1362-1375 [PubMed] O'Donnell, A.F., Stevens, J.R., Kepkay, R., Barnes, C.A., Johnston, G.C. and Singer, R.A., (2009) New mutant versions of yeast FACT subunit Spt16 affect cell integrity. Mol. Genet. Genomics 282:487-502 [PubMed] Robinson, M., Poon, P.P., Schindler, C., Murray, L.E., Kama, R., Gabriely, G., Singer, R.A., Spang, A., Johnston, G.C. and Gerst, J.E., (2006) The Gcs1 Arf-GAP mediates Snc1,2 v-SNARE retrieval to the Golgi in yeast. Mol. Biol. Cell 17:1845-1858 [PubMed] Wong, T., Fairn, G.D., Poon, P.P., Shmulevitz, M., McMaster, C.R., Singer, R.A. and Johnston, G.C., (2005) Membrane metabolism mediated by Sec14 family members influences Arf GTPase activating protein activity for transport from the trans-Golgi. Proc. Natl. Acad. Sci. USA 102:12777-12782 [PubMed] Singer, R.A. and Johnston, G.C., (2004) The FACT of chromatin modulator: genetic and structure/function relationships. Biochem. Cell Biol. 82:419-427 [PubMed] O'Donnell, A.F., Brewster, N.K., Kurniawan, J., Minard, L.V., Johnston, G.C. and Singer, R.A., (2004) Domain organization of the yeast histone chaperone FACT: the conserved N-terminal domain of FACT subunit Spt16 mediates recovery from replication stress. Nucl. Acids Res. 32:5894-5906 [PubMed] Gray, J.V., Petsko, G.A., Johnston, G.C., Ringe, D., Singer, R.A. and Werner-Washburne, M., (2004) 'Sleeping beauty' quiescence in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 68:187-206 [PubMed] Lewis, S.L., Poon, P.P., Singer, R.A., Johnston, G.C. and Spang, A., (2004) The ArfGAP Glo3 is required for the generation of COPI vesicles. Mol. Biol. Cell 15:4064-4072 [PubMed] Johnston, G.C. and Singer, R.A., (2003) Resumption of cell proliferation from stationary phase. Dormancy and Low-Growth States in Microbial Disease, A.R.M. Coates (ed.) Adv. Mol. Cell. Microbiol., vol. 3, Cambridge Univ. Press:223-233 Yanagisawa, L.L., Marchena, J., Xie, Z., Li, X., Poon, P.P., Singer, R.A., Johnston, G.C., Randazzo, P.A. and Bankaitis, V.A., (2002) Activity of specific lipid-regulated ADP ribosylation factor-GTPase activating proteins is required for Sec14p-dependent Golgi secretory function in yeast. Mol. Biol. Cell 13:2193-2206 [PubMed] Brewster, N.K., Johnston, G.C. and Singer, R.A., (2001) A bipartite yeast SSRP1 analog comprised of Pob3 and Nhp6 proteins modulates transcription. Mol. Cell. Biol. 21:3491-3502 [PubMed] Poon, P.P., Nothwehr, S.F., Singer, R.A. and Johnston, G.C., (2001) The Gcs1 and Age2 ArfGAP proteins provide essential overlapping function for transport from the yeast trans-Golgi network. J. Cell Biol. 155:1239-1250 [PubMed] Poon, P.P., Cassel, D., Huber, I., Singer, R.A. and Johnston, G.C., (2001) Expression, analysis, and properties of yeast ADP-ribosylation factor (ARF) GTPase activating proteins (GAPs) Gcs1 and Glo3. Methods Enzymol. 329:317-324 [PubMed] Poon, P.P., Cassel, D., Spang, A., Rotman, M., Pick, E., Singer, R.A. and Johnston, G.C., (1999) Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function. EMBO J. 18:555-564 [PubMed] Evans, D.R.H., Brewster, N.K., Xu, Q., Rowley, A., Altheim, B.A., Johnston, G.C. and Singer, R.A., (1998) The yeast protein complex containing Cdc68 and Pob3 mediates core-promoter repression through the Cdc68 N-terminal domain. Genetics 150:1393-1405 [PubMed] Brewster, N.K., Johnston, G.C. and Singer, R.A., (1998) Characterization of the CP complex, an abundant dimer of Cdc68 and Pob3 proteins that regulates yeast transcriptional activation and chromatin repression. J. Biol. Chem. 273:21972-21979 [PubMed] Murray, L.E., Rowley, N., Dawes, I.W., Johnston, G.C. and Singer, R.A., (1998) A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation. Proc. Natl. Acad. Sci. U.S.A. 95:8619-8624 [PubMed] Wang, X., Hoekstra, M.F., DeMaggio, A.J., Dhillon, N., Vancura, A., Kuret, J., Johnston, G.C. and Singer,R.A., (1996) Prenylated isoforms of yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage of cell proliferation from stationary phase. Mol. Cell. Biol. 16:5375-5385 [PubMed] Poon, P.P., Wang, X., Rotman, M., Huber, I., Cukierman, E., Cassel, D., Singer, R.A. and Johnston, G.C., (1996) Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein. Proc. Natl. Acad. Sci. USA 93:10074-10077 [PubMed] Xu, Q., Singer, R.A. and Johnston, G.C., (1995) Sug1 modulates yeast transcription activation by Cdc68. Mol. Cell. Biol. 15:6025-6035 [PubMed] Prendergast, J.A., Singer, R.A., Rowley, N., Rowley, A., Johnston, G.C., Danos, M., Kennedy, B. and Gaber, R.F., (1995) Mutations sensitizing yeast cells to the Start inhibitor nalidixic acid. Yeast 11:537-547 [PubMed]