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Cells have evolved strategies that allow appropriate responses to environmental changes. Important among these is gene expression responses via transcription switches, or gene switches. Many disease states are known to be due to defective gene switches. Thus, we can learn much about how normal cells work and the basis of many diseases if we understand the mechanistic principles of gene switch operation.
A gene switch that is under investigation in my laboratory is the genetically well-characterized multi-component gene switch that regulates 10 genes (GAL genes) that specify galactose utilization functions in the yeast, S. cerevisiae. At the heart of this so-called GAL-gene switch are the Gal4, Gal80 and Gal3 proteins that constitute a molecular mechanism for galactose-responsive transcriptional activation (galactose induction) of the GAL genes. The generally accepted broad schematic of the GAL gene switch is as follows. All GAL gene promoters contain a 17-base pair UASgAL sequence that is recognized specifically by the Gal4 protein, a typical eukaryotic DNA-binding transcriptional activator. Gal4 binds UASgAL elements in both the presence and the absence of galactose. In the absence of galactose Gal4 is unable to activate transcription because its transcriptional activation domain (Gal4AD) is inhibited through an interaction with the Gal80 protein. In response to galactose, the Gal3 protein binds to the Gal80 protein, relieving the Gal80-inhibition of transcription activation by Gal4AD. This general view of how the GAL gene switch works leaves much unknown concerning the mechanisms of action of the Gal4, Gal80 and Gal3 proteins.
Our goal is to understand how Gal4 functions to recruit RNA Polymerase II and mechanistically how such activities are regulated. As indicated above, a key feature in the overall regulation of Gal4’s capacity to recruit RNA Pol II is Gal80. Over the past few years our experiments have led us to propose a novel view of how the Gal80 inhibition of Gal4 is regulated. In our newly proposed Gal80 Shuttle model we incorporate discoveries made by other workers in the field as well as those made by us. The key observations that our Gal80 Shuttle model takes into consideration are as follows: 1) Gal3 resides exclusively in the cytoplasm of the cell; 2) Gal80 shuttles rapidly between the cytoplasmic and nuclear compartments of the cell; 3) Gal80 exists as a monomer, dimer and perhaps a tetramer; 4) a Gal80 dimer is the unit of Gal80 that binds to and inhibits a dimer of Gal4 that is located at the UASgAL sites within the nucleus; 5) a Gal80 monomer is the unit of Gal80 that binds to Gal3; 6) Gal80 binds to Gal3 exclusively in the cytoplasm and in the presence of galactose; and 7) in response to galactose the binding of Gal80 to UASgAL-associated Gal4 decreases. We have integrated these observations to constitute a model for the GAL gene switch wherein two distinct mechanistic elements work in concert to cause the activation of Gal4 in response to galactose. One is the binding of Gal80 to Gal3 exclusively in the cytoplasm in response to galactose. This will effect a mass redistribution of Gal80 causing the steady state concentration of Gal80 to increase in the cytoplasm and decrease in the nucleus. The second is the binding of Gal3 to exclusively a monomer of Gal80. This will effect a reduction in total cellular concentration of the dimer form of Gal80, including its nuclear pool, providing that the Gal80 nuclear-cytoplasmic exhange is rapid. Both mechanistic elements will decrease the probability that a Gal80 dimer is complexed with Gal4 within the nucleus and increase the probability of Gal4 interactions that elicit recruitment of RNA polymerase to the GAL gene promoters in response to galactose.
The Gal80 Shuttle model provides a framework for our current efforts that aim to rigorously test many facets of the model employing experiments that are different from those that led to the model. The Gal80 Shuttle model is illustrated below.
The Gal80 Shuttle model:
Diep CQ, Peng G, Bewley M, Pilauri V, Ropson I, Hopper JE. (2006) Intragenic suppression of Gal3C interaction with Gal80 in the Saccharomyces cerevisiae GAL gene switch. Genetics. 172(1), 77-87.
Adams CA, Kar SR, Hopper JE, Fried MG. (2004) Self-association of the amino-terminal domain of the yeast TATA-binding protein. J Biol Chem. 279(2), 1376-82.
Peng G, Hopper JE. (2002) Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein. Proc Natl Acad Sci U S A. 99(13), 8548-53.
Sandt CH, Hopper JE, Hill CW. (2002) Activation of prophage eib genes for immunoglobulin-binding proteins by genes from the IbrAB genetic island of Escherichia coli ECOR-9. J Bacteriol. 2002 Jul;184(13), 3640-8.
Carrozza MJ, John S, Sil AK, Hopper JE, Workman JL. (2002) Gal80 confers specificity on HAT complex interactions with activators. J Biol Chem. 277(27), 24648-52.
Gribenko AV, Hopper JE, Makhatadze GI. (2001) Molecular characterization and tissue distribution of a novel member of the S100 family of EF-hand proteins. Biochemistry. 240(51), 15538-48.
Peng G, Hopper JE. (2000) Evidence for Gal3p's cytoplasmic location and Gal80p's dual cytoplasmic-nuclear location implicates new mechanisms for controlling Gal4p activity in Saccharomyces cerevisiae. Mol Cell Biol. 20(14), 5140-8.
Sil AK, Xin P, Hopper JE. (2000) Vetors allowing amplified expression of the Saccharomyces cerevisiae Gal3p-Gal80p-Gal4p transcription switch: Applications to galactose-regulated high-level production of proteins. Protein Expr Purif. 18(2), 202-12.