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Transcriptional Regulation in Yeast
The main focus of our
research is on the regulation of transcription in the yeast Saccharomyces cerevisiae. Specifically, we are investigating how different regulatory proteins
interact to control gene expression and how these interactions influence the
regulatory function of the proteins. We have chosen three systems to study this
problem: 1) examining the protein-protein and protein-DNA interactions of the
yeast Mata2 protein, a cell-type specific repressor involved in mating-ype
regulation, 2) examining the molecular interactions of the Mcm1 protein, a
MADS-Box protein that is involved in the transcriptional regulation of cell
type, cell-cycle and metabolic pathways, and 3) investigating the factors
involved in the regulation of transcription at the middle stages of meiosis. Protein-Protein and
Protein-DNA Interactions of the Yeast a2 Repressor In comparison to higher
eukaryotes, most promoters in yeast are relatively small and usually contain
only a few regulatory sites. However the yeast HO promoter is almost 2000 bp and contains more than
20 different regulatory sites, making it a good model system to study complex
regulatory interactions. The yeast a2 and a1 proteins, members of the homeodomain family of
DNA-binding proteins, bind in combination to specific DNA sites to repress
haploid-specific genes in the diploid yeast cell type. There are ten different a1/a2 binding sites in the HO promoter and we have shown that these sites have
very different affinities for the a1/a2 complex in vitro and
in vivo. Interestingly, we have
shown that sites with low affinity play an important role in regulation. We
have also shown that repression by the a1/a2 complex does not block DNA-binding by the some of the activator
proteins that are required for activation of HO expression. This result suggests that the a1/a2 complex does not repress transcription by
sterically blocking access to the promoter. Protein-Protein and
Protein-DNA Interactions of the Yeast Mcm1 MADS-Box Protein Mcm1 is an essential
protein in yeast that, in complex with its cofactors, is involved in the
regulation of cell-type specific and cell-cycle genes. To determine which
residues in Mcm1 are required for DNA binding, bending, protein interactions
and transcriptional regulation we have substituted residues of the MADS-Box
domain with alanine residues and examined the effects of these mutations in
vivo and in vitro. We have used these mutants to determine how Mcm1
interacts with the Mata1 protein to activate a-specific genes. We are now also
investigating how a1 binds DNA and which region of the protein interacts with
Mcm1. In addition, we are investigating how Mcm1 interacts with the ArgR
complex of proteins to regulate arginine metabolic and anabolic genes. We have
shown that Mcm1 directly interacts with Arg80, another MADS-box protein, and
are currently investigating whether these proteins form heterodimers or
tetramers to regulate transcription. Many
of the substitutions that we have constructed in Mcm1 are lethal. Some of these
substitutions are at residues which are on the surface of the protein and may
define positions which interact with the co-factors that are required for
Mcm1-mediated transcriptional regulation. The Mcm1 protein interacts with the
SFF factor to activate the expression of genes such as CLB2 and SWI5 in the G2/M phase of the cell cycle. We have shown that many of the
mutations in Mcm1 significantly decrease the expression of CLB2 and SWI5, suggesting they are defective in complex with SFF for activating genes
at the G2/M stage of the cell cycle. Mcm1 is also involved in the activation of
genes expressed at the M/G1 phase of the cell cycle. Many of these same
mutations also decrease activation of genes expressed at the M/G1 stage of the
cell cycle. However, we have also found that some mutations increase the level
of expression of these M/G1 genes, suggesting that Mcm1 may be working as both
an activator and a repressor at these promoters. Transcriptional
Regulation of Meiosis-Specific Promoters in Yeast. Meiosis and sporulation in Saccharomyces
cerevisiae are characterized by the
specific and sequential expression of large numbers of genes. We are interested
in identifying the factors that control the temporal regulation of these genes
in the meiotic pathway and determining how they function. The MSE is a
regulatory element found in the promoters of many mid-sporulation genes that
sets the timing of their expression during meiosis. The Ndt80 protein binds to
MSEs during middle sporulation to activate gene expression. We have determined
that the Sum1, Rfm1 and Hst1 proteins are required to repress transcription
during both vegetative growth and early meiosis. We have shown that Sum1 binds
MSEs and interacts with Rfm1, which in turn recruits Hst1 to the promoters of
middle-sporulation genes. We have shown that Hst1, a protein with sequence
similarity to the Sir2 histone deacetylase, is required for deacetylation of a
subset of promoters that are regulated by Sum1 and that the state of
deacetylation correlates with repression. This result suggests that other
deacetylases may be working in combination with Sum1. We are currently in the
process of identifying these other cofactors. We have also performed genomic
DNA microarray analysis in sum1, rfm1 and hst1 mutant strains to
identify which genes are specifically regulated by these proteins. We have mapped the DNA-binding domains of Ndt80 and Sum1 and determined that neither protein shares homology with any other known DNA-binding motif. In collaboration with Millie Georgiadis's lab we have determined by X-ray crystallography that the DNA-binding domain of Ndt80 contains a novel structural motif. We have modeled how Ndt80 binds DNA and tested this model through mutational analysis of the protein. We have identified residues along one surface of the protein which are required for DNA-binding activity in vitro and transcriptional activation of sporulation genes in vivo. Interestingly, residues in Ndt80 that are modeled to contact DNA share significant sequence similarity with proteins from higher eukaryotes, including a human protein that has been shown to be associated with cancer. These proteins may therefore be members of a novel family of transcription factors. Publications Moore, M., Shin, M., Schindler, K., Bruning, A., Vershon, A.K. and Winter E. (2006)
Arg-Pro-X-Ser/Thr-Ala is a consensus phosphoacceptor sequence for the meiosis-specific
Ime2 protein kinase in Saccharomyces cerevisiae. Carr, E.A., Charney, J., Sofer, W., and Vershon, A.K. (2006) Abraham, D. and Vershon, A.K. (2005) The N-terminal arm of Mcm1
affects the transcription of a subset of genes associated with the cell
wall. Mathias, J.R. Hanlon, S.E., Flanagan, R.O., Sengupta, A. M. and
Vershon, A.K. (2004) Repression of the yeast HO gene by the MATa2 and MATa1
homeodomain proteins. Nucleic Acid Res. 32, 6469-6478. Nagaraj, V.H., Flanagan, R.O., Bruning, A.R., Mathias, J.R. Vershon, A. K., and Sengupta, A. M. (2004)
Identification of a1-a2 binding sites inthe yeast genome. BMC Genomics 5, 59 Hanlon, S.E., Xu, Z., Norris, D.N., and Vershon, A. K. (2004)
Analysis of the Meiotic Role of the Mitochondrial Ribosomal Proteins Mrps17
and Mrpl37. Fingerman, I., Sutphen, K., Montano, S. P., Georgiadis, M.M., and Vershon, A. K. (2004)
The yeast Ndt80 protein binds DNA through a series of flexible loops.
Carr, E. A., Mead, J. and Vershon, A. K. (2004)
a1-induced DNA bending is required for transcriptional activation
by the Mcm1-a1 complex. Hanlon, S.E. Norris, D.N. and Vershon, A. K. (2003)
Depletion of H2A-H2B dimers in Saccharomyces cerevisiae triggers
meiotic arrest by reducing IME1 expression and activating the BUB2-dependent
branch of the spindle checkpoint. Genetics (in press) Pierce, M., Benjamin, K.R., Montano, S. P., Georgiadis, M.M., Winter, E., and Vershon, A. K. (2003)
Sum1 and Ndt80 Proteins Compete for Binding to MSE Sequences that Control Meiotic
Gene Expression. Mol. Cell. Biol. 23, 4814-4825. McCord, R, Pierce, M., Xie, J., Wonkatal, S., Mickel, C. and Vershon, A. K. (2003)
Rfm1, a novel factor required to recruit the Hst1 histone deacetylase
for repression of middle sporulation genes. Mol. Cell. Biol. 23, 2009-2016. S. Wontakal, M. Pierce, and A.K. Vershon. (2002)
Identification of Factors Involved in MSE-Mediated Repression of SMK1.
The Rutgers ScholarVol 4 pp. 1-7
http://scils.rutgers.edu/~weyang/ejournal/volume04/wontvers/wontvers.htm
Montano, S.P., Cote, M., Fingerman, I., Pierce, M.,
Vershon, A. K. Georgiadis, M. M. (2002) The crystal
structure of a novel DNA-binding domain from Ndt80,
a transcriptional activator required for meiosis in yeast.
Proc. Natl. Acad. Sci. 99, 14041-14046. Montano, S.P., Cote, M., Pierce, M., Vershon, A. K.
Georgiadis, M. M. (2002) Crystallographic studies of a
novel DNA-binding domain from the yeast transcriptional
activator Ndt80. Acta. Crys. D. 58, 2127-2130. Jamail, A., Dubois, E, Vershon, A.K.,
and Messenguy, F. (2002) Swapping functional
pecificity of a MADS-box protein: Residues required
for Arg80 regulation of arginine metabolism.
Mol. Cell. Biol. 22, 5741-5752. Mead, J., Bruning, A., Gill, M.K., Steiner, A.M., Acton, T.B.
and Vershon, A.K. (2002) Interactions of the Mcm1 MADS-box Protein
with Cofactors that Regulate Mating in Yeast. Mol. Cell. Biol. 22,
4607-4621. Ke, A., Mathias, J. R., Vershon, A. K. and C. Wolberger (2002).
Structural and Thermodynamic Characterization of the DNA
Binding Properties of a Triple Alanine Mutant of MATa2
Structure 10, 961-971. Hart, B., Mathias, J., Ott, D. McNaughton, L., Anderson, J.,
Vershon, A.K., and Baxter, S. M. (2002).
Engineered improvements in DNA-binding function
of the MATa1 homeodomain reveal structural changes
involved in combinatorial control J. Mol. Biol. 316, 247-256. Mathias, J.R., Zhong, H., Jin, Y. and Vershon, A.K (2001)
Altering the DNA-binding Specificity of the Yeast Mata2
Homeodomain Protein. J. Biol. Chem. 276, 32696-703. Lindgren, A., Bungard, D., Pierce, M., Xie, J,
Vershon, A.K,. and Winter, E. (2000) The pachytene
checkpoint in Saccharomyces cerevisiae requires the
Sum1 transcriptional repressor. EMBO J 19, 6489-97. Kim, J., Bortz, E. Zhong, H., Leeuw T., Leberer, E,
Vershon, A.K. and Hirsch, J.P. (2000) Localization and
signaling of GB subunit Ste4p are controlled by the a-factor
receptor and the novel a-specific protein Asg7p Mol. Cell.
Biol. 20, 8826-8835. Vershon, A.K. and Pierce, M. (2000)
Transcriptional Regulation of Meiosis in Yeast.
Curr. Op. Cell Biol. 12, 334-339. Acton, T.B., Mead, J., Steiner, A.M., and Vershon, A.K.
(2000) Scanning mutagenesis of Mcm1: Residues required for
DNA binding, bending and transcriptional activation by a
MADS box. Mol. Cell. Biol. 20, 1-11. Li, T., Jin, Y., Vershon, A.K., and Wolberger, C. (1999) Crystal structure of the MATa1/MATa2 homeodomain heterodimer in complex with DNA containing an A-tract. Nucleic Acids Res 26, 5707-5718. Jin, Y., Zhong, H. and Vershon, A.K. (1999) Operator mutagenesis and altered DNA sequence specificity of the yeast a1/a2 homeodomain complex. Mol. Cell. Biol. 19, 585-593. Zhong, H., McCord, R., and Vershon, A.K.. (1999) A search of the yeast genome for all potential target sites of the a2/Mcm1 repressor complex. Genome Research 9, 1040-1047. Xie, J., Pierce, M., Gailus-Durner, V., Wagner, M.,
Winter, E., Vershon, A.K. (1999) Sum1 and Hst1 repress
middle sporulation specific gene expression during mitosis
in Saccharomyces EMBO J. 18, 6448-6454. Justice M.C., Hogan B.P., Vershon A.K. (1997).
Homeodomain-DNA interactions of the Pho2 protein are
promoter-dependent. Nucleic
Acids Res. 25: 4730-4739. Lab Support Janet Mead Parent, Research Associate Adrian Bruning, Research Associate Diane Imburgio, Postdoctoral Fellow Deepu Abraham, Graduate Assistant Edward Carr, Graduate Assistant Ian Fingerman, Graduate Assistant Sean Hanlon, Graduate Assistant Jonathon Mathias, Graduate Assistant Laura Youngster, Graduate Assistant Jennifer Chen, Undergraduate Student |
Research
Summary