Robert T. Simpson
	Professor and Holder of the Verne M. Willaman Chair in Biochemistry and Molecular Biology
	308 Althouse Laboratory, University Park, PA 16802 Phone: (814) 863-0276 Fax: (814) 863-7024 Email: rts4@psu.edu
	B.A., Swarthmore College M.D., Harvard University Ph.D. in biological chemistry, Harvard University
	Simpson Lab Web Site

Press Release of April 28, 2004

How do chromosomal proteins control eukaryotic DNA transcription, recombinationand replication?

Background: The role of chromatin structure in control of the function of DNAin transcrip-tion, replication, recombination and repair has becomeincreasingly obvious. To study this area, we use a highly tractablesystem, Saccharomyces cerevisiae, which offers the potential for geneticmanipulation of both the gene under examination and the backgroundgenotypes for proteins of interest. The ability to place a gene on aminichromosome and isolate that structure, as native chromatin, invarious functional states, offers an unprecedented opportunity to relatecomposition, structure and function of chromatin. We also isolate proteins implicated in regulation of DNA function and study their interactions with each other and with cis-acting DNA sequences. Another project develops methods for mapping chromatin structure in vivo and extends these for long range (hundreds of kilobases) analysis of protein-DNA interactions in the yeast genome.

Transcriptional Repression: Our studies focus on genes regulated by the mating type locus. Yeastexist in two mating types, a and alpha. Specific genes expressed in a-cellsare repressed in alpha-cells. We have shown that the alpha2 repressor organizesa repressive chromatin structure on the promoters of a-cell specificgenes in alpha-cells. In addition to its promoter, the entire codingsequence of the STE6 gene is packaged as precisely positionednucleosomes. Genetic evidence implicated several other proteins inrepression of a-cell specific genes. We have now shown that, in additionto Mcm1p and the alpha2 repressor, the amino terminal regions of histone H4and the products of the SSN6 and TUP1 genes are required to organizechromatin on a-cell specific genes. We find an absolute concordance ofthe presence of the organized chromatin structure with completerepression of a-cell specific promoters. Another yeast gene also controlled by Ssn6p and Tup1p is SUC2. It has highly organized chromatin over the promoter region, although positioned nucleosomes only extend ~500 bp into the structural gene. Regulation of this gene and its chromatin structure involves a complex interplay between the repressive SSN6/TUP1 complex and the chromatin remodeling machinery present in the SWI/SNF complex and another, as yet unidentified, protein group.

Transcriptional Silencing: We have determined the chromatin structure of the silenced HMLalpha mating type locus; positioned nucleosomes occupy most of the 4 kb region between the I and E silencer elements. Surprisingly, the promoter between the alpha1 and alpha2 genes is nucleosome free, in contrast to the a-cell specific genes where the TATA box is in the middle of a positioned nucleosome. Mutations in the genes for proteins required for silencing, SIR1, SIR3, and SIR4 lead to distinctive alterations in chromatin organization at HML. We are now comparing this structure with chromatin organization at HMRa. Additionally, we have begun investigation of the changes in structure of the silent mating type loci as recombination with the active mating type locus takes place during mating type switching.

Mating Type Switching: Activation of the HO endonuclease in homothallic yeast leads to a double strand DNA break at the MAT locus near the center of chromosome III; the break is repaired by recombination with one of the silent mating type loci located near the ends of the same chromosome. A twenty year mystery has been the reason why MATa cells use HMLalpha and MATalpha cells use HMRa for recombination with a fidelity of ~90%. James Haber's laboratory has recently found that a 700 bp region located ~20 kb from HML serves as a recombination enhancer; it is sufficient to activate the left arm of chromosome III for recombination in a-cells and to repress this region in alpha-cells. We have determined the chromatin structure of this region and find distinctive features suggesting protein binding and altered DNA geometry in a-cells. In alpha-cells, positioned nucleosomes flanking an alpha2 operator span ~4 kb of the genome, precluding access of trans-acting factors to the enhancer. We are using a number of mutants to elucidate important features of the enhancer in collaborative studies with the Haber laboratory. Using the yeast one hybrid system, we search for proteins that bind to enhancer sequences and are candidates for the master control gene that determines directionality in yeast mating type interconversion.

Minichromosomes: We have developed methods for isolation of yeast minichromosomes as native chromatin. By introducing a specific gene into a minichromosome, we can determine proteins associated with the gene in its active or inactive state. Physicochemical studies of the regulatory proteins and their interactions are then carried out in vitro. This laboratory uses in vitro analysis of protein-proteininteractions in reconstructed chromatin to explore the mechanisms ofcontrol of gene expression. As a benchmark for assessing the fidelity ofthe artifically assembled chromatin, we use the composition andstructure of the isolated minichromosome.

Long Range Structure Mapping: Other studies in our laboratory involve development of methods forin vivo mapping of chromatin stucture. We used the minichromosome systemto establish a method of assessing chromatin organization usingexpression of prokaryotic methyl-transferases. We now extend this to morepromiscuous methyltransferases and development of methods that use automated, multiplex analysis of modification by these enzymes.We take advan-tage of an unprecedented opportunity for long rangechromatin structure study afforded by the recently reported completesequence of the entire yeast genome.

Representative Publications:

• Cooper, J.P., Roth, S.Y. and Simpson, R.T. The global transcriptional regulators, SSN6 and TUP1, play distinct roles in the establishment of a repressive chromatin structure. Genes Dev 8,1400-1410, 1994.

• Patterton, H-G. and Simpson, R.T. Modified curved DNA that could allow local DNA underwinding at the nucleosomal pseudodyad fails to position a nucleosome in vivo. Nucleic Acids Res 23, 4170-4179, 1995.

• Kladde, M.P., Xu, M. and Simpson, R.T. Direct study of DNA-protein interactions in repressed and active chromatin in living cells. EMBO J 15, 6290-6300, 1996.

• Kladde, M.P. and Simpson, R.T. Chromatin structure mapping in vivo using methyltransferases. Meth Enzymol 274B, 214-233, 1996.

• Weiss, K. and Simpson, R.T. Cell type-specific chromatin organization of the region that governs directionality of yeast mating type switching. EMBO J 16, 4352-4360, 1997.

• Gavin, I.M. and Simpson, R.T. Interplay of yeast global transcriptional regulators Ssn6p-Tup1p and Swi-Snf and their effect on chromatin structure. EMBO J 16, 6263-6271, 1997.

• Patterton, H.G., Landel, C.C., Landsman, D., Peterson, C.L. and Simpson, R.T. The biochemical and phenotypic characterization of Hho1p, the putative linker histone H1 of Saccharomyces cerevisiae. J Biol Chem; 273, 7268-7276, 1998.

• Xu, M., Simpson, R.T. and Kladde, M.P. Gal4p-mediated chromatin remodeling depends on binding site position in nucleosomes but does not require DNA replication. Mol Cell Biol, 18, 1201-1212, 1998.

• Wu, C., Weiss, K., Yang, C., Harris, M.A., Tye, B-K., Newlon, C.S., Simpson, R.T. and Haber, J.E.: Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching. Genes Dev 12, 1726-1737 (1998).

• Weiss, K. and Simpson, R.T.: High resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating type locus HMLalpha. Mol Cell Biol 18,  5392-5403 (1998).

• Kladde, M.P. and Simpson, R.T.: Rapid detection of functional expression of C-5-DNA methyltransferases in yeast. Nucleic Acids Res 26,1354-1355 (1998). Full text reproduced with permission from NAR Online http://www.oup.co.uk/nar.

• Xu, M., Kladde, M.P., Van Etten, J.L. and Simpson, R.T.: Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC. Nucleic Acids Res 26, 3961-3967 (1998).

• Simpson, R.T.: Chromatin structure and analysis of mechanisms of activators and repressors.  Methods 15:283-94 (1998).

• Simpson, R.T.: In vivo methods to analyze chromatin structure. Curr Opin Genet Dev 9:225-9.(1999).

• Kladde,M.P., Xu, M. and Simpson, R.T.: DNA methyltransferases as probes of chromatin structure in vivo. Methods Enzymol 304:431-47 (1999).

• Ravindra, A., Weiss, K. and Simpson, R.T.: High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating-type locus HMRa. Mol Cell Biol 19:7944-50 (1999).

• Ducker, C.E. and Simpson, R.T.: The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome. EMBO J 19:400-409 (2000). (Full Text).

• Gavin, I.M., Kladde, M.P. and Simpson, R.T.: Tup1p represses Mcm1p transcriptional activation and chromatin remodeling of an a-cell-specific gene. EMBO J 19:5875-5883 (2000). (Full Text).

• Wang, X. and Simpson, R.T.: Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I. Nucleic Acids Res 29:1943-1950 (2001). (Full Text)

Search the MEDLINE database at PubMed for articles by R. Simpson

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