Frank Pugh

Biochemistry and genomics of eukaryotic transcription regulation

Summary

Our research is devoted to understanding how genes are controlled in eukaryotic cells. We are focusing our attention on how the RNA polymerase transcription machinery and its regulatory proteins assemble at all 6,000 genes of the yeast Saccharomyces cerevisiae. Since the transcription machinery is fundamentally the same in all eukaryotes, lessons learned from yeast provide the foundation for understanding how genes are regulated in humans, and how mis-regulation of genes leads to diseases such as cancer.

Eukaryotic transcriptional regulation involves hundreds of proteins. DNA sequence-specific regulators read the transcriptional regulatory code in the DNA by binding to promoter elements and orchestrating the assembly and disassembly of the transcription machinery. At an early stage during transcriptional activation resident chromatin isaltered, and this regulates access of general transcription factors (GTFs) TFIIA, -B, -D, -E, -F, and -H to the underlying promoter DNA. These GTFs, RNA polymerase II (pol II), and other associated regulators assemble into a pre-initiation complex (PIC). Our recent work suggests that PICs assemble via two primary GTF pathways involving TFIID which predominates at TATA-less promoters and SAGA which predominates at TATA-containing promoters. The TFIID pathway is central to most genes, whereas the SAGA pathway is tailored for stress-induced gene expression. Once recruited into a PIC, pol II initiates transcription, then subsequently transcribes the entire gene to produce mRNA. This transcription phase is also subjected to a variety of regulatory controls.

Our research utilizes biochemistry to understand gene regulatory mechanism and genomic methods to integrate such mechanisms into the global gene regulatory network. Our genomic methods include genome-wide location (chIP-chip and ChiP-sequencing) assays, genome-wide expression profiling, and bioinformatic analyses. These approaches allow us to measure the chromatin structure and occupancy of transcription proteins at all promoters, their contribution to gene expression, and their relationship to each other. This approach involves millions of data points, each measuring the plasticity of the transcription machinery as it operates throughout the entire genome. Computational modeling of the data allows us to integrate biochemically-defined regulatory mechanisms with the goal of generating a unified gene regulatory network.

Please see our developing Genome Cartography Project .

Representative Publications:

• Chitikila, C., K. L. Huisinga, J. D. Irvin, M. Mitra, and B. F. Pugh 2002. Interplay of TBP inhibitors in global transcriptional control. Mol. Cell 10, 871-882.
• Kou, H., Irvin, J. D., Huisinga, K. L., and Pugh, B. F. 2003. Structural and functional analysis of mutations along the crystallographic dimer interface of the yeast TATA binding protein. Mol. Cell. Biol. 23, 3186-3201.
• Huisinga, K. L., and Pugh, B. F. 2004. A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. Mol. Cell 13, 573-585.
• Basehoar, A. D., Zanton, S. J., and Pugh, B. F. 2004. Identification and distinct regulation of yeast TATA box-containing genes. Cell 116, 699-709.
• Pugh, B. F. 2004. Is acetylation the key to opening locked gates? Nature Struct. Mol. Biol. 11, 298-300.
• Kou, H., and Pugh, B. F. 2004. Engineering dimer stabilizing mutations in the TATA binding protein that reverse biological defects associated with dimer destabilizing mutants. J. Biol. Chem. 279, 20966-73.
• Zanton, S. J., and Pugh, B. F. 2004. Changes in genome-wide occupancy of core transcriptional regulators during heat stress. Proc. Natl Acad. Sci. 101, 16843-16848.
• Alexander, D. E., Kaczorowski, D., Lowry, D. M., Jackson-Fisher, A. J., Zanton, S. J. and Pugh, B. F. 2004. The Brf1 subunit of RNA polymerase III transcription factor TFIIIB induces TBP dimers to dissociate. J. Biol. Chem. 279, 32401-32406.
• Irvin, J.D. and Pugh, B.F. 2006. Genome-wide transcriptional dependencies on TAF1 functional domains.  J. Biol. Chem. 281, 6404-12.
• Durant, M. and Pugh, B.F. 2006. Genome-wide relationships between TAF1 and histone acetyltransferases in S. cerevisiae. Mol Cell Biol. 26,2791-802.
• Zanton, S. J., and Pugh, B. F. 2006. Full and partial genome-wide assembly and disassembly of the yeast transcription machinery in response to heat shock. Genes Devel. 20, 2250-2265.
• Ioshikhes, I., Albert, I., Zanton, S. J., and Pugh, B. F. 2006. Nucleosome positions predicted through comparative genomics. Nature Genet. 38, 1210-1215.  Subject of commentary.
• Pugh, B. F. 2006. HATs off to PIC assembly. Mol Cell. 23, 776-777.
• Albert, I., Mavrich, T. N., Tomsho, L. P, Qi, J., Zanton, S. J., Schuster, S. C., and Pugh, B. F. 2007. Translational and rotational settings of H2A.Z nucleosomes across the S. cerevisiae genome. Nature;. 446, 572-576.
• Huisinga, K.L. and Pugh, B.F. 2007.   A TATA Binding Protein regulatory network that governs transcription complex assembly. Genome Biol. 8(4): R46.
• Durant, M. and B. F. Pugh 2007. NuA4-directed chromatin transactions throughout the Saccharomyces cerevisiae genome.  Mol Cell Biol. 2007 15, 5327-35.

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