Transcription regulation mechanisms across eukaryotic genomes
Research in the Pugh laboratory is devoted to understanding how genes are controlled in eukaryotic cells. The lab consists of a team of postdocs, graduate students, technicians, and undergraduates. They are devising experiments to determine how the RNA polymerase transcription machinery and its regulatory proteins work with chromatin to regulate the thousands of genes of model organisms including the bakers yeast Saccharomyces cerevisiae, the fruit fly Drosophila melanogaster, and mammalian tissue culture cells. Since the transcription machinery is fundamentally the same in all eukaryotes, most experiments are conducted on yeast, as they allow for the most efficient means of discovering transcriptional regulatory mechanisms. Lessons learned from these model organisms provide the foundation for understanding how genes are regulated in humans, and how mis-regulation of genes leads to diseases such as cancer.
The eukaryotic transcription machinery and its surrounding chromatin mileux consist of hundreds of distinct proteins, each having a specific function. 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 is altered, 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). Research in the Pugh lab has shown 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.
This entire process takes place in the context of chromatin, whose fundamental building block is the nucleosomes. The Pugh lab was the first lab to apply to “ChIP-seq” technology to define at the highest possible resolution where each nucleosome is located in the yeast and fly genomes. The lab has used this technology to map at the same resolution where proteins that interact with nucleosomes bind across the genome. More recently the Pugh lab has developed an ultra-high resolution mapping technique to determine the precise genomic location of any gene regulatory protein. We expect this technique to provide greater detail and insight into how proteins bind to their target sites in living cells.
Our research utilizes biochemistry, molecular biology, and genomics to understand gene regulatory mechanisms and how they are integrated into the global gene regulatory network. Our ChIP-seq technology involves the collection of millions of data points, each measuring the plasticity of the transcription machinery as it operates throughout the genome. Computational modeling of the data allows us to integrate biochemically-defined regulatory mechanisms with the goal of generating a unified gene regulatory network.