Stress induced gene expression and UV resistance pathways
Cancer results from of an accumulation of genetic and cellular damage and the ensuing uncontrolled cell proliferation. Eukaryotic cells respond to these challenges by activating DNA damage sensing, signaling and repair pathways that are stimulated by damage to DNA and other stresses. Activation of these pathways cause the execution of cell cycle delays, referred to as checkpoints, and the expression of DNA repair genes. Alterations in these functions are known to predispose people to cancer and other diseases. Our laboratory uses a combination of biochemistry, genetics, genomics and molecular biology to study the mechanisms controlling stress-induced changes in gene expression in eukaryotes. The two main focus areas of the lab are the role of transcription and mRNA decay factors in regulating the DNA damage response and the control of transcription elongation by protein complexes and chromatin.
Regulation of mRNAs from birth to death during stress responses
Gene expression is controlled at multiple levels, requiring highly regulated and integrated events including chromatin remodeling, initiation, elongation, processing, transport and ultimately the destruction of mRNA. Determining how these events are coordinated is essential to understanding gene expression mechanisms. Our research exploits the powerful genetic systems of budding yeast and Drosophila melanogaster to examine the function of a complex proposed to perform multiple steps in gene regulation: the Ccr4-Not complex. The Ccr4-Not complex is highly conserved across the eukaryotic kingdom. Initially purposed to be a nuclear transcription regulatory complex in yeast, it has since been identified as the major mRNA deadenylase in the cytoplasm and implicated in protein destruction and micro RNA (miRNA) processing. Mutations in subunits of this complex cause altered DNA damage checkpoint functions, impaired cell cycle progression and sensitivity to stress. The human orthologues of these proteins are putative oncogenes and are implicated in regulating hepatitis C virus replication in human cells; thus, understanding the function of this complex is directly relevant to human disease. The goals of our project studying Ccr4-Not are to (1) uncover how multiple steps in gene expression are coordinated and regulated; (2) define the functions of a highly conserved eukaryotic transcription factor complex implicated in human disease; (3) identifying how specific mRNAs are marked for post-transcriptional control during transcription.
Control of RNA Polymerase II elongation and regulation of chromatin structure during elongation
Transcription of a gene requires the movement of RNA polymerase II (RNAPII) across many thousands of bases in the genome. Throughout the elongation process, RNAPII acquires and releases up to 29 different multisubunit complexes to allow it to transverse a gene. Elongation factors assist RNAPII in transcribing through chromatin and transcription blocks, and successful completion of the gene requires the coordinated action of elongation factors and the remodeling of chromatin ahead of RNAPII. We are analyzing the recruitment and actions of elongation factors during RNAPII elongation in vitro and in vivo. Using highly purified factors and reconstitution biochemistry, we can monitor the assembly and disassembly of elongation complexes to gain mechanistic insights into how elongation factors promote transcription elongation. Furthermore, we can exploit the highly tractable genetic system of yeast to monitor elongation in vivo and to interrogate the role of the structure of the nucleosome in regulating elongation.
Figure 1. Gene expression is regulated at multiple levels in the nucleus and cytoplasm.
Figure 2. Ccr4-Not functions in the nucleus and cytoplasm to control the synthesis and destruction of mRNAs during the stress response