My lab is using functional genomics, molecular biology, biochemistry, and advanced imaging in both the yeast model and cultured human cells to study fundamental mechanisms of genome maintenance and stability. Failure to maintain genome integrity leads to mutations that can promote tumour formation. Normal genome maintenance mechanisms can be overwhelmed by carcinogen exposure, or the presence of germline or somatic variants that induce genomic instability. Our work is aimed at determining the causes of genomic instability as an enabling characteristic of tumour formation, and exploring the potential of these early events to suggest novel therapeutic targets. Currently, we are working on several projects, briefly outlined below.
Roles of RNA processing in genome maintenance
Functional genomic screening of the yeast model, Saccharomyces cerevisiae, and of mammalian cells has revealed that most steps in RNA metabolism (including transcription, processing, and decay) can cause genome instability when disrupted. In some cases it is clear that mutations in RNA processing factors lead to transcription-coupled DNA:RNA hybrids that form R-loops. R-loop structures expose damage-prone single-stranded DNA, and can also impede DNA replication fork progression, causing breaks. Importantly, not all genome destabilizing RNA processing mutants create R-loops, and we are also interested in defining these alternative mechanisms.
RNA processing mutations are now recognized to occur in various tumours. These include widely distributed and frequent mutations of the spliceosome (e.g. SF3B1 in hematopoietic malignancies), mutations in the RNA degrading exosome (e.g. DIS3), and mRNA processing genes (e.g. FIP1L1). The importance of these cancer-associated mutations for genome maintenance and the potential for R-loop formation to contribute to cancer cell phenotypes is also under investigation. Finally, some yeast RNA processing mutants exhibit synthetic lethality with cancer-associated genome maintenance genes, and therefore represent candidate therapeutic targets. We are exploring the conservation of such interactions and the potential for inhibiting aspects of RNA metabolism for therapeutic benefit.
DNA repair: mutation signatures and transcription-replication conflicts
Mutational processes leave a characteristic pattern of mutations on tumour genomes based on the underlying biology that is disrupted or the causative environmental agent. While elegant computational methods can extract such mutation ‘signatures’ from tumour genomes, the mechanisms are usually unknown. We have established a yeast model for linking specific genetic changes in conserved genome integrity pathways, and their interactions with genotoxic drugs, to patterns of accumulated mutations using whole genome sequencing. Yeast provides a clean and tractable genetic system whose genome can be sequenced in an efficient, cost-effective manner. These signatures have led to new insights to biochemical mechanisms of DNA repair (e.g. slippage and realignment in DNA translesion synthesis polymerases) and to an appreciation that defects in DNA repair can increase transcription-replication conflicts.
Our observation that some DNA repair defects increase transcription-replication conflicts, likely due to R-loops impairing DNA replication, connects to a growing body of literature which links genome maintenance factors (e.g. BRCA2, XPF/XPG, the Fanconi Anemia pathway and p53) to the suppression of R-loop-mediated genome instability. Our group is actively pursuing various DNA repair proteins that can suppress R-loop formation in yeast and human cells. We are developing tools to directly probe the molecular mechanisms of these effects and screen for conditions which enhance R-loop formation genetically or chemically.
The global cellular response to genotoxic stress
We have a long-standing interest in how cells respond to environmental stress dating back to Dr. Stirling’s earliest research on molecular chaperones (c. 2002-2008). Currently, this work is focused on the ways in which RNA and protein quality control machinery respond to genotoxic chemicals. These pathways may serve as chemoresistance mechanisms in the face of genotoxic cancer therapies. Using the yeast model, we have found that transcriptome changes following genotoxic stress directly regulate the constituents of protein aggregate structures. We believe that factors ejected from chromatin due to the transcriptional upheaval of a stress response are captured by protein quality control machinery in order to promote adaptation and recovery. The interplay between transcriptome dynamics, protein quality control, and RNA quality control (i.e. through P-bodies and Stress Granules) is an active area of interest in the lab.
Sorgjerd KM, Zako T, Sakono M, Stirling PC, Leroux MR, Saito T, Nilsson P, Sekimoto M, Saido TC and Maeda M. Human prefoldin inhibits Amyloid β (Aβ) fibrillation and contributes to formation of non-toxic Aβ aggregates. Biochemistry. 52, 3532-3542, 2013. View Abstract
van Pel DM*, Stirling PC*, Minaker SW, Sipahimalani P and Hieter P. Saccharomyces cerevisiae genetics predicts candidate therapeutic genetic interactions at the mammalian replication fork. G3 (Bethesda) 3, 273-282, 2013 *Authors contributed equally. View Abstract
Minaker SW, Filiatrault MC, Ben-Aroya S, Hieter P and Stirling PC. Biogenesis of RNA polymerases II and III requires the conserved GPN small GTPases in S. cerevisiae. Genetics 193, 853-864, 2013. View Abstract
Stirling PC*, Chan YA*, Minaker SW*, Aristizabal MJ, Barrett I, Sipahimalani P, Kobor, MS and Hieter P. R-loop mediated genome instability in mRNA cleavage and polyadenylation mutants. Genes Dev. 26, 163-175, 2012 *Authors contributed equally. View Abstract
Stirling PC, Crisp MJ, Basrai MA, Tucker CM, Dunham MJ, Spencer FA and Hieter P. Mutability and mutational spectrum of chromosome transmission fidelity (Ctf) genes. Chromosoma 121, 263-275, 2012. View Abstract
Stirling PC, Bloom MS, Solanki-Patil T, Smith S, Sipahimalani P, Li Z, Kofoed M, Ben-Aroya S, Myung K and Hieter P. The complete spectrum of yeast chromosome instability genes identifies candidate CIN cancer genes and functional roles for ASTRA complex components. PLoS Genet. 7, e1002057, 2011. *Faculty of 1000 rated. View Abstract
Lundin VF, Leroux MR* and Stirling PC*. Quality control of cytoskeletal proteins and human disease. Trends Biochem. Sci. Review. 35, 288-297, 2010. *Corresponding authors. View Abstract
Carroll SY*, Stirling PC*, Stimpson HE, Giesselman E, Schmitt MJ and Drubin DG. A yeast killer toxin screen provides insights into A/B toxin entry, trafficking and killing mechanisms. Dev. Cell 17, 552-560, 2009. *Authors contributed equally. *Faculty of 1000 rated. View Abstract
Dekker C*, Stirling PC*, Filmore H, McCormack EA, Pappenberger G, Paul A, Brost RL, Costanzo M, Boone C, Leroux MR and Willison KR. The interaction network of the chaperonin CCT. EMBO J. 27, 1837-1839, 2008.*Authors contributed equally. *Faculty of 1000 rated. View Abstract
Stirling PC, Srayko M, Takhar KS, Pozniakovsky A, Hyman AA and Leroux MR. Functional interaction between phosducin-like protein 2 and cytosolic chaperonin is essential for cytoskeletal protein function and cell cycle progression. Mol. Biol. Cell 18, 2336-2345, 2007. View Abstract
Martín-Benito J, Gómez-Reino J, Stirling PC, Lundin VF, Gómez-Puertas P, Boskovic J, Chacón P, Fernández JJ, Berenguer J, Leroux MR and Valpuesta JM. Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart. Structure 15, 101-110, 2007. View Abstract
Stirling PC, Bakhoum SF, Feigl AB and Leroux MR. Convergent evolution of clamp-like binding sites in diverse chaperones. Nat. Struct. Mol. Biol. 13, 865-870. 2006. Review. View Abstract
Stirling PC, Cuellar J, Alfaro GA, El Khadali F, Beh CT, Valpuesta JM, Melki R and Leroux MR. PhLP3 modulates CCT-mediated actin and tubulin folding via ternary complexes with substrates. J. Biol. Chem. 281, 7012-7021, 2006. View Abstract
Lundin VF*, Stirling PC*, Gomez-Reino J, Mwenifumbo JC, Obst JM, Valpuesta JM and Leroux MR. Molecular clamp mechanism of substrate binding by hydrophobic coiled coil residues in the archaeal chaperone prefoldin. Proc. Natl. Acad. Sci. USA 101, 4367-4372, 2004. *Authors contributed equally. *Faculty of 1000 rated. View Abstract
Stirling PC*, Lundin VF* and Leroux MR. Getting a grip on non-native proteins. EMBO Reports 4, 565-570, 2003. *Authors contributed equally. Review. View Abstract