My lab is using functional genomics, molecular biology, biochemistry, and 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.
R-loops and Transcription-Replication conflicts
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. These transcription-replication conflicts (TRCs), and their association with R-loops, are potentially a major endogenous source of DNA replication stress, a common phenotype in cancer.
We began work in this area with a focus on splicing factors such as SF3B1 and mRNA 3’-end processing factors like FIP1L1 (PMID: 22279048; 30462576). However, we quickly realized that many cellular pathways impinge upon transcription replication conflicts. Now the work has expanded to include studies on fork associated DNA repair proteins that oppose the damaging effects of TRCs, including published work on BLM, the Mre11-Rad50-Nbs1 complex, and PCNA regulators (PMID: 29042409; 31537797; 32542338). We also recognize that chromatin remodelers influence TRCs and have reported such observations for cells with mutations in the BAF complex subunit ARID1A (PMID: 33826602). Our work on specific candidate genes implicated in R-loop processing by our screens is ongoing.
More broadly, we have now begun to profile R-loop binding proteins (RLBPs) in unbiased ways using characteristics of RLBPs to predict more, CRISPR screens and proximity proteomics (unpublished work and https://www.biorxiv.org/content/10.1101/2021.08.09.454968v1). Together these approaches are expanding our view of cellular pathways influencing R-loop associated genome instability. In addition, we have become interested in the association of R-loops with other non-B-DNA structures such as G-quadruplexes and have work collaboratively on G4 ligands and rolls of G4 and R-loops at ALT telomeres (PMID: 34316718; 28211448).
Mutation signatures, DNA damage and synthetic lethality
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: PMID: 24336748; 28223526). We have since extended this approach to human cell lines where we are studying the effects of CRISPR generated TLS polymerase deletions, with a current focus on genotoxins and cannabis smoke byproducts.
Synthetic lethality describes a mutual dependency for cell viability between two individually non-essential genes. This concept has been proposed to apply to selection of unlinked cancer therapeutic target genes based on tumour genotypes for nearly 25 years. To date only PARP inhibitors represent this class of therapy in the clinic. We are using screens in yeast and human cells to enrich the network of synthetic lethal interactions, and developing synthetic biology tools to hone in on the best candidates for therapeutic translation among the dense networks of genetic interactions in the literature.
Protein Quality Control as a 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 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. We are particularly interested in a peri-nucleolar and SUMO regulated protein sequestration site called the intranuclear quality control site (INQ). We provided the first links of this site to splicing regulation (PMID: 28978642) and control by the Cdc48/VCP protein disaggregase (PMID: 33172985). If and how sequestration in INQ is conserved to the reported protein quality control function of the human nucleolus is an area of active investigation.
Associate Professor, Medical Genetics, University of British Columbia
With the exception of valuable training in California, Dr. Stirling is a life-long British Columbian. He grew up in Port Alberni, British Columbia and now resides in East Vancouver. He has a long-standing interest in the natural world and has been pursuing a career in the basic sciences for decades. Dr. Stirling is a CIHR New Investigator and a Michael Smith Foundation for Health Research Scholar. Details of his training are listed below.
• PhD (Molecular Biology and Biochemistry), Simon Fraser University, 2007
• BSc (Biochemistry), University of Victoria, 2002
• Postdoctoral Research Fellow (Supervisor: Dr. Philip Hieter)
Michael Smith Laboratories, University of British Columbia (UBC), 2009 – 2013
• Postdoctoral Research Fellow (Supervisor: Dr. David Drubin)
Molecular and Cell Biology, University of California, 2007 – 2008
• Scientist, Terry Fox Laboratory, BC Cancer Agency (BCCA), Jan 2014 –
• Assistant Professor, Medical Genetics, UBC, Jan 2014 – June 2019
• Associate Professor, Medical Genetics, UBC, July 2019 –
• Cell biology
• Functional genomics
• Molecular mechanism
• Translational and therapeutic anti-cancer application
• Budding yeast
• Synthetic lethality