Roles of R-Loop Structures in Cellular Function and Disease Progression
Our group's research focuses on R-loops, dynamic and fascinating three-stranded nucleic acid structures that form within the genomic DNA of all cells. The majority of R-loops occur co-transcriptionally when newly transcribed RNA invades its template DNA to form a DNA:RNA structure and a displaced single strand of DNA, which can further fold into secondary structures such as G-quadruplexes. Both R-loops and G-quadruplexes pose barriers to DNA replication, and their improper accumulation or persistence is thus thought to contribute to disease by causing DNA damage and genome instability. At the same time, however, an abundance of evidence points to these structures playing essential roles in normal cellular homeostasis as well. Our group aims to further uncover these cellular functions and explore how their dysregulation contributes to pathogenesis and disease progression. We approach this broad field of inquiry by interfacing molecular biology techniques with high-throughput sequencing and bioinformatics, using and generating cell lines to model R-loop dynamics in disease including breast and ovarian cancer.
Finding R-Loop "Readers" in DNA Repair
One fundamental aspect of our research is identifying cellular interactors of R-loops. R-loop formation must be tightly controlled and resolved to avoid conflicts with replication machinery, and a large and growing suite of regulators has been identified over decades of research. Many of these regulators are helicases or RNases that act to unwind and remove the RNA component of R-loops from genomic DNA. In contrast, we are particularly interested in R-loop "readers": enzymes that bind and/or recognize R-loops in order to connect them to downstream pathways such as gene regulation, chromatin remodeling, or DNA repair. Identifying these readers is crucial to understanding how R-loops are integrated into their cellular roles; elucidating how mutations in readers can impact R-loop mediated function; and developing therapies that target R-loop related vulnerabilities in disease. We utilize innovative proximity labeling strategies, proteomic screens, and cell knockout models to answer these questions.
Exploring R-Loop Mediated Genome Rewiring
We are also exploring an emerging and exciting role for R-loops in the regulation of genome organization. In addition to mediating gene expression as well as the establishment of epigenetic marks such as DNA methylation, R-loops and G-quadruplexes can bolster the binding of CTCF, a master chromatin architectural protein that facilitates 3D organization of the genome at multiple scales. In vitro chemical stabilization of G-quadruplexes results in increased CTCF occupancy correlating with alterations to TAD boundary strength and de novo formation of chromatin loops. This recent discovery opens up the tantalizing possibility that R-loop accumulation, in addition to consequences on DNA damage and genome stability, may contribute to disease progress by rewiring the 3D genome into a configuration that supports pathogenic gene expression programs. We are examining this hypothesis by combining cutting-edge genomic sequencing techniques to probe how R-loops, CTCF, G-quadruplexes, and chromatin conformation change in tandem upon perturbation in disease models.
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