"R-loop" as a novel key mechanism of repeat contraction in diverse repeat expansion diseases and cancer
Based on our findings showing that RNA-DNA hybrid - R-loop formation is sufficient to trigger CGG contraction in FXS iPSCs and NPCs (Lee et al., Cell 2023), we will expand this repeat contraction strategy to diverse diseases of unstable repeat expansion. We will examine whether R-loop formation at the tandem repeat regions in diverse repeat expansion diseases (e.g., CGGn, CAGn, CTGn, or GAAn) can trigger repeat contraction by stabilizing secondary structures of the repetitive single-stranded DNA (ssDNA). It has been suggested that single-stranded tandem repeats can form various non-canonical secondary structures (e.g. hairpin-like structure), which might trigger the repeat contraction/expansion mechanism by recruiting MMR and other DNA repair mechanisms, as reported in many repeat expansion diseases.
Moreover, we will reveal the detailed “epi”genetic / molecular mechanisms beyond the repeat contraction by R-loop. Lee lab will pioneer a common therapeutic framework to overcome the diverse repeat expansion diseases by identifying detailed mechanisms of R-loop mediated contraction of pathogenic DNA repeats.
Lee lab aims to
1) induce the R-loop mediated repeat contraction to other relevant repeat expansion diseases
2) reveal the molecular basis of the R-loop mediated repeat contraction in FXS by identifying novel factors by establishing reporter/screening systems and examining the genetic modifiers of other repeat expansion diseases,
3) identify novel “epi”genetic mechanisms for the repeat instability, repeat expansion disease phenotypes, and transposon silencing by integrating diverse genomic data.
Project 1: Tackle diverse repeat expansion diseases by R-loop mediated repeat contraction
By ectopically inducing R-loops to the various long tandem repeats, my group will aim to tackle the etiology of many other repeat expansion diseases that have common epigenetic features with FXS by the R-loop mediated repeat contraction. Multiple repeat expansion diseases, including DM1, FRDA, and ALS, share important epigenetic aspects with FXS – e.g., involvement of DNA methylation and R-loop association with the long tandem repeats. If we can safely remove the root cause - the expanded tandem repeats – it will be the best way to treat the devastating diseases. By inducing stable R-loops at the long tandem repeats in patient-derived DM1, FRDA, and ALS cells, our lab will trigger the repeat contraction by cell-intrinsic MMR pathway.
In our recent FXS study, we successfully induced strong R-loops by recruiting dCas9. To target different tandem repeat regions in other repeat expansion diseases, we will employ engineered Cas9 variants for more universal PAM sequence recognition.
Project 2: Identify the molecular basis of repeat contraction/expansion
Currently, there is a major gap in our understanding of the mechanisms for repeat instability in FXS and other repeat expansion diseases, and one primary reason is lack of the molecular experimental settings for recapitulating the repeat contraction/expansion process robustly. We recently established well-defined experimental conditions for inducing contraction of long repeats by small molecules and R-loop formation that can provide a great molecular model system for identifying the novel mechanisms of repeat contraction/expansion. The repeat contraction models we developed will allow us to readily test the repeat instability quantitatively while perturbing candidate genes by various ways (e.g. knock-down, overexpression, knock-out, and degron). Identifying the major factors involved in repeat expansion/contraction can open a new avenue for developing therapeutic approaches. By using these very well-defined conditions for the CGG repeat contraction from our research, we aim to test the involvement of the genetic modifiers of various trinucleotide repeat disorders including myotonic dystrophy and Huntington’s disease in FXS.
For more unbiased identification of the novel factors, my lab will establish a reporter system for FMR1 reactivation by generating FMR1-GFP knock-in FXS cell line. By combining the CRISPRi/a system with the robust FMR1 reactivation protocols from my recent study, we will screen the factors positively or negatively affecting the FMR1 reactivation. Also, we will establish a dCas9-APEX-mediated proximity labeling system to identify the novel factors that are recruited specifically to the FXS cells with long CGG repeats. Selected potential factors will further be tested for the effects on the repeat expansion/contraction in the FXS and other repeat expansion diseases.
Project 3: Identify the epigenetic mechanisms of the aberrant development by long tandem repeats and their instability.
Our long-term goal is to understand how the repeat expansion diseases arise by "epi"genetic dys-regulation during development. We will investigate “epi”genetic mechanisms involved in tandem repeat expansion/contraction and disease phenotypes, ranging from non-canonical nucleic acid structures (e.g. R-loops and G-quadruplexes) to DNA and histone modifications related to DNA replication/repair and transcription at the pathogenic long tandem repeats in diverse repeat expansion diseases. Our lab will recapitulate repeat expansion processes and abnormal development in the context of epigenetic landscape changes by differentiating patient-derived iPSCs to the disease-relevant cell types (e.g. neuronal cells for FXS, cardiomyocytes for DM1) or organoids.
Importantly, tandem repeat instability coincides with 1) DNA replication and 2) transcription when the long tandem repeats become accessible/vulnerable. Notably, these two major cellular processes are also associated with R-loop formation. Our recent study emphasized the involvement of R-loop formation by transcription in repeat contraction. We will extend our study to R-loop formation during DNA replication and examine the changes in the epigenetic landscape to identify the factors for repeat instability at replication forks in disease cells.
By perturbing the identified disease-relevant epigenetic mechanisms, we will cross-examine the causality of the disease phenotypes and repeat instability. We will employ genetic knock-out, degron, and pharmacologic approaches in the context of repeat instability by ectopic induction of R-loop via dCas9 recruitment or by small molecules (e.g. 5i or Decitabine). As many enzymatic and protein interaction inhibitors have been developed for chromatin-modifying proteins, our findings on the role of epigenetic pathways will have strong potential to be drug targets.