The dynamics and impact of R-loops in epigenetic stability and aging

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PROJECT SUMMARY Introduction: A growing body of evidence supports the notion that epigenetic dysregulation is a key driver of aging. Indeed, recent studies show that epigenetic markers accurately predict chronological age, and that in vivo epigenetic reprogramming can prolong lifespan and enable tissue regeneration in aged animals. Most striking was the recent evidence from the David Sinclair group which demonstrated that induction of epigenetic “noise” through use of non-mutagenic double-strand breaks (ICE mouse model) leads to accelerated aging phenotypes at the physiological and cellular level (ICE MEFs), such as loss of cell identity. Interestingly, the epigenetic aging induced in ICE mice results from the dysregulation of key enhancers, epigenomic structures which drive gene expression via 3D interactions with target gene promoters. Recent evidence indicates that enhancers are transcribed into a non-coding RNA species, enhancer RNA (eRNA), and that eRNA supports enhancer stability. Moreover, mounting evidence reveals that eRNA forms a structure with enhancer DNA called an “R-loop” (an RNA:DNA hybrid with a displaced ssDNA strand). In a recent study, our lab demonstrated that STAG2, a protein which helps maintain enhancer stability and cell identity, also binds R-loops in vitro and co-localizes with them at enhancers, suggesting that STAG2 binds eRNA R-loops. We also find (unpublished) that STAG2 protects R- loops from degradation by the RNA:DNA helicase RNaseH1. Furthermore, I found that DHX9, a protein which regulates R-loop formation, is the top over-expressed gene in ICE mouse muscle and I found evidence of eRNA R-loops at enhancers dysregulated in ICE MEFs. Taken together, these findings suggest that STAG2 protects eRNA R-loops to maintain youthful enhancer stability with age. Therefore, I hypothesize that dysregulation of physiological R-loops drives epigenetic aging by impairing youthful enhancer stability. Aim 1: Elucidate the mechanism of eRNA R-loops in enhancer stability with epigenetic aging. I will uncover eRNA R-loops that are associated with enhancer dysregulation in epigenetic aging. I will use a CRISPR system to manipulate these R-loops and assess the impact on enhancer stability and cellular aging phenotypes. Aim 2: Determine the impact of the STAG2/R-loop interaction in preserving the youthful epigenome. I will assess cellular aging and epigenetic noise in ICE cells with manipulation of STAG2 and RNaseH1, and I will assess the differential binding of STAG2 with epigenetic aging in ICE MEFs. Conclusion and significance: With these aims, I will elucidate the role of physiological eRNA R-loops (and STAG2/R-loop interactions) in the mechanism of enhancer stability with aging. These aims are significant as they are the first to address the physiological role of R-loops in either enhancer stability or in aging, and they also have the potential to reveal novel drug targets for restoration of the youthful epigenome. Furthermore, the completion of the proposed training plan will prepare me for an independent research career in aging biology.