Recent advances in genomic medicine reveal the frequent association of mutations in splicing factors, transcription factors, and epigenetic regulators with Myelodysplastic Syndromes (MDS) and related blood disorders. Focusing on mutations in splicing factors, my research showed that mutations in different splicing factors or different mutations in the same splicing factors cause essentially non-overlapping changes in pre-mRNA splicing, raising the possibility for the existence of some common pathways affected. In my recently published work, I showed that high-risk mutations in splicing factors all induced excessive R-loops, which triggered the activation of the ATR pathway and DNA replication stress that led to cell cycle arrest, suggesting the induction of R-loops as a unifying mechanism for MDS. I now propose that this pathway may also underlie the synergy between mutations in genes involved in the regulation of gene expression at different levels. In this K99/R00 application, I propose to extend the concept of R-loops as the disease driver by testing the synergy between mutations in splicing factors and transcription factors and epigenetic regulators, given frequent co-occurrence of these mutations in MDS patients, which I will pursue in the mentored phase with Dr. Xiang-Dong Fu as my mentor and Dr. Dong-Er. Zhang as my co-mentor. During this investigation, I also observed a striking correlation between R-loop formation and DNA replication initiation. Realizing that this is related to not only the disease mechanism I am studying, but also a long-standing problem in understanding how transcription is coupled with DNA replication in mammalian cells, I propose a series of experiments to be pursued in the independent phase, through which I wish to define my own research focus to advance my career after postdoctoral training. To facilitate the development of this research program, I recruit three experts to serve on my advisory committee, including Dr. Michael G. Rosenfeld and Dr. Chris Glass who are leaders in the field of transcription and Dr. Richard Kolodner who is an authority in DNA replication/repair so that I can continue to accumulate preliminary results for my long-term research while pressing ahead on short-term studies in the mentored phase.
This proposal seeks to uncover the mechanism for the synergy between mutations in the splicing factor SRSF2 and the transcription factor RUNX1 as well as the epigenetic regulators DNMT3a and TET2. The proposed research aims to advance the concept of abnormally induced R-loops as a unifying mechanism to cause developmental defects in hematopoietic progenitor cells. I will also attack a long-standing problem in coupling between transcription and DNA replication in mammalian cells, which will provide key insights into this fundamental problem in development and cancer.