Mechanisms of transcriptional dysregulation in SF3B1 mutant MDS

About This Grant

Myelodysplastic Syndromes (MDS) is a group of heterogenous bone marrow failure syndromes often seen with advancing age. Mutations in splicing factors (SFs) such as SF3B1, U2AF1 and SRSF2 are the driving genetic alterations in over half of all MDS. These mutations have classically been linked to alternative splicing of oncogenes or tumor suppressors, but recent studies suggest broader defects including disruption of co-transcriptional splicing, which is the close functional coupling of transcription and splicing. Our group has recently shown that mutant SF3B1 impairs spliceosome assembly and slows RNA Polymerase II (Pol II) elongation, resulting in transcription-replication conflicts and replication stress. These changes reorganize chromatin, reducing promoter accessibility and histone marks. Notably, this model can explain the mutual exclusivity of SF mutations: cumulative transcriptional stress from multiple mutations is unsustainable for clonal expansion. In this application, we seek to define the role of HTATSF1, a protein with roles in both splicing and transcription, in transcriptional dysregulation in SF-mutant MDS. Our preliminary results show reduced interaction of HTATSF1 with mutant SF3B1. We hypothesize that this reduced binding of HTATSF1 to mutant SF3B1 impairs its recruitment to Pol II, disrupting the coordination between transcription and splicing. Conventional genome-wide assays lack spatial and temporal resolution to study the complexity of highly dynamic complexes such as spliceosome and Pol II. To overcome this, we will use multi-color, single-molecule imaging to resolve HTATSF1 recruitment and interaction kinetics in real time. We have leveraged CRISPR/Cas9 to introduce tags (degron for acute degradation and HaloTag for high resolution live-cell imaging in primary murine embryonic stem cells. Two aims are proposed to determine HTATSF1’s role in SF-mutant MDS. In the first aim, we will study how impairment of transcription noted in SF-mutant MDS is linked to HTATSF1. Total Internal Reflection Fluorescence (TIRF) microscopy will be used to track endogenously tagged HTATSF1 at a single-molecule resolution. In the second aim, we will determine HTATSF1’s role in altered splicing, a feature of SF-mutant We will utilize single-molecule imaging as well as differential phosphoproteomics in these studies. Ultimately, our findings may inform the development of therapies targeting transcriptional dysregulation, replication stress, and chromatin dysregulation in MDS.