Project Summary/ Abstract Recent highthroughput exome sequencing has revealed various somatic mutations in individuals with clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS) patients. Among these mutations, C-to-T mutations is among the most common type of single mutations observed in CH and MDS. It remains yet to be explained why C-to-T mutations are preferentially enriched in CH and MDS and how the C-to-T transition alters hematopoietic stem and progenitor cells (HSPCs) fitness and leads to aberrant clonal expansion. By combining in vitro biochemical and structural analyses with epigenomic profiling of bone marrow samples from MDS patients, the team discovered a previously-underappreciated mutual reinforcement between C-to-T transition and genomic binding of transcription factors (TFs), which contributes to the clonal expansion of HSPCs and aggravate CH and MDS. In Aim1, the team will use genetically-modified mouse models and CRISPR/Cas9- based genome editing methods to probe the causality between C-to-T transition and TF binding in HSPCs. In Aim 2, the team will test how the C-to-T transition and DNA methylation deregulation reshape the genomic distribution of TFs, subsequently altering downstream transcriptional outputs to cause clonal expansion in HSPCs. In parallel, the team will use HSPCs purified from CH and MDS patients to further validate findings made ex vivo and in rodent models. The proposed studies are anticipated to significantly advance our mechanistic understanding on how TFs shape the genome and how somatic mutations alter transcriptional regulation in HSPCs. From a translational perspective, discoveries made from this study will lead to the identification of therapeutic vulnerabilities for CH and MDS treatment.