Establishing CUX1 as a determinant of hematopoietic stem cell fate

PROJECT SUMMARY A longstanding question in developmental biology is how stem cells commit to a certain lineage. While cell surface markers correlate with hematopoietic stem cell (HSC) functions, less is known regarding transcriptional regulators that drive stem cell behavior. CUX1 encodes a highly conserved homeodomain-containing transcription factor. Deletions and inactivating mutations of CUX1 are recurrent in clonal hematopoiesis of indeterminate potential and myelodysplastic syndrome, and CUX1 loss is associated with cytopenias, lineage skewing, and a poor-prognosis in disease states. Typically, only one copy of CUX1 is inactivated, indicating that loss of CUX1 impacts hematopoietic development in a haploinsufficient manner. In line with this, we previously reported a dosage-dependent role for CUX1 in hematopoiesis. How CUX1 levels regulate hematopoietic stem and progenitor cell (HSPC) fate, with respect to proliferation, self-renewal, and lineage choice remains a major gap in knowledge. The overall objective of the current proposal is to determine the molecular mechanism by which CUX1 dosage regulates HSPC fate. To address this, we have now generated a novel CUX1-reporter mouse to measure CUX1 protein levels at the single-cell level during development. In preliminary data, we demonstrate that CUX1 is expressed highly in HSPCs while exhibiting heterogeneity within stem and progenitor compartments. We show that CUX1 levels correlate with hematopoietic stem cell (HSC) activity, with CUX1dim HSCs demonstrating higher multilineage, long-term hematopoiesis compared to CUX1bright HSCs. We show that CUX1 interacts with the SWI/SNF chromatin remodeling complex to regulate chromatin accessibility in primary human HSPCs. Thus, our central hypothesis is that CUX1 regulates HSC fate via dosage-dependent opening of lineage-specific enhancers through recruitment of SWI/SNF. We test this hypothesis with two Specific Aims. Aim 1: Overall hypothesis – CUX1 upregulation is necessary for HSC differentiation. In a series of in vivo experiments in mice and complementary assays with primary human cells, we will correlate CUX1 protein levels with HSC functions, including proliferation, self-renewal, and differentiation. We will perturb CUX1 levels to assess how CUX1 dosage impacts these functions. Aim 2: Overall hypothesis – CUX1 upregulation drives enhancer priming for differentiation. To determine how CUX1 impacts chromatin remodeling and enhancer activation, we will leverage cutting-edge functional genomics approaches and single-cell methodologies in HSCs using our CUX1-reporter and CUX1-knockdown mice and primary human HSPCs. Accomplishing the proposed studies will elucidate the critical epigenetic role for dosage-sensitive CUX1 transcriptional regulation in hematopoietic development. This work is critical to achieve our long-term goal of identifying therapeutic interventions for patients with CUX1 haploinsufficiency.