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.