A novel dll4 enhancer regulates sinusoidal niche-mediated HSC proliferation and maintenance

Abstract The sinusoidal vascular niche plays a critical role in maintaining hematopoietic stem and progenitor cells (HSPCs), and its dysfunction is associated with bone marrow failure, poor transplant engraftment, and impaired immune recovery. Although Notch signaling is known to influence HSPC fate, the cis-regulatory mechanisms controlling niche-specific expression of Notch ligands remain largely uncharacterized. Using single-cell multiome (scRNA-seq and scATAC-seq) analysis in zebrafish and human umbilical vein endothelial cells engineered to express adenoviral E4-ORF1 (E4-HUVECs), we identified a conserved transcriptional network centered on Notch pathway components, including dll4, notch1a, and jag2a. Motif analysis of accessible chromatin regions revealed enrichment for REL, KLF3, and FLI1 binding sites within this network. We identified a conserved enhancer located 1.7 kb downstream of the dll4 with specific activity in sinusoidal endothelial cells. CUT&Tag confirmed the presence of active enhancers (H3K27ac) in both zebrafish and E4-HUVECs. CRISPR-Cas9 editing of this enhancer in runx1:mCherry zebrafish, which label HSPCs, led to a 76.7% reduction in HSPC numbers (p.adj < 2.4e-26, n = 118), indicating impaired HSPC maintenance or proliferation. Disruption of REL and KLF3 motifs within the enhancer caused a 73.2% (p.adj < 6.5e-09, n = 31) and 33.7% (p.adj < 2.2e-05, n = 73) reduction respectively, supporting their roles in enhancer function. To address the enhancer's role in establishing niche structure, we edited the enhancer in kdrl:mCherry zebrafish, which marks vasculature. This resulted in disrupted vascular architecture, including hyperbranching and loss of vessel integrity. qPCR revealed a significant decrease in dll4 expression in sorted kdrl⁺ endothelial cells (p.adj < 0.008) after enhancer editing. These results demonstrate that this enhancer controls dll4 expression in endothelial cells, and its loss impairs vascular structure and the sinusoidal niche's ability to support HSPC proliferation. To investigate factors that activate this transcriptional network, we focused on IL-8, a pro-inflammatory cytokine elevated in bone marrow stress, chemotherapy, and myeloablative conditions. IL-8 is known to directly stimulate endothelial cells survival and influence angiogenesis, permeability and migration. CUT&RUN profiling in E4-HUVECs revealed binding of eight predicted transcription factors (NFκB, REL, FLI1, FOXO1, HEY1, STAT4, NFATC2) to the human DLL4 enhancer after IL-8 stimulation. CUT&RUN assays in sorted zebrafish kdrl:mCherry endothelial cells confirmed REL binding to within 20 bp of predicted binding sites, directly implicating REL in enhancer activation specifically in sinusoidal niche. Knockout of REL or FLI1 in E4-HUVECs significantly reduced DLL4 expression (p.adj < 8.7e-06 and p.adj < 4.9e-04 respectively), suggesting these transcription factors may directly regulate Notch signaling in sinusoidal niche. Together, our work identifies a novel gene regulatory network involving rel and dll4 as a key regulators of sinusoidal niche function, essential for both vascular integrity and HSPC maintenance. Modulating this pathway offers a potential therapeutic strategy to improve outcomes in HSC transplantation by restoring endothelial support functions.