Decoding Transcription Factor Dependent Enhancer-Gene Regulatory Networks That Define Hematopoietic Niche Function

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for patients with hematologic malignancies, including many cases of acute leukemia. The bone marrow sinusoidal vascular niche plays a crucial role in hematopoietic stem cell (HSC) engraftment and the regeneration of hematopoiesis following transplantation. However, current pre-transplantation conditioning regimens, which typically involve high-dose chemotherapy, can cause significant short- and long-term toxicity to the bone marrow niche. Thus, the repair and regeneration of the sinusoidal vascular niche are critical for hematopoietic recovery after HSCT. Notably, the expression of Notch signaling genes, such as Jagged-1 and Jagged-2, within the sinusoidal vascular niche has been shown to support hematopoietic regeneration after myelosuppression. Despite this, the role of enhancers in Notch signaling and the regulation of this process by transcription factors (TFs) remain areas requiring further exploration.

To address this, we conducted multimodal single-nuclei RNA and ATAC sequencing on both zebrafish embryos and human umbilical vein endothelial cells engineered to express the adenovirus E4ORF1 gene (E4-HUVECs). The E4-HUVECs are an off-the-shelf product currently in phase 3 clinical trials aimed at improving engraftment after HSCT (NCT05181540). Our study aimed to provide a detailed characterization of gene regulatory networks (GRNs) and sinusoidal niche interactions within the BM microenvironment.

Our analysis included 35,798 zebrafish embryonic cells at 3 days post-fertilization (dpf), which were classified into four major niche clusters: sinusoidal, mesenchymal stem cell (MSC), osteoblast, and fibroblast. To comprehensively identify GRNs involved in sinusoidal niche hematopoietic recovery, we performed the same experiment on E4-HUVECs, which support the expansion and differentiation of HSPCs into mature precursor cells. GSEA revealed a conserved transcriptional state between zebrafish sinusoidal niche cells and E4-HUVECs. Specifically, there was significant enrichment of zebrafish sinusoidal differentially expressed genes (DEGs) in E4-HUVECs sinusoidal endothelial cells (normalized enrichment score [NES] 1.57; adjusted p = 3.2e-06). Key conserved target genes included NOTCH1, DLL4, and JAG2, supporting previous studies on the role of Notch signaling and its ligands in the recovery of the sinusoidal vascular niche.

To further examine the regulatory elements of these conserved genes, we analyzed chromatin accessibility and motif enrichment in both zebrafish and E4-HUVECs, focusing on the conserved sinusoidal gene set. In the differentially accessible regions, we identified 3,273 promoters and enhancers in E4-HUVECs associated with 155 conserved genes and 7,419 promoters and enhancers in zebrafish associated with 224 conserved genes. Motif analysis predicted the binding of 31 TFs within these regions, including Fli-1 and Rel, which were expressed in both human and zebrafish cells. Notably, Fli-1 and Rel were predicted to bind enhancers of NOTCH1, DLL4, and JAG2. We used Cut and Tag on E4-HUVECs with antibodies against Fli-1 and Rel to map the binding sites of Notch/Notch ligands.

We further constructed an mClover reporter driven by a putative dll4 enhancer sequence and an empty vector as a control group, then microinjected it into zebrafish embryos. Sinusoidal-specific fluorescence was observed in the CHT region of 57% of injected zebrafish embryos at 72 hpf compared to the control group. This finding indicates that the putative dll4 enhancer is sufficient to drive dll4 expression as a cis-regulatory element, with potential implications for hematopoietic niche recovery.

In summary, our data demonstrate the function of the dll4 enhancer and the role of Fli-1 and Rel in the GRNs that activate Notch/Notch ligands within the hematopoietic niche. Understanding how these sinusoidal vascular niche Notch ligand GRNs facilitate hematopoietic regeneration may guide the development of novel therapeutics to enhance hematopoietic recovery after HSCT.