Levanon, D. | Search bioRxiv

bioRxiv2025

Resolving hematopoietic stem versus progenitor cell potential in the mouse dorsal aorta by differential Runx1 +110 enhancer activity

Hematopoietic stem cells (HSCs) are important in cell-based therapies for blood-related disorders. While progress has been made in the directed differentiation of pluripotent PSCs, such cultures promote hematopoietic progenitor cells (HPCs) over HSCs. Thus, elucidating signals, factors, and markers associated with HSC versus HPC lineage development in the embryo is imperative. During mouse embryonic development, HSCs and HPCs originate from hemogenic endothelium (HE) through a process critically dependent on the transcription factor Runx1. Here, we identified a Runx1 enhancer that distinguishes emerging dorsal aorta HSCs from HPCs. Phenotypic, functional, and transcriptomic analyses of Runx1 +110 enhancer-GFP reporter (110GFP) transgenic embryos showed that 110GFP expression marks HPCs, but not the emerging HSC lineage. Comparative transcriptomics revealed a 17-gene signature associated with in vivo long-term HSC potential. Furthermore, 110GFP- preHSCs showed increased expression of Jarid2 and other PRC2 components, suggesting a role for epigenetic regulation in establishing the HSC fate during EHT. Finally, single-cell multiome analysis of dorsal aorta EHT identified the Runx1 +3 enhancer as preferentially accessible in preHSC and underlined the specific activity of the +110 enhancer in HPCs. Our study demonstrates the power of cell-type specific enhancer-reporter models to dissect cell fate decisions in development and provides new inroads to label and/or perturb HSC versus HPC fate decisions in vivo and in vitro.

developmental biology↗

bioRxiv2019

Runx3 prevents spontaneous colitis by directingdifferentiation of anti-inflammatory mononuclear phagocytes

RUNX3 is one of three mammalian Runt-domain transcription factors (TFs) that regulate gene expression in several types of immune cells. Runx3-deficiency in mice is associated with a multitude of defects in the adaptive and innate immunity systems, including the development of early onset colitis. Our study reveals that conditional deletion of Runx3 specifically in mononuclear phagocytes (MNP) (MNPRunx3-/-) but not in T cells, recapitulates the early onset spontaneous colitis seen in Runx3-/- mice.\n\nWe show that Runx3 is expressed in colonic MNP, including resident macrophages (RM) and the dendritic cell cDC2 subsets and its loss results in impaired differentiation/maturation of both cell types. At the transcriptome level, loss of Runx3 in RM and cDC2 was associated with upregulation of pro-inflammatory genes similar to those in the early onset IBD murine model of RMIl10r-/-. The impaired RM maturation in the absence of Runx3 was associated with a marked decrease in expression of anti-inflammatory and TGF{beta}-regulated genes. Similarly, the decreased expression of {beta}-catenin signaling associated genes in Runx3-deficient cDC2 indicates their impaired differentiation/maturation. Analysis of ChIP-seq data suggests that in both MNP cell types a significant fraction of these differentially expressed genes are high confidence Runx3 directly regulated genes. Interestingly, several of these putative Runx3 target genes harbor SNPs associated with IBD susceptibility in humans. Remarkably, the impaired maturation and pro-inflammatory phenotype of MNP lacking Runx3 was associated with a substantial reduction in the prevalence of colonic lamina propria Foxp3+ regulatory T cells and an increase in IFN{gamma}-producing CD4+ T cells, underscoring Runx3 critical role in establishing tolerogenic MNP.\n\nTogether, these data emphasize the dual role of Runx3 in colonic MNP, as a transcriptional repressor of pro-inflammatory genes and an activator of maturation-associated genes including anti-inflammatory genes. Our study highlights the significance of the current MNPRunx3-/- model for understanding of human MNP-associated colitis. It provides new insights into the crucial involvement of Runx3 in intestinal immune tolerance by regulating colonic MNP maturation through TGF{beta}R signaling and anti-inflammatory functions by Il10R signaling, befitting the identification of RUNX3 as a genome-wide associated risk gene for various immune-related diseases in humans including gastrointestinal tract diseases such as celiac and Crohns disease.

genetics↗