Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate.

The hematopoietic niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses, we define a conserved gene expression signature and cis -regulatory landscape unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code involving members of the Ets, Sox and Nuclear Hormone Receptor families that is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance and division of HSPCs in vivo . These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo , and for effective therapies to modulate the endogenous niche.

Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-β-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli.

The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress. Santoriello, Sporrij et al. show that the progesterone receptor associates with RNA helicase DDX21 during nucleotide depletion, promotes its binding on chromatin and rescues efficient transcription in melanoma cells.

A healthy blood system is maintained by a diverse set of hematopoietic stem cell (HSC) clones. Transplantation of HSCs introduces a bottleneck reducing the number of clones, potentially leading to adverse outcomes. Prostaglandin E2 (PGE2) was previously identified as a potent enhancer of HSC function and has showed promise in clinical trials. We now used mouse competitive transplants to elucidate how PGE2 affects clonal dynamics. After exposure to a two hour pulse of PGE2 2500 LSK cells (Lineage-, Sca1+, cKit+) were competitively transplanted into lethally irradiated recipients. A transposon based barcoding system allowed to track clonal diversity of donor cells. Eight months post transplant donor derived LSK cells in the bone marrow were compared to donor derived peripheral blood (PB). The total number of detected LSK clones was similar between PGE2 (33 ± 20) and control (31 ± 19). However, within PGE2 treated LSKs 32% (±11%) of clones were active compared to 16% (±4%) in the control. In addition an increased overlap of barcodes between myeloid and lymphoid cells indicated that PGE2 treated LSK cells are twice as multipotent (p < 0.05) compared to control. In an independent, limit dilution transplant experiment barcodes were analyzed in single sorted PB granulocytes. PGE2 leads to more balanced clone sizes, with no single clone making up more than 25% of granulocytes sampled. In contrast both control samples had dominant clones making up more than 80% of PB granulocytes. To investigate the underlying mechanism we performed single cell RNAseq on CD150+, CD48- SLAM-LSK cells. PGE2 treated SLAM-LSKs cells form a distinct cluster that is located within the more primitive, quiescent SLAM-LSK cells. Selected cell cycle transcripts as well as immediate early genes such as Fos are enriched in this PGE2 specific cluster. In our current working model PGE2 activates immediate early genes and their downstream targets in a larger proportion of cells enhancing engraftment of a more diverse set of clones. Unravelling PGE2s effect on HSCs will further current understanding of basic HSC biology and add to clinical applications targeted at improving hematopoietic transplants.

The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine cxcl8 and its receptor, cxcr1, are expressed by zebrafish endothelial cells, and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC–endothelial cell “cuddling,” HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression. Finally, using parabiotic zebrafish, we show that cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.