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.

Single Nucleotide Polymorphisms (SNPs) identified through genome-wide association studies (GWAS) provide insight into the mechanism of human genetic diseases, and majority of functional GWAS mutations target genomic regulatory elements. During erythroid differentiation of human CD34+ cells, we mapped regulatory DNA elements (enhancers and open chromatin regions) by H3K27Ac ChIP-seq and ATAC-seq, and studied the SNPs that reside within these DNA regulatory elements. We followed genomic binding of lineage restricted GATA transcription factors and also chose to examine the binding of the BMP signal responsive transcription factor SMAD1 in CD34+ cells during erythropoiesis. By overlapping their genomic occupancy with stage-matched RNA-seq, we found that SMAD1, in association with GATA-factors, serves as marker of genes responsible for differentiation at every step of erythropoiesis. ChIP-seq for other crucial signaling transcription factors, such as WNT-responsive and TGFb-responsive factors (TCF7L2 and SMAD2, respectively) demonstrated a remarkable co-existence of such factors at GATA+SMAD1 co-bound regions nearby stage-specific genes. We defined such regions as “Transcriptional Signaling Centers (TSC)” where multiple signaling transcription factors converge with master transcription factors to determine optimum stage-specific gene expression in response to growth factors. Our bioinformatics-algorithms demonstrated that PU1 and FLI1 binding sites were present in progenitor-specific TSCs whereas KLF1 and NFE2 sites were enriched in TSCs of red blood cells. We performed CRISPR-CAS9 mediated perturbations of each of the PU1, GATA and SMAD1 motifs separately in a representative progenitor TSC in K562 and HUDEP2 cells. Similar to loss of PU1 and GATA motifs, loss of SMAD1 motif selectively inhibited expression of the associated gene and showed defects in erythroid differentiation, demonstrating that TSCs are important to provide optimum gene expression and proper erythroid differentiation. To determine if such TSCs are targeted by GWAS mutations, we have studied 1270 lead and additional 27,799 SNPs in linkage disequilibrium with lead SNPs that are associated with six critical red blood cell traits - hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). Surprisingly, we observed that, out of the 3831 “functional” SNPs that fall within non-exonic H3K27Ac enhancers, while only 5% (188) of RBC-SNPs target only blood-master-transcription-factor motifs, at least 48% (1821) of them reside on various signaling pathway associated transcription factor motifs including SMADs (BMP/TGFb signaling), RXR/ROR (nuclear receptor signaling), FOXO/FOXA (FOX signaling), CREBs (cAMP signaling) and TCF7L2 (WNT signaling). Additionally, these RBC-trait-SNPs are specifically enriched in GATA+SMAD1 co-bound TSCs and fall within signaling factor binding sites. We validated such SNPs that target SMAD-motifs. The SNP rs9467664 is associated with the MCV-trait near HIST1H4A, a gene that increases in expression during differentiation. Using gel-shift assay, we found that SMAD1 binding is compromised when the major allele T changes to minor allele A under MCV-trait. Remarkably, eQTL analysis using microarray gene expression profiles of peripheral blood obtained from the Framingham Heart Studies revealed that expression of HIST1H4A is significantly more in a population with T-allele than that with A-allele. This demonstrates that inhibition of SMAD1 binding by the SNP causes a loss of allele-specific HIST1H4A expression. Another MCV-associated SNP rs737092 targets a SMAD motif within an erythroid-specific TSC near RBM38 gene. T-allele, in comparison with C-allele, that retains SMAD1 binding showed more expression in luciferase-based reporter assays specifically under BMP stimulation suggesting that rs737092 compromise BMP-responsiveness. Taken together, our study provides the first evidence that naturally occurring GWAS variations directly impact gene expression from signaling centers by modulating binding of signaling transcription factors under stimulation. Such aberrant signaling events over time could lead to “signalopathies”, ultimately resulting in phenotypic variations of RBC traits.

Single Nucleotide Polymorphisms (SNPs) identified through genome-wide association studies (GWAS) could provide insight into the mechanism of human genetic diseases. Here we have studied SNPs that are associated with six critical red blood cell traits - hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). During erythroid differentiation of human CD34+ cells, we mapped enhancers and open chromatin regions by H3K27Ac ChIPseq and ATACseq, and studied the SNPs that reside within these DNA regulatory elements. We followed genomic binding of lineage restricted GATA transcription factors and BMP signal responsive transcription factor SMAD1 in CD34+ cells during erythropoiesis. By overlapping their genomic occupancy with stage-matched RNAseq, we found that SMAD1, in association with GATA-factors, serves as marker of genes responsible for differentiation at every step of differentiation. ATACseq and H3K27Ac patterns demonstrated that GATA+SMAD1 co-occupied regions correlate with open chromatin and super enhancers at every stage, whereas GATA-only regions are associated with genes with low/basal level of expression during differentiation. ChIPseq for other crucial signaling transcription factors, such as cAMP-responsive and TGFb-responsive factors (CREB and SMAD2, respectively) demonstrated a remarkable co-existence of such factors at GATA+SMAD1 co-bound regions nearby stage-specific genes. We defined such regions as “signaling centers” where multiple signaling transcription factors converge with master transcription factors to determine optimum stage-specific gene expression in response to growth factors. Surprisingly, we observed that while only 15% of RBC-SNPs target blood-master-transcription-factor motifs, at least 70% of them reside on various signaling pathway associated transcription factor motifs including SMADs (BMP/TGFβ signaling), RXR/ROR (nuclear receptor signaling), FOXO/FOXA (FOX signaling), CREBs (cAMP signaling) and TCF7L2 (WNT signaling). Our bioinformatics-algorithms demonstrated that, in contrast to GATA-only sites, SMAD1+GATA co-bound signaling centers harbor cis -acting motifs and display enriched binding of cell-type specific transcription factors (e.g. PU1 and FLI1 in progenitor vs. KLF1 and NFE2 in differentiated cells). Such distinct identities of signaling centers could serve as codes to distinguish progenitor-specific genes from erythroid-specific genes, and govern their stage-specific expression. We performed CRISPR-CAS9 mediated perturbations of each of the PU1, GATA and SMAD1 motifs separately in a representative progenitor signaling center in K562 cells. Similar to loss of PU1 and GATA motifs, loss of SMAD1 motif selectively inhibited expression of the associated gene. This suggests a signaling factor SMAD1 is important within signaling centers to obtain optimum gene expression. Moreover, a progenitor factor PU1 direct binding of SMAD1 to progenitor-specific signaling centers since with overexpression of PU1 in K562 cells, SMAD1 occupancy was concomitantly increased in selective genomic regions where PU1 binding was increased. More than 80% of the RBC-trait-SNPs are enriched within SMAD1-bound signaling centers. Such SNPs either destroy or create new signaling factor binding sites, e.g. SMAD motifs. We validated one such SNP associated with the MCV-trait near HIST1H4A, agene that increases in expression during differentiation. Using gel-shift assay, we found that SMAD1 binding is compromised when the major allele T changes to minor allele A under MCV-trait. Remarkably, eQTL analysis using microarray gene expression profiles of peripheral blood obtained from the Framingham Heart Studies revealed that expression of HIST1H4A is significantly more in a population with T-allele than that with A-allele. This demonstrates that inhibition of SMAD1 binding by the SNP causes a loss of allele-specific HIST1H4A expression. Taken together, our study provides the first evidence that naturally occurring GWAS variations directly impact gene expression from signaling centers by modulating binding of signaling transcription factors. Such aberrant signaling events over time could lead to “signalopathies”, ultimately resulting in phenotypic variations of RBC traits.

Angiopoietin-like proteins (angptls) are capable of ex vivo expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs). Despite this intriguing ability, their mechanism is unknown. In this study, we show that angptl2 overexpression is sufficient to expand definitive HSPCs in zebrafish embryos. Angptl1/2 are required for definitive hematopoiesis and vascular specification of the hemogenic endothelium. The loss-of-function phenotype is reminiscent of the notch mutant mindbomb (mib), and a strong genetic interaction occurs between angptls and notch. Overexpressing angptl2 rescues mib while overexpressing notch rescues angptl1/2 morphants. Gene expression studies in ANGPTL2-stimulated CD34(+) cells showed a strong MYC activation signature and myc overexpression in angptl1/2 morphants or mib restored HSPCs formation. ANGPTL2 can increase NOTCH activation in cultured cells and ANGPTL receptor interacted with NOTCH to regulate NOTCH cleavage. Together our data provide insight to the angptl-mediated notch activation through receptor interaction and subsequent activation of myc targets.

During hematopoietic differentiation there is a rapid turnover of cell stages. Our goal is to delineate how signal-responsive transcription factors get integrated in lineage choices. Thus, we followed CD34+ hematopoietic stem and progenitor cells (HSPCs) during erythropoiesis. Our previous work demonstrated that the signal-responsive factor SMAD1 co-localizes with the lineage regulators GATA2 and GATA1 at lineage specific genes in HSPCs. To discover how SMAD1 is integrated in lineage choices we performed ChIP-seq and expression analysis for GATA2, GATA1, SMAD1 and H3K27ac in HSPCs during erythroid differentiation. Expression analysis indicated that there is one major global gene expression transition during days 3-4 of differentiation. This transition is further verified by the binding events during differentiation. Indeed at day4 GATA2 is down-regulated and GATA1 replaces GATA2 in some genomic regions. These data indicate the existence of a single time point that HSPCs commit to an erythroid fate wherein dramatic changes in transcription factor binding and expression occur. Before this transition, more than 70% of GATA2 bound genes remain unchanged and after the transition, more than 70% of the GATA1 bound genes remain stably bound. In contrast, the SMAD1 binding profile differed greatly at each time point with less than 30% similarity between adjacent days along the differentiation time-course. Genomic loci bound by both GATA 1 or 2 and SMAD1 showed better correlation with H3K27ac and gene expression than genomic loci that GATA binds alone. Our results demonstrate that, although lineage regulators like GATA1 and GATA2 sculpt the transcriptional landscape that will determine cell fate decisions, fine tuning of these decisions is aided by signaling transcription factors.

The angiopoietin-like proteins (angptls) are novel growth factors that are capable of stimulating expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs) ex vivo. Although the receptor for some of the angptl family members is known, their molecular mechanism of action is undefined. Here, we show that overexpression of angptl2 in a stable heatshock inducible transgenic zebrafish line, Tg(hsp70:zangptl2), is sufficient to increase cmyb- and runx1-positive HSPCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis. Anti-sense morpholino knockdown of angptl1 and 2 resulted in a significant decrease in cmyb- and runx1-positive HSPCs in the AGM, suggesting that angptls are required for definitive hematopoiesis. These double morphants also displayed severe disruption in vascular development and differentiation prior to the defects observed in the AGM, indicating that angptl regulation of HSPC development occurs through an early specification of the hemogenic endothelium. The loss of function phenotypes in the developing aorta is reminiscent of mutant fish with defective notch signaling (mindbomb) and a tight genetic interaction occurs between angptl and notch signaling. Knocking down angptl1 and 2 decreased notch signaling in a transgenic notch reporter line while overexpression of a constitutively active intracellular notch rescues the angptl double morphant phenotype. These data imply that angptls function upstream of notch signaling. Interestingly, the absence of HSPCs and notch signaling in the mindbomb mutants are rescued by overexpression of angptl2 in Tg(hsp70:zangptl2), suggesting that angptls can regulate notch signaling. To examine the molecular mechanism of Angptl-mediated Notch activation, we stimulated cultured human CD34+ cells or endothelial cells with purified Angptl2 and observed a rapid increase in Notch receptor cleavage indicating that Angptl2 can induce Notch activation. Furthermore, we found through endogenous co-immunoprecipitation experiments that the Angptl receptor, LILRB2, interacted with Notch receptor. This strongly points to a direct regulation of Notch activation/signaling by Angptls through physical interactions between Notch and Angptl receptors. Previously, we found that angptl-mediated akt activation is important for HSPC formation in the AGM. To examine the role of Akt during Notch activation, we immunoprecipitated all Akt-phosphorylated substrates in Angptl-stimulated cells and found the presence of ADAM17/TACE, one of the cleavage enzymes that is crucial to Notch receptor activation. Together these results suggest a model in which Angptl-binding of the LILRB2 receptor enables recruitment of downstream molecules such as Akt proximal to Notch, allowing for subsequent cleavage and activation of Notch receptor. Finally, to examine downstream signaling of Angptl-mediated Notch activation, we performed chromatin immunoprecipitation for Notch followed by sequencing in Angptl2-stimulated CD34+ cells and found enrichment for Myc binding elements. Independent microarrays also revealed a strong Myc signature through gene set enrichment analysis. Thus, to confirm results from our bioinformatics analyses, we overexpressed zebrafish myc in angptl1 and 2 double morphants or mindbomb mutants, and found a significant rescue in HSPCs formation in the AGM. Collectively with these results, we propose that angptl can regulate notch signaling through receptor interaction, leading to activation of myc target genes during definitive hematopoiesis. Our data provide new insights to the previously uncharacterized Angptl signaling during HSPC development and present a novel mechanism of action for Notch activation. Disclosures: Aster: Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria. White:N-of-One: Consultancy, Equity Ownership. Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Slow hematopoietic recovery following irradiation can result in adverse complications for many patients, but the genetic basis of recovery is ill defined. Using a zebrafish model of irradiation-induced hematopoietic regeneration, we defined differences in recovery between two commonly used wild type strains (AB and Wik) and mapped quantitative trait loci (QTL) that govern these differences. Regeneration was assayed by measuring the frequency of multilineage precursors, which are the earliest hematopoietic population detectable following irradiation injury in the zebrafish. Wik fish had a higher precursor frequency (mean-35) at 7 days post irradiation (dpi) than AB fish (mean-30) indicating Wik regenerate faster. To establish the genetic determinant underlying the difference in hematopoietic regeneration, AB and Wik fish were crossed to generate F2 ABXWik hybrid animals for mapping. The F2 animals displayed a range of precursor frequencies from 4.5 to 52.9 at 7dpi. Interval mapping was performed on the fish with the 10% highest and lowest precursor frequency. Initial mapping performed with over 200 single nucleotide polymorphisms distributed across the genome indicated significant linkage on Chromosome 3 (LOD-3.258), which was then confirmed by analyses in F3 progeny. Whole genome sequencing was employed to further refine the genetic interval to a 20 Mbp region. The genes within this QTL (136) were then compared to 132 genes found in a mouse QTL (Scp2) on Chromosome 11 associated with differences between HSC proliferation in C57/Bl6 and DBA.2 mice. Four out of the 12 common genes tested displayed differential gene expression between AB and Wik fish, suggesting that variable expression in multiple genes likely contribute to fluctuations in regeneration. This study is one of the first to use zebrafish genetics to define factors that control natural fluctuations in regenerative potential and can provide targets for therapies to boost hematopoietic output during times of stress.

The zebrafish has become a commonly used model for studying hematopoiesis as a result of its unique attributes. Zebrafish are highly suitable for large-scale genetic and chemical screens compared to other vertebrate systems. It is now possible to analyze hematopoietic lineages in zebrafish and validate cell function via transplantation assays. Here, we review advancements over the past decade in forward genetic screens, chemical screens, fluorescence-activated cell sorting analysis, and transplantation assays. Integrating these approaches enables new chemical and genetic screens that assay cell function within the hematopoietic system. Studies in zebrafish will continue to contribute and expand our knowledge about hematopoiesis, and develop novel treatments for clinical applications.