A GATA-2-GPR65 Regulatory Module Controls Hematopoietic Stem Cell Generation
Authors: Gao Xin , Wu Tongyu , Lahvic Jamie , Johnson Kirby D. , Ranheim Erik A. , Zon Leonard I. , Bresnick Emery H 
Published: October 2019

Abstract
The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. We demonstrated that a deletion of a Gata2 cis-element reduced GATA-2 levels in the AGM and abrogated the capacity of hemogenic endothelium to generate HSCs. Consistent with the defective HSC generator, the mutant fetal livers were deficient in hematopoietic stem and progenitor cells (HSPCs). Using an ex vivo intact AGM culture system, we demonstrated that retrovirus-mediated GATA-2 expression in the +9.5-/- AGM rescues its hematopoietic defect. Thus, the reduced GATA-2 levels in the +9.5-/- AGM cause the HSC generation defect, and this rescue assay provides a unique system to decipher the downstream genetic network. To discover novel druggable regulators in the GATA-2 pathway to promote HSC generation, we profiled the expression pattern of all G-protein-coupled-receptors, which represent the most successful class of pharmaceutical targets, in the AGM using our RNA-seq dataset (+9.5+/+ vs. +9.5-/- AGM). This global GPCR analysis revealed four GATA-1 and GATA-2 co-regulated genes, Adora3, Gpr65, Ltb4r1, and Adora2b. Database mining revealed that only the Gpr65 expression pattern resembled that of Gata2. To evaluate GPR65 functions during HSC generation, we conducted an shRNA-based loss-of-function analysis in the AGM. While downregulating Gpr65 did not alter the abundance of the CD31+ c-Kit+ hematopoietic cell population, it significantly increased the CD31+ c-Kit+ Sca1+ HSC-containing cell population (1.4 fold, p<0.05), indicating that GPR65 suppresses HSC generation. To validate the involvement of GPR65 during the HSC generation process in vivo, we conducted a morpholino oligonucleotide (MO)-based loss-of-function study in zebrafish. In situ hybridization analysis revealed high Runx1/c-Myb expression (labeling definitive HSCs and progenitors) in 48% of embryos injected with Gpr65 MOs compared with 11% of wild type embryos. Consistent with the ex vivo AGM analysis, this increase in Runx1/c-Myb expression upon Gpr65 MO treatment suggests GPR65 is a negative regulator of HSC emergence in vivo. To dissect the molecular mechanism governing GPR65-suppressed HSC generation, we tested whether lowering Gpr65 levels altered the expression of key HSC regulators. Quantitative RT-PCR analysis revealed that downregulating Gpr65 by 60-70% in AGM CD31+ c-Kit- endothelialcells increased Gata2 mRNA by 2.9 fold (p<0.05), Gata2 primary transcripts by 3.9 fold (p<0.05), and elevated expression of the GATA-2 target gene Runx1 (2.9 fold, p<0.05). These results support a mechanism whereby GPR65-mediated Gata2 repression is an important determinant of GPR65-suppressed HSC generation. In addition to this important function in the AGM, Gpr65 knockdown studies in primary fetal liver HSPCs revealed GPR65 suppression of Gata2 transcription to the same magnitude as in the AGM. To determine if GPR65-mediated Gata2 repression requires the +9.5 site, we infected freshly isolated HSPCs from fetal livers heterozygous for the +9.5 site with retrovirus expressing shRNA targeting Gpr65. Quantitative RT-PCR with allele-specific primers revealed that Gpr65 knockdown significantly upregulates Gata2 primary transcripts from the wild type (3.1 fold, p<0.01), but not the 9.5 mutant, allele. These results establish a requirement of the +9.5 site for GPR65 to repress Gata2 transcription. As we reported that SetD8, the only enzyme known to monomethylate H4K20, represses Gata2 expression via the +9.5 site, we tested whether GPR65 represses Gata2 expression through SetD8. H4K20me1 ChIP revealed that downregulating Gpr65 significantly reduces H4K20me1 levels at the +9.5 site by 30% (p<0.005), suggesting that GPR65 repression of Gata2 transcription involves SetD8. Our studies indicate that a G-protein coupled receptor, GPR65, is negative regulator of HSC generation and establish a GATA-2-GPR65 Type Iincoherent feedforward loop that controls HSC generation, providing a foundation to develop new targets for expanding HSCs for transplantation therapies and a new druggable target to treat hematologic disorders.

Disclosures
Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.