ETV6 Represses Inflammatory Response Genes and Regulates HSPC Function During Stress Hematopoiesis in Mice

ETS Variant 6 (ETV6) encodes an essential transcriptional repressor abundantly expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with Thrombocytopenia 5 (T5), a poorly-understood genetic condition predisposing to thrombocytopenia and hematologic malignancies. To elucidate how germline ETV6 variants impact the HSPC compartment and contribute to disease, we generated a knock-in mouse harboring an Etv6R355X loss-of-function variant, which represents the mouse equivalent to the T5-associated variant ETV6R359X. All HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice under homeostatic conditions; however, these animals exhibit subtle shifts in the proportions and/or numbers of specific progenitor subtypes. To examine whether the Etv6R355X/+ mutation impacts HSPC function, we carried out serial competitive transplantation and observed that Etv6R355X/+ lineage sca1+cKit+ (LSK) cells exhibit significantly impaired reconstitution, with near complete failure to repopulate irradiated-recipients by the tertiary transplant. Mechanistic studies incorporating CUT&RUN, ATAC-Seq and Hi-C identify ETV6 binding at inflammatory gene loci, including those within the TNF pathway in Etv6+/+ HSPCs, the mouse BM-progenitor derived HPC5 cell line, and G-CSF-mobilized human CD34+ cells. Further, single-cell RNA sequencing of mouse LSK cells isolated six-weeks post-competitive transplantation reveals upregulation of inflammatory gene pathways. Corroborating these findings, we observe significantly increased production of TNF by Etv6R355X/+ versus Etv6+/+ HSPCs post-transplantation. From these studies, we conclude that ETV6 represses inflammatory response genes within HSPCs under conditions of hematopoietic stress, and that this mechanism may be critical to sustain HSPC function.