There is a worldwide shortage of matched donors for blood stem cell transfer of leukaemia or lymphoma patients. The generation of blood cells upon in vitro differentiation of embryonic stem (ES) cells or induced pluripotent stem (iPS) cells could represent a powerful approach to generate the autologous cell populations required for these transplantations. In this context, it is important to further understand the development of blood cells.
Our finding that RUNX1/AML1 is critical for generation of haematopoietic cells, suggests that RUNX1 regulates the expression of a set of genes critical for the development of the haematopoietic system from the haemogenic endothelium. Through comparison of gene expression in cell populations generated from either Runx1 deficient or Runx1 competent ES cells, we identified the Gfi1 and Gfi1b genes as direct targets of RUNX1. Gfi1 and Gfi1b genes encode two highly homologous nuclear zinc finger proteins that function as transcriptional repressors. A C-terminal domain containing six C2-H2-type zinc finger motifs mediates the DNA binding activity of these proteins while their repressional activity requires the N-terminal SNAIL/GFI-1 (SNAG) domain. GFI1 deficiency leads mainly to neutropenia and reduction in self-renewal capacity of the haematopoitic stem cells. In contrast, its paralogue Gfi1b is mostly expressed in erythroid and megakaryocytic lineages and Gfi1b knockout results in embryonic lethality at E14.5 due to a deficiency in erythroid and megakaryocyte development. Several studies have shown that the expression of Gfi1 and Gfi1b are controlled by negative feedback loops and that GFI1 and GFI1b can repress expression of each other in a cross regulatory fashion.
We first validated the differential expression of both Gfi1 and Gfi1b in the presence/absence of RUNX1 and then demonstrated a direct binding of RUNX1 to these loci by chromatin immunoprecipitation (ChIP) and Chip-seq. To investigate the potential functions of GFI1 and GFI1b in the endothelial to haematopoietic transition, we evaluated their ability to rescue defects observed in Runx1-/- differentiation cultures. We demonstrated that in the absence of RUNX1, GFI1 and GFI1B are able to trigger the loss of endothelial identity of haemogenic endothelium. Upon Gfi1 or Gfi1b expression, these cells down-regulate the expression of endothelial genes and undergo the morphological changes observed in the transition from endothelium adherent cells into haematopoietic round cells. However these cells are unable to acquire the full competence to generate haematopoietic colonies. Conversely, we established that fully committed blood progenitors generated in Gfi1 and Gfi1b double knockout embryos maintain the expression of endothelial genes and cannot be released from their cell layer within the yolk sac and therefore fail to disseminate into embryonic tissues. Taken together, our findings strongly suggest a new and unexpected role of GFI1 and GFI1B transcription factors in mediating the loss of endothelial identity in the generation of haematopoietic progenitors from the haemogenic endothelium.
Previous studies have indicated a critical requirement for RUNX1 at the onset of haematopoietic development and that RUNX1/AML1 is expressed as multiple, naturally occurring spliced isoforms that generate proteins with distinct activities on target promoters. Deletion of Runx1 in adult haematopoietic cells results in significant defects, including an expansion of peripheral blood monocytes and granulocytes, a reduction in lymphocytes and thrombocytopenia. Stringent regulation of Runx1 expression during haematopoiesis is therefore vital. The transcription of Runx1 is under the control of 2 promoters: the Distal (P1) and Proximal (P2) promoters, encoding isoforms RUNX1C and RUNX1B respectively. RUNX1B and RUNX1C differ solely in their N-terminal domains, RUNX1C being 14 amino acids longer and beginning with MASDS and RUNX1B beginning with MRIPV.
To study their expression during adult haematopoiesis, we utilised a dual P1-GFP/P2-hCD4 reporter mouse line to sort adult haematopoietic cell populations by flow cytometry and investigate their haematopoietic potential. We found that P1-GFP is expressed in all Lin-cKit+ progenitors as well as mature macrophages, granulocytes, B cells, subsets of T cells and immature erythroblast populations. By comparison, P2-hCD4 expression is highly heterogeneous, being upregulated in subsets of Lin-cKithigh Sca1high (LSK) CD48+ progenitors and in lymphoid and granulocyte-monocyte (GM) progenitors. The pre-megakaryocyte-erythroid progenitor (PreMegE) can also be separated into P2-hCD4- “pro-erythroid” and P2-hCD4+ “pro-megakaryocyte” subsets.
To elucidate the functional roles of the Runx1 isoforms, we utilised P1 null mouse models. The absence of RUNX1C results in aberrant T cell development, reduced long-term competitive repopulation and an alteration of myelo-lymphoid potential in engrafted bone marrow cells. These data support a hypothesis of distinct roles for the two RUNX1 isoforms during adult haematopoiesis.
Figure 1: Model of Activities of P1 and P2 Promoters in Adult Haematopoiesis.