RUNX3 overexpression inhibits normal human erythroid development

Abstract

RUNX proteins belong to a family of transcription factors essential for cellular proliferation, differentiation, and apoptosis with emerging data implicating RUNX3 in haematopoiesis and haematological malignancies. Here we show that RUNX3 plays an important regulatory role in normal human erythropoiesis. The impact of altering RUNX3 expression on erythropoiesis was determined by transducing human CD34+ cells with RUNX3 overexpression or shRNA knockdown vectors. Analysis of RUNX3 mRNA expression showed that RUNX3 levels decreased during erythropoiesis. Functionally, RUNX3 overexpression had a modest impact on early erythroid growth and development. However, in late-stage erythroid development, RUNX3 promoted growth and inhibited terminal differentiation with RUNX3 overexpressing cells exhibiting lower expression of glycophorin A, greater cell size and less differentiated morphology. These results suggest that suppression of RUNX3 is required for normal erythropoiesis. Overexpression of RUNX3 increased colony formation in liquid culture whilst, corresponding RUNX3 knockdown suppressed colony formation but otherwise had little impact. This study demonstrates that the downregulation of RUNX3 observed in normal human erythropoiesis is important in promoting the terminal stages of erythroid development and may further our understanding of the role of this transcription factor in haematological malignancies.

Increased expression of RUNX3 inhibits normal human myeloid development

“…We next studied whether RUNX3 knockdown (KD) affected myeloid cell development using 3 different shRNA constructs. As previously reported, RUNX3 expression was successfully reduced by approximately 50% in sorted HSPC (day 3) [ 12 ]. RUNX3 KD did not impact the myeloid growth and development of human HSPC (Supplementary Fig.…”

“…A retroviral vector co-expressing RUNX3 and Discosoma sp. red fluorescent protein (DsRed) was generated by directional cloning of RUNX3 (NM_001031680.2) into Bam H1/ Eco R1 sites of a PINCO vector modified to express DsRed [ 11 , 12 ]. PINCO co-expressing RUNX1::ETO and green fluorescent protein (GFP) was also employed in this study [ 13 ].…”

“…Human neonatal cord blood was obtained from the Maternity Unit of the University Hospital of Wales (Cardiff) in accordance with the 1964 Declaration of Helsinki. Human CD34 + HSPC were isolated, cultured and transduced with retro/lentivirus as previously described (Supplemental Materials and Methods) [ 12 , 13 ]. Following infection (day 3 of culture), cells were maintained in bulk liquid culture for growth and differentiation assessment by flow cytometry (as below).…”

“…To assess myeloid cell growth and differentiation in bulk liquid culture, human HSPC were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; Fisher Scientific, Loughborough, UK) supplemented with IL-3, SCF, G-CSF and GM-CSF (BioLegend, London, UK) at 5 ng/mL for 13 days. Transduced cultures were analyzed by flow cytometry for cell surface markers expression using a BD FACSCanto™II (Supplemental Materials and Methods) as previously described [ 12 , 14 ]. The gating strategy employed in these studies is shown in Supplemental Fig.…”

“…Myeloid colony assays were performed on day 3 using HSPC FACSorted for DsRed or GFP positivity (or both in case of double transduced cells expressing RUNX3 and RUNX1::ETO) using a BD FACSAria™III (BD Biosciences, Wokingham, UK). Sorted HSPC were plated by limiting dilution in 96-U plates (0.3 cells/well) in IMDM supplemented with IL-3, SCF, G-CSF, and GM-CSF at 5 ng/mL and incubated at 37 °C with 5% CO 2 [ 12 ]. Following 7 days of growth, individual myeloid colonies (>50 cells) and clusters (>5, <50 cells) were counted and scored.…”