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S.G. Kellaway et al.
hematopoietic stem cells10 and is also expressed through- out embryonic hematopoiesis.11 EVI1 is able to bind DNA via ten zinc-fingers, but MDS1-EVI1 additionally contains a proline-rich domain with homology to SET domains.12 RUNX1, EVI1 and MDS-EVI1 have all been associated with cell cycle regulation alongside the control of differen- tiation.13-15
Mice carrying a RUNX1-EVI1 transgene present with disrupted hematopoiesis and with varying degrees of leukemic transformation that is ultimately embryonic lethal.16-18 In a cell line model of t(3;21) it was shown that RUNX1-EVI1 blocks differentiation by binding to chro- matin at both normal RUNX1 binding sites and else- where, co-ordinating a transcriptional network that is dependent on GATA2 rather than RUNX1.19 These stud- ies suggest that RUNX1-EVI1 acts in a dominant negative fashion to RUNX1,20 but has additional effects, likely due to interference with EVI1 binding and interactions.
In order to understand the molecular effects of RUNX1- EVI1 expression in the absence of other mutations, we integrated gene expression and chromatin immunopre- cipitation followed by sequencing (ChIP-seq) data from blood precursor cells derived from a mouse embryonic stem cell line (mESC) in which we induced RUNX1-EVI1 at the onset of hematopoiesis. We show that RUNX1- EVI1 induction leads to a block in the cell cycle and inter- feres with both the EVI1 and the RUNX1 driven develop- mental programs, with cells adopting a multi-lineage gene expression pattern. Moreover, RUNX1-EVI1 orchestrates redistribution and increased binding of endogenous RUNX1, and increases chromatin accessibility at sites enriched in PU.1 motifs. Taken together we show that RUNX1-EVI1 expression is incompatible with normal hematopoietic stem cell function.
Methods
Mouse RUNX1-EVI1 embryonic stem cell line generation RUNX1-EVI1 from the pME18s-RUNX1-EVI1 plasmid (a gift from Kinuko Mitani, Dokkyo Medical University, Japan) was cloned into the p2lox-targeting vector (a gift from Michael Kyba, University of Minnesota). A2lox mESC (a gift from Michael Kyba) were transduced with 20 mg of p2lox-RUNX-EVI1 using the 4D-Nucleofector (Lonza) with the mouse ES program, with
the P3 primary cell kit.
Embryonic stem cell line differentiation
ESC were differentiated as previously described. Briefly, cells were plated into bacterial-grade dishes, after 3.25 days the resulting embryoid bodies were dispersed using TrypLE express (Gibco) to single cells and FLK1+ cells were purified by magnetic cells sorting. These FLK1+ cells were then cultured in gelatin- coated flasks with mouse vascular endothelial growth factor and mouse interleukin 6. After 1 day 0.5 μg/ml doxycycline was added where appropriate and cells cultured for a further 18 hours.
Fluorescence associated cell sorting
Cell populations were identified and sorted on day 2 of blast culture based on surface markers. For experiments including hemogenic endothelium (HE) the floating and adherent cells were pooled. These cells were stained with KIT-APC (BD pharmingen), Tie2-PE (eBioscience) and CD41-PE-Cy7 (eBioscience) and analyzed on a Cyan ADP flow cytometer
(Beckman Coulter) with data analysis using FlowJo, or sorted on a fluorescence associated cell sorting (FACS) Aria cell sorter (BD Biosciences). Progenitors matured in liquid culture were stained with CD11b-PE (eBioscience) and F4/80-APC (eBioscience).
Gene expression analysis
RNA was isolated from sorted cells using the NucleoSpin RNA kit (Macherey-Nagel). RNA sequencing (RNA-seq) libraries were prepared from two biological replicates using the True-Seq strand- ed total RNA kit (Illumina).
DNaseI-sequencing
DNase I hypersensitive sites sequencing (DNaseI-seq) was per- formed as previously described.21 3x105 sorted cells were added directly to DNaseI (Worthington Biochemical Corporation) used between 6 and 13 U/mL for 3 minutes at 22°C. The reaction was terminated by addition of sodium dodecyl sulfate to 0.5% and cell lysates treated with 0.5 mg/mL proteinase K. DNA was isolated by phenol/chloroform extraction and used to generate a library using the KAPA hyper prep kit, according to the manufacturer’s instructions.
Chromatin immunoprecipitation sequencing
ChIP-seq was performed essentially as described.22 KIT+ float- ing progenitor cells were double crosslinked, nuclei prepared22 then sonicated for eight cycles of 30 seconds (s) off using a Picoruptor (Diagenode). Immunoprecipitation was carried out overnight at 4°C, washed and eluted. Extracted DNA was then used to generate a library using the KAPA hyper prep kit.
Data availability
All sequencing data have been deposited at National Center for Biotechnology Information under the number GSE143460. Further methods, including bioinformatic analysis are detailed in the Online Supplementary Appendix.
Results
RUNX1-EVI1 disrupts hematopoietic growth and differentiation
In order to understand the direct effects of RUNX1-EVI1 fusion protein induction on hematopoietic specification we needed to express RUNX1-EVI1 in primary cells in the absence of other mutations. Initial attempts to express the protein in purified human CD34+ cells by retroviral trans- duction were unsuccessful indicating that expressing uncontrolled levels of this protein may be toxic for the cells (unpublished data). In order to circumvent these problems, we generated a mESC line expressing a human RUNX1-EVI1 cDNA under the control of a doxycycline (dox)-inducible promoter (Figure 1A). The RUNX1-EVI1 transgene was derived from the t(3;21) SKH1 cell line23 and comprised amino acids from the N-terminus of RUNX1 translocated to the MDS1-EVI1 isoform (Online Supplementary Figure S1A). We used a well characterized in vitro differentiation system that recapitulates the different steps of embryonic hematopoietic specification in vitro,11,24 namely mesoderm specification into endothelial cells, fol- lowed by endothelial-hematopoietic transition (EHT) which gives rise to multipotent hematopoietic progenitor (HP) cells (Figure 1A, lower panel) during blast culture. The EHT is crucially dependent on the expression of Runx1,6,25 and is also the stage at which Evi1 expression is maximal before being downregulated.11 We therefore
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haematologica | 2021; 106(6)