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Leukemic GATA1s delays megakaryocyte differentiation
Introduction
The X-chromosome-encoded hematopoietic transcrip- tion factor GATA1 is essential for normal erythroid and megakaryocytic differentiation.1-3 Clonal mutations acquired in fetal life, leading to loss of the N-terminal 84 amino acids of GATA1, occur in approximately 28% of newborns with Downs syndrome (DS) and are associated with either a clinically overt, or clinically silent, myelo- proliferative disorder known as transient myeloprolifera- tive disorder (TMD).4-7 The mutant truncated GATA1 pro- tein is known as GATA1short or GATA1s. In most neonates with DS the mutant fetal GATA1s clone disap- pears by 3 months of age7 (and Roberts and Vyas unpub- lished data). In approximately 3% of all neonates the TMD clone acquires additional mutations8,9 that trans- form the clone resulting in megakaryoblast-erythroid leukemia known as myeloid leukemia of Down syn- drome (ML-DS). Germline mutations resulting in GATA1s, in disomic individuals and families also cause disease, but rather than being oncogenic cause cytope- nia10 including the clinical phenotype of Diamond- Blackfan anemia.11
In order to begin to understand how GATA1s perturbs hemopoiesis, a mouse model of GATA1s has been stud- ied.12 These mice develop a transient megakaryoblastic myeloproliferative disorder that resolves in utero and like- ly originates from yolk sac hemopoiesis. Interestingly, these mice are anemic in utero leading to embryonic loss. Mice that survive then have minimal hemopoietic defects in adult life. Consistent with this human induced pluripo- tent stem cells (iPSC) derived from GATA1s-expressing TMD cells failed to complete erythropoiesis.13
This suggests that the N-terminal of GATA1 has a spe- cific developmental role in restraining megakaryocyte production and is required for terminal red cell matura- tion. However, it is unclear which developmental hemo- poietic cell populations require the N-terminus of GATA1 and the cellular and molecular mechanisms responsible for perturbed hemopoiesis in GATA1s cells.
In order to identify the cellular populations most per- turbed by GATA1s, we studied hemopoietic differentia- tion from both ESC culture-derived embryoid bodies (that recapitulate yolk sac hemopoiesis) and murine yolk sacs in GATA1s and control wild-type GATA1 mice. We define specific stages in megakaryocyte maturation, where GATA1s megakaryocytic cells are significantly increased in overall number, exhibit decreased apoptosis, have increased numbers of cells in S-phase, exhibit a delay in terminal maturation and mature abnormally. Importantly, this population affected by GATA1s muta- tions is also observed in human TMD samples.
Methods
Creation of gene targeted embryonic stem cells (ESC), growth and differentiation of murine ESC, characterisation of ESC, flow cytometry, gene expression analysis, cell staining and microscopy, acetylcholinesterase staining quantitation, cell cycle and apoptosis assays
Details are stated in the Online Supplementary Appendix. Antibody clones and colours are listed in the Online Supplementary Table S1. Raw RNA sequencing data have been deposited in Arrayexpress (https://www.ebi.ac.uk/arrayexpress/) with accession
number E-MTAB-8968. Western blotting was performed as previ- ously described.14
Mice
Animal studies were approved by the University of Oxford’s Ethics Committee and conducted in accordance with the UK Home Office regulations (PPL n°PA7C92A40). Embryos were processed as set out in the Online Supplementary Appendix.
Human samples
Parents gave written informed consent in accordance with the Declaration of Helsinki, and the study was approved by the Thames Valley Research Ethics Committee (06MRE12-10; NIHR portfolio no. 6362).
Statistical analyses
All experiments were performed using at least three different cultures or animals in independent experiments. The Student’s t-test was used for statistical analyses. P<0.05 was considered sig- nificant.
Results
Differentiation of bioGATA1 (bioG1) and bioGATA1s (bioG1s) cells
Murine bioGata1 and bioGata1s alleles were created in male BirA ligase-expressing ESC15 by gene targeting of X- chromosome encoded Gata1 (Online Supplementary Figure 1A-B). Correct targeting was verified by Southern blot analysis (Online Supplementary Figure 1C) and PCR (Online Supplementary Figure 1D-E). We generated three ESC types: BirA-bioGATA1s (hereafter, bioG1s) and as controls parental BirA (hereafter, BirA) and BirA-bioGATA1 (here- after, bioG1).
In order to study the mGATA1s megakaryocyte pheno- type, we used a 12 day megakaryocyte in vitro ESC differ- entiation protocol16 (Figure 1A). ESC were differentiated into embryoid bodies (EB), EB disaggregated at day 6 (d6), then CD41+ hemopoietic cells isolated by bead-enrich- ment and kithiCD41+ cells fluorescence-activated cell sort- ing (FACS)-purified (Online Supplementary Figure S1F-G) for further 6-day culture on OP9 stromal cells with cytokines to promote megakaryocyte differentiation. Western blot analysis of d6 CD41+ cells confirmed bioG1 cells expressed only a single higher molecular weight full- length bioGATA1 isoform, whereas bioG1s cells only expressed a single lower weight bioGATA1s isoform (Figure 1B). We next confirmed expression of Gata1 exon 3 (common to both Gata1 and Gata1s) in BirA, bioG1 and bioG1s cells and appropriately detected cDNA spanning Gata1 exon 2-3 only in BirA and bioG1 and not bioG1s cells (Figure 1C).
Next, we tested the lineage characteristics of cells pro- duced by the 12 day culture. First, we took all cells at day 12 (d12) and confirmed expression of megakaryocyte genes gpIIB, gpVI, mpl and p-selectin in BirA, bioG1 and bioG1s cells but not in ESC (Figure 1C). Next, by staining d12 cells with megakaryocyte-specific acetylcholinesterase stain (Figure 1D) we confirmed megakaryocyte produc- tion. Interestingly, bioG1s cultures produced significantly fewer megakaryocytes providing a first clue that megakaryocyte differentiation is impaired by GATA1s.
In order to obtain a more complete initial view of megakaryocyte differentiation we analysed kit (marker of
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