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G. Juban et al.
by high CD41 expression and low level of kit expression (kitloCD41hi). Importantly, we showed the accumulation of a similar immature megakaryocytic population in samples from TMD patients, though its immunophenotype slight- ly differs between mouse and human.
A number of mechanisms may contribute to increased number of GATA1s P3 cells. Hemopoietic cells have a range of cell fate options including differentiation (with or without entering cell cycle), entering cell cycle without differentiating, apoptosis and quiescence. Our data shows that GATA1s P3 cells have increased number of cells in S-phase, reduced number in G0/G1 and a lower number of apoptotic cells compared to GATA1 P3 cells. Detailed kinetic studies of ES-derived hemopoiesis demonstrate a delay in exiting the P3 compartment into the next, more mature megakaryocyte compartment (termed P4) where cells have lost kit expression and presumably lost the pro- liferative drive afforded by kit signaling. For a 10-fold increase in cell number there need only just over three more cell divisions to account for the increase in GATA1s cell number.
Three major, open questions arise out of our work that provides a platform for future studies. The first two relat- ed questions are, what molecular mechanisms explain how GATA1s causes differentiation delay and why does differentiation delay specifically occur in the megakary- ocyte lineage? Though the answers to these questions are unclear, prior data suggests that sustained elevated expres- sion of GATA2 in GATA1s cells may play a role.22 Chromatin occupancy by GATA2/E-box proteins/LMO2/FLI1/ERG/RUNX1 heralds megakary- ocyte lineage priming and sustained GATA2 repression of specific loci is correlated with terminal megakaryocyte maturation23 and indirectly modulates megakaryocyte cell progression in GATA1 deficient megakaryocytes.24 However, proof that GATA2 is pivotal for GATA1s onco- genicity is still required and if GATA2 is needed, the mechanism by which it delays megakaryocyte differenti- ation in GATA1s cells requires further work.
Prior work has also suggested that GATA1 may directly interface with cell cycle.25 Consistent with this, one report has shown that GATA1 directly binds pRB/E2F2 via amino acid residues in the N-terminal GATA1 domain that is delet- ed in GATA1s.26 Normally, GATA1/pRB/E2F2 restrain uncommitted murine hemopoietic cell proliferation where- as GATA1s fails to bind pRB/E2F2 and fails to do this.
Our data also confirm that erythroid maturation is reduced in GATA1s cells consistent with prior work.13 One
mechanism for this may be reduced GATA1s binding to cis-elements of erythroid genes which was demonstrated in an erythroid-megakaryocyte cell line model27 that may cause a failure of terminal erythroid maturation, resulting in activation of an apoptotic program which is normally forestalled by GATA1 and erythropoietin.28
The third question is why does GATA1s exert a devel- opmental-stage specific myeloproliferative effect? One possible explanation is that fetal liver-restricted IGF-1 sig- naling promotes E2F-induced erythro-megakaryocyte pro- liferation and that the extent of this proliferation is restrained by GATA1, but not GATA1s.29 Additionally, post-natal bone marrow-specific type 1 interferon signal- ing may actively suppress GATA1s megakaryocyte-ery- throid progenitor growth promoting resolution of TMD in the post-natal period.30
In summary, our work now establishes the stage to test the role of previously identified molecular players (GATA1s, GATA2, E2F proteins, pRB, IGF-1 and interferon signaling) and possibly new determinants that regulate transition into and out of P3-like compartment in vivo and regulate the commitment of P2-like cells into either megakaryocytic or non-megakaryocytic paths of differen- tiation.
Disclosures
No conflicts of interest to disclose.
Contributions
GJ and NS conceived, designed and performed experiments, analyzed and interpreted the data, wrote the manuscript; HC and DCH performed experiments and analyzed the data; QC performed modeling analyses; KS, BS, CG assisted with exper- iments; EK, MA generated ESC and mouse line bioG1; DW wrote the script to analyze staining data; GO performed compu- tational analyses for RNAseq; JD, BU managed mouse colonies; QC, HC, DCH, BS and DW contributed to editing the manu- script; EM, IR, JS, CP, and PV designed the study, analyzed and interpreted the data, wrote the manuscript and academically drove the project.
Funding
PV and IR are supported by Bloodwise Specialist Programme Grant 13001 and by the NIHR Oxford Biomedical Centre Research Fund. PV and CP are supported by programme grants from the MRC Molecular Haematology Unit (MC_UU_12009/11). CG is supported by a Wellcome Trust Clinical Training Fellowship.
References
1. Fujiwara Y, Browne CP, Cunniff K, Goff SC, Orkin SH. Arrested development of embry- onic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc Natl Acad Sci U S A. 1996;93(22):12355- 12358.
2.Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J. 1997; 16(13):3965-3973.
3.Vyas P, Ault K, Jackson CW, Orkin SH, Shivdasani RA. Consequences of GATA-1
deficiency in megakaryocytes and platelets.
Blood. 1999;93(9):2867-2875.
4. Wechsler J, Greene M, McDevitt MA, et al.
Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syn- drome. Nat Genet. 2002;32(1):148-152.
5. Rainis L, Bercovich D, Strehl S, et al. Mutations in exon 2 of GATA1 are early events in megakaryocytic malignancies associated with trisomy 21. Blood. 2003; 102(3):981-986.
identification of a population at risk of
leukemia. Blood. 2013;122(24):3908-3917. 8. Yoshida K, Toki T, Okuno Y, et al. The land- scape of somatic mutations in Down syn- drome-related myeloid disorders. Nat
Genet. 2013;45(11):1293-1299.
9. Labuhn M, Perkins K, Papaemmanuil E, et
al. Mecanisms of progression of myeloid preleukemia to transformed myeloid leukemia in children with Down syndrome. Cancer Cell. 2019; 36(2):123-138.
6.Ahmed M, Sternberg A, Hall G, et al. 10.HollandaLM,LimaCS,CunhaAF,etal.An
Natural history of GATA1 mutations in Down syndrome. Blood. 2004;103(7):2480- 2489.
7. Roberts I, Alford K, Hall G, et al. GATA1- mutant clones are frequent and often unsus- pected in babies with Down syndrome:
inherited mutation leading to production of only the short isoform of GATA-1 is associ- ated with impaired erythropoiesis. Nat Genet. 2006;38(7):807-812.
11. Sankaran VG, Ghazvinian R, Do R, et al. Exome sequencing identifies GATA1 muta-
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