HGATA1 Under the Control of a μLCR/β-Globin Promoter Rescues the Erythroid but Not the Megakaryocytic Phenotype Induced by the Gata1 low Mutation in Mice.

Fabrizio Martelli,Giuseppe Sarli,Maria Zingariello,Alessandro M Vannucchi,Paola Verachi,Annalisa Lonetti,Barbara Bacci,Maria Mazzarini,Anna Rita Migliaccio

doi:10.3389/fgene.2021.720552

Abstract

The phenotype of mice carrying the Gata1 low mutation that decreases expression of Gata1 in erythroid cells and megakaryocytes, includes anemia, thrombocytopenia, hematopoietic failure in bone marrow and development of extramedullary hematopoiesis in spleen. With age, these mice develop myelofibrosis, a disease sustained by alterations in stem/progenitor cells and megakaryocytes. This study analyzed the capacity of hGATA1 driven by a μLCR/β-globin promoter to rescue the phenotype induced by the Gata1 low mutation in mice. Double hGATA1/Gata1 low/0 mice were viable at birth with hematocrits greater than those of their Gata1 low/0 littermates but platelet counts remained lower than normal. hGATA1 mRNA was expressed by progenitor and erythroid cells from double mutant mice but not by megakaryocytes analyzed in parallel. The erythroid cells from hGATA1/Gata1 low/0 mice expressed greater levels of GATA1 protein and of α- and β-globin mRNA than cells from Gata1 low/0 littermates and a reduced number of them was in apoptosis. By contrast, hGATA1/Gata1 low/0 megakaryocytes expressed barely detectable levels of GATA1 and their expression of acetylcholinesterase, Von Willebrand factor and platelet factor 4 as well as their morphology remained altered. In comparison with Gata1 +/0 littermates, Gata1 low/0 mice contained significantly lower total and progenitor cell numbers in bone marrow while the number of these cells in spleen was greater than normal. The presence of hGATA1 greatly increased the total cell number in the bone marrow of Gata1 low/0 mice and, although did not affect the total cell number of the spleen which remained greater than normal, it reduced the frequency of progenitor cells in this organ. The ability of hGATA1 to rescue the hematopoietic functions of the bone marrow of the double mutants was confirmed by the observation that these mice survive well splenectomy and did not develop myelofibrosis with age. These results indicate that hGATA1 under the control of µLCR/β-globin promoter is expressed in adult progenitors and erythroid cells but not in megakaryocytes rescuing the erythroid but not the megakaryocyte defect induced by the Gata1 low/0 mutation.

Highlights

GATA1 is a member of the GATA family of transcription factors that ensures appropriate differentiation in hematopoietic cells of multiple lineages including erythroid (Tsai et al, 1989), megakaryocytic (Martin et al, 1990; Romeo et al, 1990), eosinophil (Yu et al, 2002a) and mast cells (Migliaccio et al, 2003)
The consequent reduced content of GATA1 protein results in the following cell specific abnormalities: it increases proliferation of MEP altering their differentiation potential making the cells able to generate mast cells under erythroid-specific conditions (Ghinassi et al, 2007), it increases the apoptotic rates of the erythroblasts (McDevitt et al, 1997; Vannucchi et al, 2001) and blocks megakaryocyte maturation retaining these cells in a proliferative state (Vyas et al, 1999; Vannucchi et al, 2002)
The mice are born anemic and thrombocytopenic (McDevitt et al, 1997; Vyas et al, 1999). They recover from their anemia by recruiting the spleen as extramedullary site (Migliaccio et al, 2009; Spangrude et al, 2016) but retain the abnormalities at the level of MEP (Ghinassi et al, 2007) and megakaryocytes (Vyas et al, 1999; Vannucchi et al, 2002) for all their life developing myelofibrosis with age (Vannucchi et al, 2002)

Summary

Introduction

GATA1 is a member of the GATA family of transcription factors that ensures appropriate differentiation in hematopoietic cells of multiple lineages including erythroid (Tsai et al, 1989), megakaryocytic (Martin et al, 1990; Romeo et al, 1990), eosinophil (Yu et al, 2002a) and mast cells (Migliaccio et al, 2003). Gata deficient mice die of severe anemia between day 10.5 and 11.5 of gestation with sign of intraembryonic hemorrhage (McDevitt et al, 1997). In human, both inherited and acquired GATA1 mutations have been described. Based on extensive information indicating that in mice the ratio between the GATA1 and GATA2 content (Gata2/Gata switch) controls the proliferation of murine stem/progenitor cells (Bresnick et al, 2010), it is not surprising that acquired frame shift and point mutations impairing GATA1 functions induce leukemia in humans. Ribosomopathies affecting the efficiency of translation of GATA1 mRNA are associated, in addition to Diamond Blackfan Anemia (Ludwig et al, 2014), with primary myelofibrosis, the most severe of the Philadelphia-negative myeloproliferative disorders (Vannucchi et al, 2005; Gilles et al, 2017)

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