Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition
Kamaleldin E. Elagib, Chih-Huan Lu, Goar Mosoyan, Shadi Khalil, Ewelina Zasadzińska, Daniel R. Foltz, Peter Balogh, Alejandro A. Gru, Deborah A. Fuchs, Lisa M. Rimsza, Els Verhoeyen, Miriam Sansó, Robert P. Fisher, Camelia Iancu-Rubin, Adam N. Goldfarb
Kamaleldin E. Elagib, Chih-Huan Lu, Goar Mosoyan, Shadi Khalil, Ewelina Zasadzińska, Daniel R. Foltz, Peter Balogh, Alejandro A. Gru, Deborah A. Fuchs, Lisa M. Rimsza, Els Verhoeyen, Miriam Sansó, Robert P. Fisher, Camelia Iancu-Rubin, Adam N. Goldfarb
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Research Article Hematology

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition

Abstract

Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.

Authors

Kamaleldin E. Elagib, Chih-Huan Lu, Goar Mosoyan, Shadi Khalil, Ewelina Zasadzińska, Daniel R. Foltz, Peter Balogh, Alejandro A. Gru, Deborah A. Fuchs, Lisa M. Rimsza, Els Verhoeyen, Miriam Sansó, Robert P. Fisher, Camelia Iancu-Rubin, Adam N. Goldfarb

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Figure 2

Neonatal progenitors fail to execute specialized megakaryocytic P-TEFb activation pathway.

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Neonatal progenitors fail to execute specialized megakaryocytic P-TEFb a...
(A) Neonatal failure to upregulate megakaryocytic P-TEFb targets. Left panel: primary adult and neonatal progenitors, either undifferentiated (Un) or cultured 6 days in erythroid (Ery) or megakaryocytic (Mk) medium were immunoblotted for indicated factors. Top arrow, full-length FLNA; bottom arrow, approximately 190-kDa cleavage fragment. Right panel: densitometry comparing adult and neonatal megakaryocyte levels from 3 independent experiments conducted as in the right panel. Graphs show mean ± SEM for signals normalized to tubulin, with PB values set at 1. *P < 0.05; **P < 0.01; ***P < 0.005, t test. Rel, relative. (B–D) Evidence for diminished P-TEFb activation in neonatal versus adult megakaryocytes. Adult and neonatal progenitors cultured 6 days in megakaryocyte medium underwent IB for RNAPII subunit RPB1 (B), SPT5 phospho-threonine 806 (pT806) and total (SPT5) (C), and histone H2Bub1 and total H2B (D). II0 and IIA designate hyper- and hypophosphorylated forms for RNAPII, respectively. Graphs show mean ± SEM for scanning densitometry values derived from 3 independent experiments. *P < 0.05; **P < 0.01, t test. (E) Neonatal megakaryocytes downregulate 7SK-stabilizing factors to a degree similar to that of adult megakaryocytes. (F) Neonatal block in megakaryocytic downregulation of 7SK snRNA. 7SK levels relative to CB megakaryocytes in progenitors cultured as in A. Graphs show mean ± SEM of 7SK normalized to GAPDH in 3 independent experiments. *P < 0.05, t test. Note that panels A and E as well as Figure 3A and Supplemental Figure 4A all derive from the same IB membrane and therefore share the same tubulin control. Note that panel D and Supplemental Figure 4B derive from the same IB membrane and share the same tubulin control. See also Supplemental Figures 2 and 3.