Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition
Kamaleldin E. Elagib, … , Camelia Iancu-Rubin, Adam N. Goldfarb
Kamaleldin E. Elagib, … , Camelia Iancu-Rubin, Adam N. Goldfarb
Published May 8, 2017
Citation Information: J Clin Invest. 2017;127(6):2365-2377. https://doi.org/10.1172/JCI88936.
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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 3

IGF2BP3 functions as a neonatal ontogenic determinant in megakaryocytes.

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IGF2BP3 functions as a neonatal ontogenic determinant in megakaryocytes....
(A) Specific and abundant expression of IGF2BP3 in neonatal hematopoietic progenitors. Experiments and quantitations were conducted as in Figure 2A. Graph depicts mean ± SEM from 3 independent experiments. *P < 0.05; ***P < 0.005, t test. (B) Block in adult megakaryocyte enlargement by ectopic IGF2BP3 expression. Adult progenitors transduced with retrovirus expressing GFP ± IGF2BP3 underwent megakaryocyte culture for 6 days, followed by flow cytometry with gating on viable CD41+GFP+ cells. Graph depicts relative mean FSC ± SEM from 3 independent experiments. ***P < 0.005, t test. (C) Enhancement of adult megakaryocyte proliferation by ectopic IGF2BP3. Cells transduced and cultured as in B were subjected to PKH dye dilution assay, with quantitation by flow cytometry with gating on viable CD41+GFP+ cells. Graph depicts mean ± SEM for PKH mean fluorescence intensity relative to control vector–transduced cells from 3 independent experiments. ***P < 0.005, t test. (D) Erythroid antigen induction in adult megakaryocyte by ectopic IGF2BP3. Cells transduced and cultured as in B underwent flow cytometry with gating on viable GFP+ cells. Graph depicts mean ± SEM for percentage of CD41+GPA+ double positives from 3 independent experiments. *P < 0.05, t test. Note that panel A, Figure 2A, Figure 2E, and Supplemental Figure 4A all derive from the same IB membrane and therefore share the same tubulin control. See also Supplemental Figures 4 and 5.