Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus
Marta Byrska-Bishop, Daniel VanDorn, Amy E. Campbell, Marisol Betensky, Philip R. Arca, Yu Yao, Paul Gadue, Fernando F. Costa, Richard L. Nemiroff, Gerd A. Blobel, Deborah L. French, Ross C. Hardison, Mitchell J. Weiss, Stella T. Chou
Marta Byrska-Bishop, Daniel VanDorn, Amy E. Campbell, Marisol Betensky, Philip R. Arca, Yu Yao, Paul Gadue, Fernando F. Costa, Richard L. Nemiroff, Gerd A. Blobel, Deborah L. French, Ross C. Hardison, Mitchell J. Weiss, Stella T. Chou
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Research Article

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

Abstract

Germline GATA1 mutations that result in the production of an amino-truncated protein termed GATA1s (where s indicates short) cause congenital hypoplastic anemia. In patients with trisomy 21, similar somatic GATA1s-producing mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia. Here, we demonstrate that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential, but enhanced megakaryopoiesis and myelopoiesis, recapitulating the major phenotypes of the associated diseases. Similarly, in developmentally arrested GATA1-deficient murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs), expression of GATA1s promoted megakaryopoiesis, but not erythropoiesis. Transcriptome analysis revealed a selective deficiency in the ability of GATA1s to activate erythroid-specific genes within populations of hematopoietic progenitors. Although its DNA-binding domain was intact, chromatin immunoprecipitation studies showed that GATA1s binding at specific erythroid regulatory regions was impaired, while binding at many nonerythroid sites, including megakaryocytic and myeloid target genes, was normal. Together, these observations indicate that lineage-specific GATA1 cofactor associations are essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes.

Authors

Marta Byrska-Bishop, Daniel VanDorn, Amy E. Campbell, Marisol Betensky, Philip R. Arca, Yu Yao, Paul Gadue, Fernando F. Costa, Richard L. Nemiroff, Gerd A. Blobel, Deborah L. French, Ross C. Hardison, Mitchell J. Weiss, Stella T. Chou

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Figure 2

GATA1s mutations impair erythropoiesis and enhance the production of myeloid cells and megakaryocytes in iPSC-derived CD43+CD41+CD235+ progenitors.

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GATA1s mutations impair erythropoiesis and enhance the production of mye...
(A) Methylcellulose colony assays containing SCF, IL-3, EPO, GM-CSF, and (B) colony-forming megakaryocyte (CFU-Mk) assays with TPO, IL-3, and IL-6 of CD43+CD41+CD235+ progenitors. Results show mean values ± SEM of all lines performed in triplicate (n = 4 euploid/WT GATA1, 2 euploid/GATA1s, 4 T21/WT GATA1, and 5 T21/GATA1s lines). (C) Methylcellulose colony assays and (D) CFU-Mk formation from CD43+CD41+CD235+ progenitors from 2 pairs of isogenic iPSC clones with the indicated GATA1 alleles, performed as in A and B. (E) Schematic and representative flow-cytometry analysis of WT GATA1 and GATA1s CD43+CD41+CD235+ progenitors grown in liquid culture to preferentially support erythroid (CD71+CD235+), megakaryocyte (CD41+CD42+), and myeloid (CD45+CD18+) differentiation. Numbers denote percentage of total cells in the indicated gate. (F) Fold expansion of erythroid, megakaryocyte, and myeloid cells after 6 days in lineage-specific liquid cultures of euploid or T21 progenitors expressing WT GATA1 or GATA1s (n = 4 independent assays for each bar with the exception of n = 3 for euploid/GATA1s in erythroid culture, n = 5 for T21/WT GATA1 in erythroid culture, and n = 5 for T21/WT GATA1 and T21/GATA1s in megakaryocyte culture). *P < 0.05 (2-tailed Student’s t test).