Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus
Marta Byrska-Bishop, … , Mitchell J. Weiss, Stella T. Chou
Marta Byrska-Bishop, … , Mitchell J. Weiss, Stella T. Chou
Published March 2, 2015; First published January 26, 2015
Citation Information: J Clin Invest. 2015;125(3):993-1005. https://doi.org/10.1172/JCI75714.
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Categories: Research Article Stem cells

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 7

Loss of the N-terminus selectively inhibits GATA1 binding to erythroid genes in Gata1– MEPs.

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Loss of the N-terminus selectively inhibits GATA1 binding to erythroid g...
(A) Flow cytometry of G1ME cells at 42 and 96 hours after transduction with HA-GATA1fl or HA-GATA1s. Numbers denote percentage of total cells in the indicated quadrant. (B) May-Grünwald-Giemsa staining of G1ME-derived erythroblasts (left) and megakaryocytes (right) 96 hours after transduction. Scale bars: 20 μm. (C) Western blot at 42 hours after transduction showing expression of HA-GATA1fl and HA-GATA1s relative to β-actin. ND, not determined. (D) Genome-wide ChIP-seq binding signals of GATA1fl and GATA1s at 42 hours after transduction. Plotted are mean read counts (n = 2 replicates each). non-DB, nondifferentially bound (gray dots); DB, differentially bound sites (blue and cyan; FDR < 0.1). (E) Functional enrichme­­­nt analysis using GREAT. Plotted are significance values for top 10 mouse phenotype and GO biological process enrichment terms, classified as erythroid, megakaryocytic, myeloid, other hematopoietic, or cardiovascular and other. List of terms can be found in Supplemental Table 5. (F) GATA1 binding at the β-globin locus. ChIP-seq tracks, top to bottom: GATA1fl and GATA1s in G1MEs, GATA1fl in primary mouse erythroblasts and megakaryocytes (42). Rectangles above tracks: size of binding sites analyzed in differential binding analysis; gray, non-DB; blue, DB. (G) Anti-HA ChIP at selected GATA1-binding sites in G1MEs shown as ratio of GATA1s to GATA1fl occupancy ± SEM (n = 4). *Ratio significantly different than 1 at P < 0.05 (2-tailed Student’s t test). IgG GATA1s and IgG GATA1fl controls for nonspecific binding.