GATA-1 Mice Research Articles

Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis

AbstractMutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.

Global Chromatin Occupancy and Epigenetic Signature Analysis Reveal New Insights into the Function of GATA1 N-Terminus in Erythropoiesis

Mutations in GATA1 are seen in rare cases of dyserythropoietic anemia and in a subset of patients with Diamond Blackfan Anemia (DBA). Of note the truncation mutations in DBA, known as GATA1s, closely resemble those that are more commonly associated with acute megakaryoblastic leukemia in children with Down syndrome (DS). Studies with a mouse model of the Gata1s mutation revealed that replacement of the full-length protein by the shortened isoform led to a marked yet transient enhancement in megakaryopoiesis, similar in some respects to transient myeloproliferative disorder in DS. Furthermore, these mutant mice displayed impaired embryonic erythropoiesis but ostensibly no defects in adult hematopoiesis. In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in the Gata1s mouse strain. We observed a striking impairment in erythropoiesis in fetuses at E10.5 though E12.5, but saw improvement as the animals progressed through E14.5 and beyond. Defects included impaired terminal maturation and reduced numbers of erythroid progenitors, likely at the expense of expanded megakaryopoiesis. RNA-sequencing revealed that both erythroid genes and megakaryocytic genes were altered by the Gata1s mutation. Epiproteomic histone modification analysis further revealed there was an accumulation of H3K27 methylation in the R3 (CD71hiTer119hi) erythroid progenitor population, which suggests that GATA1 has a link to the epigenetic machinery that is altered in Gata1s mutant cells. Despite a global increase in H3K27me3, critical Gata2 regulatory elements in Gata1s mutant erythroid progenitors were marked by substantially less H3K27me3 than in wild-type littermates. Given that overexpression of GATA2 has been reported to impair erythropoiesis, we investigated whether reducing the GATA2 levels would ameliorate the phenotype. Indeed, we observed that haploinsufficiency for Gata2 rescued the erythroid defects of Gata1s fetuses. Next, to comprehensively study the effect of absence of the GATA1 N-terminus genome-wide, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with H3K27me3, GATA1 or GATA1s antibodies on wild-type versus Gata1s expressing fetal erythroid cells. Our data indicated that there is a substantial reduction in H3K27me3 along regulatory elements of the Runx1 gene at the late stage (R3) of fetal erythropoiesis in Gata1s mice. Along with an increase in Runx1 expression we observed strong downregulation of Klf1, a repressive target of RUNX1. Thus, failure of GATA1s to facilitate trimethylation of Runx1 and Gata2 regulatory elements appears to cause the defects in erythroid cell and megakaryocyte development. In parallel, we performed an in-depth analysis of the phenotype of adult Gata1s mice and discovered that they have reduced red cell counts, lower hemoglobin and hematocrit, increased extramedullary hematopoiesis and impaired stress erythropoiesis compared to control littermates. Although there were significantly more megakaryocyte erythrocyte progenitors (MEPs, Lin-c-Kit+Sca-1-CD34-FcgR-) in Gata1s mouse bone marrow, there were fewer pre-colony-forming unit erythroid cells (preCFU-E, Lin-c-Kit+Sca-1-CD41-FcgR-CD150hiCD105hi), likely at the expense of expanded megakaryocyte progenitors (MkP, Lin-c-Kit+Sca-1-CD41+CD150hi).Gata1s mice also developed an MDS-like disease with age. Together, our integrated genomic analysis of transcriptome, GATA1/GATA1s chromatin binding profile and chromatin signature reveal that, although Gata1s mice do not precisely model DBA, they provide novel insights into the role of the N-terminus of GATA1 in gene transcriptional regulation, lineage determination and red blood cell maturation. DisclosuresCrispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation.

About 10% of Down syndrome (DS) infants are born with a transient myeloproliferative disorder (DS-TMD) that spontaneously resolves within the first few months of life. About 20%-30% of these infants subsequently develop acute megakaryoblastic leukemia (DS-AMKL). Somatic mutations leading to the exclusive production of a short GATA1 isoform (GATA1s) occur in all cases of DS-TMD and DS-AMKL. Mice engineered to exclusively produce GATA1s have marked megakaryocytic progenitor (MkP) hyperproliferation during early fetal liver (FL) hematopoiesis, but not during postnatal BM hematopoiesis, mirroring the spontaneous resolution of DS-TMD. The mechanisms that underlie these developmental stage-specific effects are incompletely understood. Here, we report a striking upregulation of type I IFN-responsive gene expression in prospectively isolated mouse BM- versus FL-derived MkPs. Exogenous IFN-α markedly reduced the hyperproliferation FL-derived MkPs of GATA1s mice in vitro. Conversely, deletion of the α/β IFN receptor 1 (Ifnar1) gene or injection of neutralizing IFN-α/β antibodies increased the proliferation of BM-derived MkPs of GATA1s mice beyond the initial postnatal period. We also found that these differences existed in human FL versus BM megakaryocytes and that primary DS-TMD cells expressed type I IFN-responsive genes. We propose that increased type I IFN signaling contributes to the developmental stage-specific effects of GATA1s and possibly the spontaneous resolution of DS-TMD.

Increased Interferon-αa Signaling During Megakaryocyte Ontogeny: Implications for the Spontaneous Resolution of Down Syndrome Transient Myeloproliferative Disorder.

Abstract 1272Poster Board I-294About ten percent of infants with Down syndrome (DS) are born with a transient myeloproliferative disorder (DS-TMD), which spontaneously resolves within the first few months of life. However, the basis for this resolution remains unknown. Acquired mutations leading to exclusive production of a short isoform of the transcription factor GATA-1 (GATA-1s) occur in all cases of DS-TMD, and knock-in mice that exclusively produce GATA-1s have hyperproliferation of megakaryocytes during early fetal liver hematopoiesis, but not during later developmental stages. In this study, we found striking upregulation of the interferon-αa (IFN-αa) receptor and multiple IFN-αa responsive genes, including Ifi203, Ifi205, Irf-1, Irf-8, and Ifitm6, in immunophenotypically isolated megakaryocyte progenitor cells (MkPs) from bone marrow versus embryonic day 13.5 (e13.5) fetal liver of wild type mice. These differences were confirmed at the protein level in megakaryocytes by in situ immunohistochemistry. Addition of IFN-αa to GATA-1s containing e13.5 fetal liver MkPs reduces their hyperproliferation in vitro in a dose-dependent manner. Conversely, injection of neutralizing IFN-αa/β antibodies, but not control IgG, into adult GATA-1s mice markedly increases the percentage of bone marrow CD41+ cells and morphologically recognizable megakaryocytes, in contrast to wild type mice. We propose that increases in IFN-αa signaling during megakaryocyte ontogeny may account for the developmental stage-specific effects of GATA-1s on megakaryocyte hyperproliferation, and possibly the spontaneous resolution of DS-TMD. Interestingly, the genes encoding the IFN-αa/β receptor are located on human chromosome 21 and are expressed at 1.6 times that in trisomy versus disomy 21 cells. We speculate that increased interferon alpha signaling during embryogenesis may be the basis for the strong selective pressure for GATA-1s producing mutations in trisomy 21 fetuses in the first place. DisclosuresNo relevant conflicts of interest to declare.

GATA1s Mutant Protein Contributes to “Down” Syndrome Megakaryoblastic Leukemia by Derepression of E2F Targets.

Somatic mutations in the hematopoietic transcription factor GATA1, leading to the production of a GATA1 variant (GATA1s) lacking its N-terminal domain, are a critical event in the development of Down syndrome (DS) associated transient myeloproliferative disease (TMD) and myeloid leukemia (ML-DS). Mice carrying Gata1s germline mutation exhibit a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors, hyperproliferating both in vitro and in vivo. However, either Gata1s mice alone or in conjunction with two DS mouse models (Ts65dn, Ts1Cje) fail to recapitulate human TMD or leukemia. Using a retroviral insertional mutagenesis screening in Gata1s fetal progenitors, we generated murine acute megakaryoblastic leukemia (AMKL) closely resembling human ML-DS. We demonstrate that retroviral integrations at the Evi1 and Plag1 loci cooperated with Gata1s in leukemogenesis. Through an integrated genomic approach, we show that restoration of full length Gata1 expression in these murine Gata1s AMKL blasts leads to downregulation of gene sets containing E2F target genes. In addition, most of these same gene sets are also enriched in Gata1s primary fetal liver-derived megakaryocytes compared to wild type megakaryocytes, in flow sorted human ML-DS leukemic blasts compared to sorted non-DS M7 leukemic blasts, and in ML-DS CMK cell line compared to other leukemic cell lines. Using a genetic approach, we could demonstrate that the hyperproliferative phenotype of Gata1s FL megakaryocytic progenitor cells can be rescued by broadly repressing the function of E2F factors. Additionally, the CMK cell line ceased to proliferate upon E2F inactivation. Thus, we discovered a new mechanism of GATA1 mediated growth regulation by controlling E2F function. GATA1s mutant protein contributes to leukemogenesis through insufficient repression of E2F targets, perturbing the coordination of proliferation and terminal differentiation. Our study reveals a previously unknown function of the N-terminal domain of GATA1 and adds a significant step towards our understanding of the role of GATA1 mutants in leukemogenesis.

Chromosome 21-Encoded miR-125b and Its Role in the Development of Myeloid Leukemia in Children with Down's Syndrome.

Children with Down's syndrome (DS) are at high risk to develop transient leukemia (TL) or acute myeloid leukemia (ML-DS), primarily the megakaryoblastic subtype (AMKL). Recently, acquired mutations of the hematopoietic transcription factor GATA1 - encoded on chromosome X - have been identified in leukemic blasts from virtually all patients with ML-DS or TL leading to the expression of a shorter isoform of GATA1 (GATA1s). During embryonic/fetal development mice carrying the GATA1s mutation show a hyperproliferation of a transient population of yolk sac/fetal liver megakaryocytic progenitors. However, GATA1s mice as well as GATA1s mice bred to mouse DS models (Ts65Dn and Ts1Cje) have normal adult hematopoiesis and do not develop leukemia. Hence, the factors on chromosome 21 that predispose to TL or ML-DS in cooperation with oncogenic GATA1s remain largely unknown. To date, four miRNAs (miR-99a, miR-125b-2, miR-155 and let-7c) are known to be encoded on chromosome 21. Three out of four miRNAs (miR-99a, miR-125b-2, let-7c) are not trisomic in the DS mouse model. To elucidate their role in the development of TL and ML-DS, we first measured their expression level in sorted cells from patient samples [ML-DS (n = 4), TL (n = 4), non-DS AMKL (n = 4)], and in megakaryocytes from healthy donors (n = 3) by qRT-PCR. The expression of miR-125b was clearly elevated in ML-DS (10-fold), TL (5-fold) and non-DS AMKL (3-fold) compared to normal megakaryocytes (ppatients vs. control <0.01). Using Gene Set Enrichment Analysis (GSEA), we confirmed the downregulation of putative target genes of miR-125b - predicted by Targetscan and Pictar - in the gene expression profile of ML-DS in comparison to non-DS AMKL. By retroviral overexpression using the Mouse Stem Cell Virus (MSCV), we could show that ectopic expression of miR-125b markedly enhanced proliferation of myeloid progenitor cells (MPC, CD34+CD33+) (MPCmiR-125b) in vitro. In colony-forming assays, the number of MPCmiR-125b derived colonies exceeded the number of MPCvector derived colonies (empty vector) by 4-fold (χ2 < 0.01). All MPCmiR-125b derived colonies contained a mixture of immature cells (CFU-GEMM) indicating an affected differentiation. This could also be confirmed by immunophenotyping of MPC cultured in liquid medium containing various cytokines (percentage CD34+CD33+ 26% vs 9%). In GATA1s fetal liver progenitors retroviral overexpression of miR-125b-2 could further enhance the proliferation rate and mediate the formation of large megakaryocytic colonies. These data support the hypothesis that miR-125b is involved in development of trisomy 21 associated leukemia by mediating the proliferation of MPC while repressing their differentiation. Therefore, miR-125b might be potential target in the therapy of ML-DS.

A Genome-Wide Retroviral Insertional Mutagenesis Screen for Genes Cooperating with Truncated, Oncogenic GATA1s.

Somatic mutations in the hematopoietic transcription factor GATA1 are found in megakaryoblasts of Down Syndrome (DS) patients with transient myeloproliferative disorder (TMD, or transient leukemia or TL) and the related acute megakaryoblastic leukemia (DS-AMKL, or DS- AML M7). These mutations lead to production of a GATA1 variant (GATA1s) lacking its N-terminal domain. Mice carrying GATA1s mutation have normal adult hematopoiesis. However, during embryonic/fetal development, we have identified a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors in mutant mice. We proposed that these progenitor cells are the target for transformation in DS-AMKL/TMD. GATA1s mice (either during development or as adults) do not develop myeloproliferative disorder or leukemia. To recapitulate human DS TMD in mice, we bred GATA1s mice to mouse DS models (Ts65Dn and Ts1Cje) and generated GATA1s/DS double mutants. The phenotype of GATA1s/DS mice is not different from that of GATA1s mice, suggesting that these mouse DS models (representing ~166 and 112 trisomic genes on human chromosome 21, respectively, including Runx1, Ets2, and Erg) do not accurately recapitulate the effects of trisomy in DS. To search for genes that cooperate with GATA1s in an unbiased fashion, we established a genome-wide retroviral insertional mutagenesis screen. GATA1s mutant fetal liver progenitors proliferate in culture in the presence of thrombopoietin (Tpo) for about 4–5 weeks. We infected mutant fetal progenitors with MSCV retrovirus and selected in vitro in the presence of Tpo for immortalized cell lines. Retroviral integration sites in these cell lines were determined by Splinkerette PCR, and confirmed by genomic PCR. Genes that were affected by retroviral integration were confirmed by real-time PCR for their elevated expression or knock-down. From the genetic screen performed thus far, we identified two common retroviral integration sites, Evi1 and Prdm16 (PR domain containing 16). Interestingly, Evi1 is also overexpressed in M7 leukemias, though its expression in non-DS M7 leukemia is higher than in DS M7 leukemia. By retroviral overexpression, we have confirmed that ectopic expression of Evi1 in GATA1s mutant fetal progenitors further enhanced proliferation. Currently we are testing the in vivo leukemogenic abilities of these cell lines by transplantation. By this approach, we will identify genes that cooperate with GATA1s in cellular transformation and, thereby, gain insights into the mechanism of leukemogenesis in DS-AMKL/TMD.