Hematopoietic Transcription Factor GATA1 Research Articles

Enhancement of PRMT6 binding to a novel germline GATA1 mutation associated with congenital anemia.

Mutations in the master hematopoietic transcription factor GATA1 are often associated with functional defects in erythropoiesis and megakaryopoiesis. In this study, we identified a novel GATA1 germline mutation (c.1162delGG, p.Leu387Leufs*62) in a patient with congenital anemia and occasional thrombocytopenia. The C-terminal GATA1, a rarely studied mutational region, undergoes frameshifting translation as a consequence of this double-base deletion mutation. To investigate the specific function and pathogenic mechanism of this mutant, in vitro mutant models of stable re-expression cells were generated. The mutation was subsequently validated to cause diminished transcriptional activity of GATA1 and defective differentiation of erythroid and megakaryocytes. Using proximity labeling and mass spectrometry, we identified selective alterations in the proximal protein networks of the mutant, revealing decreased binding to a set of normal GATA1-interaction proteins, including the essential co-factor FOG1. Notably, our findings further demonstrated enhanced recruitment of the protein arginine methyltransferase PRMT6, which mediates histone modification at H3R2me2a and represses transcription activity. We also found an enhanced binding of this mutant GATA1/PRMT6 complex to the transcriptional regulatory elements of GATA1's target genes. Moreover, treatment of the PRMT6 inhibitor MS023 could partially rescue the inhibited transcriptional and impaired erythroid differentiation caused by the GATA1 mutation. Taken together, our results provide molecular insights into erythropoiesis in which mutation leads to partial loss of GATA1 function, and the role of PRMT6 and its inhibitor MS023 in congenital anemia, highlighting PRMT6 binding as a negative factor of GATA1 transcriptional activity in aberrant hematopoiesis.

A transcriptional network governing ceramide homeostasis establishes a cytokine-dependent developmental process

Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.

Heme as a differentiation-regulatory transcriptional cofactor.

The hematopoietic transcription factor GATA1 induces heme accumulation during erythropoiesis by directly activating genes mediating heme biosynthesis. In addition to its canonical functions as a hemoglobin prosthetic group and enzyme cofactor, heme regulates gene expression in erythroid cells both transcriptionally and post-transcriptionally. Heme binding to the transcriptional repressor BACH1 triggers its proteolytic degradation. In heme-deficient cells, BACH1 accumulates and represses transcription of target genes, including α- and β-like globin genes, preventing the accumulation of cytotoxic free globin chains. A recently described BACH1-independent mechanism of heme-dependent transcriptional regulation is associated with a DNA motif termed heme-regulated motif (HERM), which resides at the majority of loci harboring heme-regulated chromatin accessibility sites. Progress on these problems has led to a paradigm in which cell type-specific transcriptional mechanisms determine the expression of enzymes mediating the synthesis of small molecules, which generate feedback loops, converging upon the transcription factor itself and the genome. This marriage between transcription factors and the small molecules that they control is predicted to be a canonical attribute of regulatory networks governing cell state transitions such as differentiation in the hematopoietic system and more broadly.

The polymorphic landscape analysis of GATA1 exons uncovered the genetic variants associated with higher thrombocytopenia in dengue patients

The current study elucidated an association between gene variants and thrombocytopenia through the investigation of the exonic polymorphic landscape of hematopoietic transcription factor—GATA1 gene in dengue patients. A total of 115 unrelated dengue patients with dengue fever (DF) (N = 91) and dengue hemorrhagic fever (DHF) (N = 24) were included in the study. All dengue patients were confirmed through detection of NS1 antigen, IgM, and IgG antibodies against the dengue virus. Polymerase chain reaction using specific primers amplified the exonic regions of GATA1 while Sanger sequencing and chromatogram analyses facilitated the identification of variants. Variants G>A (at chX: 48792009) and C>A (at chX: 4879118) had higher frequency out of 13 variants identified (3 annotated and 10 newly recognized). Patients carrying either nonsynonymous or synonymous variants had significantly lower mean values of platelets compared to those harboring the reference nucleotides (NC_000023.11). Further analyses revealed that the change in amino acid residue leads to the altered three-dimensional structure followed by interaction with neighboring residues. Increased stability of the protein due to substitution of serine by asparagine (S129N at chX: 48792009) may cause increased rigidity followed by reduced structural flexibility which may ultimately disturb the dimerization (an important prerequisite for GATA1 to perform its biological activity) process of the GATA1 protein. This, in turn, may affect the function of GATA1 followed by impaired production of mature platelets which may be reflected by the lower platelet counts in individuals with such variation. In summary, we have identified new variants within the GATA1 gene which were found to be clinically relevant to the outcome of dengue patients and thus, have the potential as candidate biomarkers for the determination of severity and prognosis of thrombocytopenia caused by dengue virus. However, further validation of this study in a large number of dengue patients is warranted.Trial Registration: number SLCTR/2019/037.

Congenital anemia reveals distinct targeting mechanisms for master transcription factor GATA1

AbstractMaster regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

Modeling Transient Abnormal Myelopoiesis Using Induced Pluripotent Stem Cells and CRISPR/Cas9 Technology

Approximately 1%–2% of children with Down syndrome (DS) develop acute myeloid leukemia (AML) prior to age 5 years. AML in DS children (ML-DS) is characterized by the pathognomonic mutation in the gene encoding the essential hematopoietic transcription factor GATA1, resulting in N-terminally truncated short form of GATA1 (GATA1s). Trisomy 21 and GATA1s together are sufficient to induce transient abnormal myelopoiesis (TAM) exhibiting pre-leukemic characteristics. Approximately 30% of these cases progress into ML-DS by acquisition of additional somatic mutations. We employed disease modeling in vitro by the use of customizable induced pluripotent stem cells (iPSCs) to generate a TAM model. Isogenic iPSC lines derived from the fibroblasts of DS individuals with trisomy 21 and with disomy 21 were used. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system was used to introduce GATA1 mutation in disomic and trisomic iPSC lines. The hematopoietic stem and progenitor cells (HSPCs) derived from GATA1 mutant iPSC lines expressed GATA1s. The expression of GATA1s concomitant with loss of full-length GATA1 reduced the erythroid population, whereas it augmented megakaryoid and myeloid populations, characteristic of TAM. In conclusion, we have developed a model system representing TAM, which can be used for modeling ML-DS by stepwise introduction of additional mutations.

Modeling Transient Myeloproliferative Disorder Using Induced Pluripotent Stem Cells and CRISPR/Cas9 Technology

Down syndrome (DS) is recognized as one of the most important leukemia-predisposing syndromes. Specifically, 1-2% of DS children develop acute myeloid leukemia (AML) prior to age 5. AML in DS children (DS-AML) is characterized by the pathognomonic mutation in the gene encoding the essential hematopoietic transcription factor GATA1, resulting in N-terminally truncated mutant GATA1 (GATA1s). Trisomy 21 and GATA1s together induce a transient myeloproliferative disorder (TMD) exhibiting pre-leukemic characteristics. Approximately thirty percent of these cases progress into DS-AML by acquisition of additional somatic mutations in a step-wise manner. We employed disease modeling in vitro by the use of customizable induced pluripotent stem cells (iPSCs) (7, 8) to generate a TMD model. Isogenic iPSC lines derived from the fibroblasts of a DS patient with trisomy 21 and with disomy 21 were used. We also obtained DS2-iPS10 (iPSCs derived from DS patient fibroblast) from Prof. George Daley, Children's Hospital, Harvard University (Boston, MA). CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system with the indicated guide sequence (Fig. 1A) was used to introduce clinically relevant GATA1 mutation in both disomic and trisomic iPSC lines. A representative plot of TIDE (Tracking of Indels by Decomposition) analysis showing 98% allelic mutation frequency of a clone with 2 bp deletion at chromosomal level (Fig. 1B) correlated with sequence analysis using Basic Local Sequence Alignment Tool (BLAST) and Sanger sequencing chromatogram (Fig. 1C). This mutation resulted in the disruption of first initiation codon and thus prevented the production of full length GATA1 protein, while allowing the usage of second initiation codon at 84th position to generate GATA1s. GATA1 and GATA1s are not expressed in iPSCs. To determine the expression of GATA1s, we differentiated these mutant iPSC lines into hematopoietic stem cell progenitors (HSPCs) using hematopoietic differentiation kit (StemCell Technologies) following a protocol depicted in Fig. 1D. The HSPCs derived from two distinct clones of trisomic iPSCs showed expression of full-length GATA1 protein and GATA1 mutant HSPCs lacked the expression of full-length GATA1 as expected (Fig. 1E). These HSPCs expressed GATA1s. Given that trisomy 21 promotes hematopoietic differentiation, an increase in the percentage of erythroid, megakaryoid and myeloid population was observed in trisomy 21 HSPCs with full length GATA1 (Fig. 1F, compare bars 1 and 3 in each category). The expression of GATA1s reduced erythroid lineage cells whereas it augmented megakaryoid and myeloid lineages in both disomy 21 (compare red and blue bars 1 and 2) and trisomy 21 backgrounds (compare bars 3 and 4). HSPCs derived from trisomy 21 iPSCs with GATA1s exhibited more megakaryoid expansion compared to the GATA1s in disomy 21 background (Fig. 1F, compare bars 2 and 4), in agreement with the synergistic function of trisomy 21 and GATA1s in promoting TMD. Transplantation of HSPCs derived from GATA1 mutated trisomic iPSCS into NSG-SGM3 mice showed the presence of human CD45+ cells in peripheral blood at 12 weeks post cell injection (Fig. 1G). In conclusion, we have developed a model system representing TMD, which can be used for step-wise modeling of Down-syndrome AML by introducing additional mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

Generation, Characterization and Pre-Clinical Drug Evaluation of Patient-Derived Xenograft Models of Pediatric Down Syndrome AML

Children with Down syndrome (DS) are at a 500-fold increased risk for developing acute myeloid leukemia (AML) before they reach five years of age. DS-AML blasts have hypersensitivity to chemotherapeutic drugs such as cytarabine and daunorubicin. However, therapy-induced toxicity results in greater morbidity and remains a major barrier in attaining higher survival rate. Thus, alternate therapy approaches to minimize toxicity and increase efficacy are needed. Patient-derived xenograft (PDX) models of DS-AML are very limited and have not been used for preclinical studies. We generated three distinct disseminated PDX models by successful engraftment and serial passage of primary DS-AML blasts in NSG-B2m mice. DS-AML is characterized by the pathognomonic mutation in the gene encoding the essential hematopoietic transcription factor GATA1, resulting in N-terminally truncated mutant GATA1s protein. Accordingly, we identified GATA1 mutations and GATA1s protein (Fig. 1) in the DS-AML PDX lines. In addition to GATA1 mutation, whole exome sequencing also identified mutations in one or more co-operating mutations reported previously in DS-AML (Table 1). These PDX lines were extensively characterized and authenticated to be highly concordant with the primary patient sample with respect to immunophenotype (Table 2) and transcriptome analysis (by targeted sequencing using Archer FusionPlex HemeV2 panel), signifying the validity of the PDX models. We evaluated the efficacy of cytotoxic chemotherapy and epigenetic therapy in mice transplanted with DS-AML PDX lines. Cytotoxic chemotherapy comprised daunorubicin 1.5 mg/Kg Qxd3 i.v. and cytarabine 50 mg/Kg Qxd5 i.p. Epigenetic therapy consisted of DNA hypomethylating agent azacitidine and HDAC inhibitor panobinostat, alone or in combination. NSG-B2m mice were injected with DS-AML PDX lines intravenously. Disease progression and engraftment of human cells was monitored by periodic determination of the percentage of human leukemic cells versus mouse cells in the peripheral blood by staining with species-specific fluorescent antibodies followed by flow cytometry. The mice were randomized into 5 treatment groups - 1) cytotoxic chemotherapy, 2) vehicle (5% dextrose), 3) azacitidine (2.5 mg/Kg), 4) panobinostat (2.5 mg/Kg), and 5) combination of azacitidine + panobinostat (2.5 mg/Kg each). The epigenetic drugs were dosed with four cycles lasting five days a week with two days rest. One-week rest period was included between cycles 2 and 3. Chemotherapy prolonged survival by 11 and 23 days respectively in mice injected with NTPL-386 and NTPL-662. Single agent azacitidine and panobinostat prolonged survival by 48 and 31 days respectively in NTPL-60 mice. Mice transplanted with NTPL-386 showed enhanced survival by 29 and 21 days when treated with azacitidine and panobinostat alone. NTPL-662 study is ongoing and so far panobinostat treated mice showed 10 day increase in cell survival, while both azacitidine and azacitidine + panobinostat groups are alive and healthy. Azacitidine was more efficient than panobinostat in inducing remission and increasing the median survival compared to vehicle treated mice in all 3 models (Fig. 2A-C). Mice treated with the combination of azacitidine and panobinostat showed the greatest benefit and survived 127 days (for NTPL-60) and 55 days (for NTPL-386). Although azacitidine and panobinostat synergized to reduce leukemic burden and prolong survival in both PDX lines, there was a remarkable difference between the responses. Epigenetic therapy appeared to be more effective than chemotherapy. In conclusion, using DS-AML PDX models generated and characterized in-house, we show the efficacy of epigenetic therapy in comparison to chemotherapy. Disclosures Druley: Washington University: Employment; ArcherDX Inc.: Employment, Equity Ownership.

GATA‐1: A potential novel biomarker for the differentiation of essential thrombocythemia and myelofibrosis

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. ABSTRACT: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

Sphingolipid-mediated inflammatory signaling leading to autophagy inhibition converts erythropoiesis to myelopoiesis in human hematopoietic stem/progenitor cells

Elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) inhibit erythropoiesis and cause anemia in patients with cancer and chronic inflammatory diseases. TNFα is also a potent activator of the sphingomyelinase (SMase)/ceramide pathway leading to ceramide synthesis and regulating cell differentiation, proliferation, apoptosis, senescence, and autophagy. Here we evaluated the implication of the TNFα/SMase/ceramide pathway on inhibition of erythropoiesis in human CD34+ hematopoietic stem/progenitor cells (CD34/HSPCs) from healthy donors. Exogenous synthetic C2- and C6-ceramide as well as bacterial SMase inhibited erythroid differentiation in erythropoietin-induced (Epo)CD34/HSPCs shown by the analysis of various erythroid markers. The neutral SMase inhibitor GW4869 as well as the genetic inhibition of nSMase with small interfering RNA (siRNA) against sphingomyelin phosphodiesterase 3 (SMPD3) prevented the inhibition by TNFα, but not the acid SMase inhibitor desipramine. Moreover, sphingosine-1-phosphate (S1P), a ceramide metabolite, restored erythroid differentiation, whereas TNFα inhibited sphingosine kinase-1, required for S1P synthesis. Analysis of cell morphology and colony formation demonstrated that erythropoiesis impairment was concomitant with a granulomonocytic differentiation in TNFα- and ceramide-treated EpoCD34/HSPCs. Inhibition of erythropoiesis and induction of granulomonocytic differentiation were correlated to modulation of hematopoietic transcription factors (TFs) GATA-1, GATA-2, and PU.1. Moreover, the expression of microRNAs (miR)-144/451, miR-146a, miR-155, and miR-223 was also modulated by TNFα and ceramide treatments, in line with cellular observations. Autophagy plays an essential role during erythropoiesis and our results demonstrate that the TNFα/neutral SMase/ceramide pathway inhibits autophagy in EpoCD34/HSPCs. TNFα- and ceramide-induced phosphorylation of mTORS2448 and ULK1S758, inhibited Atg13S355 phosphorylation, and blocked autophagosome formation as shown by transmission electron microscopy and GFP-LC3 punctae formation. Moreover, rapamycin prevented the inhibitory effect of TNFα and ceramides on erythropoiesis while inhibiting induction of myelopoiesis. In contrast, bafilomycin A1, but not siRNA against Atg5, induced myeloid differentiation, while both impaired erythropoiesis. We demonstrate here that the TNFα/neutral SMase/ceramide pathway inhibits erythropoiesis to induce myelopoiesis via modulation of a hematopoietic TF/miR network and inhibition of late steps of autophagy. Altogether, our results reveal an essential role of autophagy in erythroid vs. myeloid differentiation.

Abstract 1952: Epigenetic modifiers outperform chemotherapy in prolonging survival in patient-derived xenograft models of Down syndrome AML

Abstract Children with Down syndrome (DS) are at a 500-fold increased risk for developing acute myeloid leukemia (AML) before they reach five years of age. DS-AML blasts have somatic mutations in the gene encoding the essential hematopoietic transcription factor GATA-1 resulting in hypersensitivity to chemotherapeutic drugs such as cytarabine and daunorubicin. However, therapy-induced toxicity results in greater morbidity and remains a major barrier in attaining higher survival rate. Thus, alternate therapy approaches to minimize toxicity and increase efficacy are needed. Trisomy 21 and GATA-1 mutations in DS-AML are known to alter the epigenetic landscape in multiple ways. Therefore, we evaluated the efficacy of epigenetic drugs in comparison to chemotherapy in two patient-derived xenograft (PDX) models of DS-AML. We developed two distinct PDX models by successfully engraftment and serial passage of primary DS-AML cells in NSG-B2m mice. Both PDX lines possessed GATA-1 mutation resulting in the expression of a truncated form of GATA-1. NTPL-60 had a nonsense mutation generating a premature stop codon after the initiation codon, while NTPL-386 had a 136 bp deletion in exon 2 resulting in the loss of the initiation codon. The mouse passaged cells were intravenously injected into 6-8 week old NSG-B2m mice. Once disease establishment was confirmed based on the presence of human cells in mouse peripheral blood, five mice per group were treated with vehicle or DNA methylation inhibitor azacitidine and histone deacetylase inhibitor panobinostat either singularly or in combination at a previously determined maximally tolerated dose of 2.5 mg/Kg each. NTPL-386 xenografted mice treated with azacitidine or panobinostat survived 29 and 21 days longer than the vehicle-treated mice respectively, while the mice treated with the combination survived the longest (35 days). Similarly, azacitidine and panobinostat extended the survival of mice transplanted with NTPL-60 by 48 and 31 days respectively compared to the vehicle-treated mice. NTPL-60 mice treated with the epigenetic drug combination are alive at 57 days. Thus, we observed that the azacitidine-panobinostat combination showed statistically significant (p < 0.0001) differences in leukemic burden and mouse survival compared to treatments with either drug alone in both NTPL-386 and NTPL-60 PDX models. We also tested the efficacy of epigenetic therapy followed by chemotherapy. The inclusion of epigenetic therapy before chemotherapy prolonged survival by 39 days compared to vehicle-treated mice. Taken together, our data indicate that epigenetic therapy may be of benefit for the treatment of children with DS-AML. Citation Format: Sonali P. Barwe, E A. Kolb, Anilkumar Gopalakrishnapillai. Epigenetic modifiers outperform chemotherapy in prolonging survival in patient-derived xenograft models of Down syndrome AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1952. doi:10.1158/1538-7445.AM2017-1952

Hematopoietic transcription factors and differential cofactor binding regulate PRKACB isoform expression.

Hematopoietic differentiation is controlled by key transcription factors, which regulate stem cell functions and differentiation. TAL1 is a central transcription factor for hematopoietic stem cell development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation. Knowledge of the target genes controlled by a given transcription factor is important to understand its contribution to normal development and disease. To uncover direct target genes of TAL1 we used high affinity streptavidin/biotin-based chromatin precipitation (Strep-CP) followed by Strep-CP on ChIP analysis using ChIP promoter arrays. We identified 451 TAL1 target genes in K562 cells. Furthermore, we analysed the regulation of one of these genes, the catalytic subunit beta of protein kinase A (PRKACB), during megakaryopoiesis of K562 and primary human CD34+ stem cell/progenitor cells. We found that TAL1 together with hematopoietic transcription factors RUNX1 and GATA1 binds to the promoter of the isoform 3 of PRKACB (Cβ3). During megakaryocytic differentiation a coactivator complex on the Cβ3 promoter, which includes WDR5 and p300, is replaced with a corepressor complex. In this manner, activating chromatin modifications are removed and expression of the PRKACB-Cβ3 isoform during megakaryocytic differentiation is reduced. Our data uncover a role of the TAL1 complex in controlling differential isoform expression of PRKACB. These results reveal a novel function of TAL1, RUNX1 and GATA1 in the transcriptional control of protein kinase A activity, with implications for cellular signalling control during differentiation and disease.

Toward RNA Repair of Diamond Blackfan Anemia Hematopoietic Stem Cells.

Diamond blackfan anemia (DBA) is a well-known inherited bone marrow failure syndrome mostly caused by mutations in ribosomal protein (RP) genes but also rarely in the hematopoietic transcription factor gene, GATA1, or TSR2, a ribosomal protein (Rps26) chaperone gene. About 25% of patients have heterozygous mutations in the RPS19 gene, which leads to haploinsufficiency of Rps19 protein in most cases. However, some RPS19 missense mutations appear to act in a dominant negative fashion. DBA typically leads to a hypoplastic anemia that becomes apparent during the first year of life, and standard treatment includes steroids or red blood cell transfusions, each modality having attendant side effects. The only curative therapy is allogeneic stem-cell transplantation, but this option is limited to patients with a histocompatible donor. DBA-mutant embryonic, induced pluripotent, and hematopoietic stem cells all exhibit growth abnormalities that can be corrected by DNA gene transfer, suggesting the possibility of ex vivo autologous gene therapy. The authors have been interested in the application of spliceosome-mediated mRNA trans-splicing (SMaRT) technology to RNA repair of DBA stem cells. Compared with gene replacement or other RNA re-programming approaches, SMaRT has several potential advantages. First, delivery of the entire normal cDNA is unnecessary, thus minimizing the overall size of the construct for packaging into a viral delivery vector. Second, RNA transcription of the corrected gene relies on the cell's endogenous transcriptional, processing, and regulatory machinery, thereby ensuring faithful and contextual expression. Third, RNA trans-splicing employs the endogenous spliceosome enzymatic machinery present in nearly all cells. Fourth, RNA trans-splicing converts mutant transcripts into therapeutically useful mRNA, and thus may be capable of treating disorders caused by dominant negative mutations. This review critically assesses prospects for both gene and RNA repair in DBA stem cells.

Myeloid and erythroid hematopoietic transcription factor expression decline after knockdown of lmna genes in zebrafish embryos

Objective: To investigate the effect of lmna gene down-regulation on early hematopoietic development of zebrafish. Methods: Phosphorodiamidate morpholino oligomer (PMO) technology was used to downregulate lmna gene expression in Zebrafish. Zebrafish embryos injected phosphorodiamidate morpholino antisense oligonucleotide of lmna gene mRNA by microinjection at unicellular stage were taken as the experimental group, and those injected meaningless phosphorodiamidate morpholino antisense oligonucleotide were taken as the control. The embryos were collected at 18, 24, 30 and 36 hpf after the fertilization. The real-time fluorescent quantitative PCR (RT-PCR) and whole embryo in situ hybridization methods were used to detect the expression of myeloid hematopoietic transcription factor pu. 1 and erythroid hematopoietic transcription factor gata1 in zebrafish. Results: RT-PCR showed that the expressions of pu. 1 and gata1 decreased in the experimental group compared with the control group (all P<0.05). Whole embryo in situ hybridization showed that the blue-black positive hybridization signals of pu. 1 and gata1 in experimental group were shallow than those in the control group. Conclusion: Myeloid hematopoietic and erythroid hematopoietic of zebrafish are blocked with the downregulation of lmna gene.

Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptionalcis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.

Loss of GATA‐1 full length as a cause of Diamond–Blackfan anemia phenotype

Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform.

Characterization Of Oncogenes On Chromosome 21 Identified By shRNA-Based Viability Screening

Children with trisomy 21 (Down syndrome, DS) are predisposed to develop acute megakaryoblastic leukemia (DS-AMKL) as well as the antecedent transient leukemia (DS-TL). Mutations in the hematopoietic transcription factor GATA1 have been found in nearly all children with DS-AMKL and DS-TL, but not in other malignancies. Recent whole genome sequencing efforts strongly supported the hypothesis that the triad of fetal origin, trisomy 21 and GATA1s-mutation are sufficient to cause DS-TL. Thus, the presence of an extra copy of hsa21 perturbs fetal hematopoiesis to provide a GATA1s sensitive background during leukemogenesis. To decipher the deregulated oncogenic gene network on hsa21, we conducted a shRNA-based viability screening.GATA1s-mutated DS-AMKL cell line CMK as well as non-DS-AML cell lines (K562, M-07) as control were lentivirally transduced and the effect of the knock-down was evaluated by cell viability and proliferation assays. Upon knock-down we found 42 genes conferring a profound selective growth disadvantage in DS-AMKL cell lines. Interestingly, 31 out of those candidate genes are located in one particular chromosomal region (21q22.1-21q22.3) and in addition 11 (out of 14 tested) are overexpressed in DS-AMKL compared to non-DS-AMKL.In a secondary functional validation screening the effects of the knock-down on the cell lines were analyzed by competition assays, apoptosis assays and cell viability assays as well as colony forming assays. Furthermore, differentiation and morphology were characterized using immunophenotyping and cytospins, respectively. We could demonstrate that the potential oncogenes participate in different cellular processes affecting proliferation, cell viability, apoptosis or differentiation.To further delineate the impact of 11 selected candidates on normal hematopoiesis, we characterized their effects in gain- and loss-of-function studies (confirmed by qRT-PCR) using CD34+ hematopoietic stem and progenitor cells (HSPCs). Four of those genes (USP25 [ubiquitin specific peptidase 25], BACH1 [BTB and CNC homology 1, basic leucine zipper transcription factor 1], U2AF1 [U2 small nuclear RNA auxiliary factor 1] and C21orf33) inhibited megakaryocytic and erythroid in vitro differentiation upon knockdown. The fraction of cells expressing early and late megakaryocytic markers CD41 and CD42b or early erythroid marker CD36 was reduced by 2-20-fold (P<0.001). Inversely, the knock-down of those four genes and two other genes (ATP5O [ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit] and C21orf45) enhanced the myeloid differentiation propensity of CD34+-HSPCs (2.1-13.4-fold increase of CD14+-monocytic cells, P<0.001). The opposite effect was observed in gain-of-function studies. Ectopic expression of six genes (hU2AF1, mC21orf33, hIFNGR2 [interferon gamma receptor 2], hWDR4 [WD repeat domain 4] or mGABPA [GA binding protein transcription factor, alpha subunit 60kDa]) resulted in a radical switch in lineage commitment with a drastic change from erythroid to megakaryocytic differentiation (CD41+ 1.7-2.4-fold increase, P<0.001, CD235a+ [late erythroid marker] 3-300-fold reduction, P<0.001).Thus, we found a remarkable number of genes regulating erythroid and megakaryocytic differentiation as well as proliferation in normal hematopoiesis. Given the genetic background during trisomy 21-mediated leukemogenesis, we propose a complex interactive network located in one particular region on hsa21. Deregulation of this network might result in synergistic effects on hematopoietic differentiation, which promotes transformation of GATA1s-mutated fetal hematopoietic progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Species-Specific Functions For Long Noncoding RNAs During Erythropoeisis

Mammalian genomes encode thousands of long noncoding (lnc) RNAs, although the extent to which lncRNAs regulate biological functions is not known. Indeed, most lncRNAs show low levels of conservation among mammals, which could suggest limited or species-restricted functions. We used RNA-seq to identify 683 and 1189 lncRNAs expressed during mouse and human erythropoiesis respectively, of which ∼25% are erythroid-specific. Primary cell chromatin immunoprecipitation (ChIP) studies indicate that most mouse erythroid lncRNAs arise from classical promoters, and their genomic loci are bound by the hematopoietic transcription factors GATA1 and SCL/TAL1. Using the G1E-ER4 cell line, which has an inducible form of GATA1, we found that lncRNAs whose genomic loci are bound by GATA1 in primary cells are upregulated on GATA1 induction in the cell line, similar to coding genes.We compared RNA-Seq transcriptomes of erythroblasts from 8 different mice strains and showed that lncRNA expression is highly conserved within strains of the same species. A comparison of the human and mouse lncRNA transcriptomes showed that only 15% of mouse erythroblast lncRNAs have detectable syntenic transcripts in human erythroblasts, and that most lncRNAs are not detectable in humans.To identify functional lncRNAs, we used RNA interference in primary erythroblasts to analyze the functions of 21 abundantly expressed murine erythroid lncRNAs, 15 of which are mouse specific and 6 of which have orthologous transcripts in humans. Suppression of seven different lncRNAs inhibited various aspects of erythroid maturation including loss of cell volume, down-regulation of the surface marker CD44, and enucleation. The suppression of enucleation ranged from 25% reduction to 75% reduction of enucleation demonstrated with at least 2 separate shRNAs for each of the 7 lncRNAs (p<0.0005) (Figure 1). Of the seven lncRNAs that have a demonstrable function in murine erythropoiesis, none are expressed in human erythroblasts, indicating that lack of conservation between different species does not necessarily predict a lack of function. These results reflect marked evolutionary differences between protein-coding genes and lncRNAs, and indicate that the latter exert tissue- and species-specific roles in mammalian development. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Zinc Finger Protein 148 Is Dispensable for Primitive and Definitive Hematopoiesis in Mice

Hematopoiesis is regulated by transcription factors that induce cell fate and differentiation in hematopoietic stem cells into fully differentiated hematopoietic cell types. The transcription factor zinc finger protein 148 (Zfp148) interacts with the hematopoietic transcription factor Gata1 and has been implicated to play an important role in primitive and definitive hematopoiesis in zebra fish and mouse chimeras. We have recently created a gene-trap knockout mouse model deficient for Zfp148, opening up for analyses of hematopoiesis in a conventional loss-of-function model in vivo. Here, we show that Zfp148-deficient neonatal and adult mice have normal or slightly increased levels of hemoglobin, hematocrit, platelets and white blood cells, compared to wild type controls. Hematopoietic lineages in bone marrow, thymus and spleen from Zfp148 gt/gt mice were further investigated by flow cytometry. There were no differences in T-cells (CD4 and CD8 single positive cells, CD4 and CD8 double negative/positive cells) in either organ. However, the fraction of CD69- and B220-positive cells among lymphocytes in spleen was slightly lower at postnatal day 14 in Zfp148 gt/gt mice compared to wild type mice. Our results demonstrate that Zfp148-deficient mice generate normal mature hematopoietic populations thus challenging earlier studies indicating that Zfp148 plays a critical role during hematopoietic development.

GATA-1 genome-wide occupancy associates with distinct epigenetic profiles in mouse fetal liver erythropoiesis

We report the genomic occupancy profiles of the key hematopoietic transcription factor GATA-1 in pro-erythroblasts and mature erythroid cells fractionated from day E12.5 mouse fetal liver cells. Integration of GATA-1 occupancy profiles with available genome-wide transcription factor and epigenetic profiles assayed in fetal liver cells enabled as to evaluate GATA-1 involvement in modulating local chromatin structure of target genes during erythroid differentiation. Our results suggest that GATA-1 associates preferentially with changes of specific epigenetic modifications, such as H4K16, H3K27 acetylation and H3K4 di-methylation. Furthermore, we used random forest (RF) non-linear regression to predict changes in the expression levels of GATA-1 target genes based on the genomic features available for pro-erythroblasts and mature fetal liver-derived erythroid cells. Remarkably, our prediction model explained a high proportion of 62% of variation in gene expression. Hierarchical clustering of the proximity values calculated by the RF model produced a clear separation of upregulated versus downregulated genes and a further separation of downregulated genes in two distinct groups. Thus, our study of GATA-1 genome-wide occupancy profiles in mouse primary erythroid cells and their integration with global epigenetic marks reveals three clusters of GATA-1 gene targets that are associated with specific epigenetic signatures and functional characteristics.