Human GATA-1 Research Articles

GATA6 regulates WNT and BMP programs to pattern precardiac mesoderm during the earliest stages of human cardiogenesis.

Haploinsufficiency for GATA6 is associated with congenital heart disease (CHD) with variable comorbidity of pancreatic or diaphragm defects, although the etiology of disease is not well understood. Here, we used cardiac directed differentiation from human embryonic stem cells (hESCs) as a platform to study GATA6 function during early cardiogenesis. GATA6 loss-of-function hESCs had a profound impairment in cardiac progenitor cell (CPC) specification and cardiomyocyte (CM) generation due to early defects during the mesendoderm and lateral mesoderm patterning stages. Profiling by RNA-seq and CUT&RUN identified genes of the WNT and BMP programs regulated by GATA6 during early mesoderm patterning. Furthermore, interactome analysis detected GATA6 binding with developmental transcription factors and chromatin remodelers suggesting cooperative regulation of cardiac lineage gene accessibility. We show that modulating WNT and BMP inputs during the first 48 hours of cardiac differentiation is sufficient to partially rescue CPC and CM defects in GATA6 heterozygous and homozygous mutant hESCs. This study provides evidence of the regulatory functions for GATA6 directing human precardiac mesoderm patterning during the earliest stages of cardiogenesis to further our understanding of haploinsufficiency causing CHD and the co-occurrence of cardiac and other organ defects caused by human GATA6 mutations.

Dysregulated expression of GATA2 and GATA6 transcription factors in adenomyosis: implications for impaired endometrial receptivity

To study the effect of adenomyosis on the localized expression of the GATA binding proteins 2 and 6 (GATA2 and GATA6) zinc-finger transcription factors that are involved in proliferation of hematopoietic and endocrine cell lineages, cell differentiation, and organogenesis, potentially leading to impaired endometrial implantation. Laboratory based experimental study. Academic hospital and laboratory. Human endometrial stromal cells (HESCs) of reproductive age patients, 18-45 years of age, with adenomyosis were compared with patients with no pathology and leiomyomatous uteri as controls (n = 4 in each group, respectively). Additionally, midsecretory phase endometrial sections were obtained from patients with adenomyosis and control patients with leiomyoma (n = 8 in each group, respectively). GATA2 and GATA6 immunohistochemistry and H-SCORE were performed on the midsecretory phase endometrial sections from adenomyosis and leiomyoma control patients (n = 8 each, respectively). Control and adenomyosis patient HESC cultures were treated with placebo or 10-8 M estradiol (E2), or decidualization media (EMC) containing 10-8 M E2, 10-7 M medroxyprogesterone acetate, and 5 × 10-5 M cAMP for 6 and 10 days. Additionally, control HESC cultures (n = 4) were transfected with scrambled small interfering RNA (siRNA) (control) or GATA2-specific siRNAs for 6 days while adenomyosis HESC cultures (n = 4) were transfected with human GATA2 expression vectors to silence or induce GATA2 overexpression. Immunohistochemistry was performed to obtain GATA2 and GATA6 H-SCORES in adenomyosis vs. control patient endometrial tissue. Expression of GATA2, GATA6, insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), progesterone receptor (PGR), estrogen receptor 1 (ESR1), leukemia inhibitory factor (LIF), and Interleukin receptor 11 (IL11R) messenger RNA (mRNA) levels were analyzed using by qPCR with normalization to ACTB. Silencing and overexpression experiments also had the corresponding mRNA levels of the above factors analyzed. Western blot analysis was performed on isolated proteins from transfection experiments. Immunohistochemistry revealed an overall fourfold lower GATA2 and fourfold higher GATA6 H-SCORE level in the endometrial stromal cells of patients with adenomyosis vs. controls. Decidual induction with EMC resulted in significantly lower GATA2, PGR, PRL and IGFBP1 mRNA levels in HESC cultures from patients with adenomyosis patient vs. controls. Leukemia inhibitory factor and IL11R mRNA levels were also significantly dysregulated in adenomyosis HESCs compared with controls. . Silencing of GATA2 expression in control HESCs induced an adenomyosis-like state with significant reductions in GATA2, increases in GATA6 and accompanying aberrations in PGR, PRL, ESR1 and LIF levels. Conversely, GATA2 overexpression via vector in adenomyosis HESCs caused partial restoration of the defective decidual response with significant increases in GATA2, PGR, PRL and LIF expression. In-vivo and in-vitro experiment results demonstrate that there is an overall inverse relationship between endometrial GATA2 and GATA6 levels in patients with adenomyosis who have diminished GATA2 levels and concurrently elevated GATA6 levels. Additionally, lower GATA2 and higher GATA6 levels, together with aberrant levels of important receptors and implantation factors, such as ESR1, PGR, IGFBP1, PRL, LIF, and IL11R mRNA in HESCs from patients with adenomyosis or GATA2-silenced control HESCs, support impaired decidualization. These effects were partially restored with GATA2 overexpression in adenomyosis HESCs, demonstrating a potential therapeutic target.

Single-nucleotide variants within heart enhancers increase binding affinity and disrupt heart development.

Transcriptional enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with phenotypic diversity, evolutionary adaptations, and disease. Pinpointing which enhancer variants contribute to changes in gene expression and phenotypes is a major challenge. Here, we find that suboptimal or low-affinity binding sites are necessary for precise gene expression during heart development. Single-nucleotide variants (SNVs) can optimize the affinity of ETS binding sites, causing gain-of-function (GOF) gene expression, cell migration defects, and phenotypes as severe as extra beating hearts in the marine chordate Ciona robusta. In human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a SNV within a human GATA4 enhancer increases ETS binding affinity and causes GOF enhancer activity. The prevalence of suboptimal-affinity sites within enhancers creates a vulnerability whereby affinity-optimizing SNVs can lead to GOF gene expression, changes in cellular identity, and organismal-level phenotypes that could contribute to the evolution of novel traits or diseases.

Abstract 3155: Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse

Abstract The purpose of this study is to identify key regulatory pathways that potentially drive abnormal gene expression program in relapsed Acute Myeloid Leukemia (AML) patients, by integrative computational analyses on multi-omics molecular profiles. Relapsed AML remains a clinical challenge. Epigenetic heterogeneity may contribute to transcriptional dysregulation and disease progression in AML. However, what specific transcriptional programs and potential regulatory mechanisms contribute to disease relapse are not yet well understood. To characterize the global transcriptional landscapes in relapsed AML, we integrated genomics data from two cohorts of matched diagnosis and relapse patient specimens. We identified 5,416 differentially expressed genes (DEGs) between diagnosis and relapse in Cohort I. Unsupervised clustering yielded three distinct DEG groups: group A, B and C genes that were predominantly (88%) down-regulated, divergently regulated, or predominantly (65%) up-regulated, respectively, upon relapse. The expression pattern of all DEGs separated the patients into two clusters, most robustly by Group B genes. Interestingly, the majority of DEGs did not associate with changes in gene promoter methylation. Similar patterns were observed in Cohort II. We used Binding Analysis for Regulation of Transcription (BART) to identify transcriptional regulators (TRs) that potentially regulated the DEGs not associated with DNA methylation changes, and assessed the differential expression of identified TRs during disease progression. PU.1 was identified as a potential TR for Group A genes and was down-regulated upon relapse. GATA1 and TAL1 were identified as regulating Group B genes and were up-regulated in patient cluster1 and down-regulated in cluster2, consistent with the expression pattern of Group B genes. RBBP5 was a top predicted TR for Group C genes and was up-regulated upon relapse. We next validated the potential functionality of those predicted factors. In NSG mice transplanted with a human AML specimen, TAL1 and GATA1 were downregulated in AML cells collected four weeks after chemotherapy treatment, and were inferred as TRs for the down-regulated genes, similar to the patient data. PU.1 was inferred as regulating the up-regulated genes. Furthermore, we found that the level of differential expression of TAL1, GATA1, and PU.1 in each patient specimen associated with the correlation of DEG profiles between the patient specimen and TR perturbation in human-derived hematopoietic cell lines. Our results support the possibility that in some AML patients, TRs with roles in hematopoiesis and leukemia might contribute to disease relapse. Further mechanistic studies deciphering the molecular and phenotypic events facilitated by these TRs will yield significant insight into disease biology and possible therapeutic targeting approaches in relapsed AML. Citation Format: Zhenjia Wang, Yaseswini Neelamraju, Cem Meydan, Nicholas Dunham, Jorge Gandara, Tak Lee, Subhash Prajapati, Franck Rapaport, Caroline Sheridan, Paul Zumbo, Michael Becker, Lars Bullinger, Martin Carroll, Richard D’Andrea, Richard Dillon, Ross Levine, Christopher E. Mason, Ari Melnick, Donna Neuberg, Stefan Bekiranov, Chongzhi Zang, Francine E. Garrett-Bakelman. Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3155.

Regulated Expression of GATA1 As a Gene Therapy Cure for Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a bone marrow failure disorder characterized by impaired red blood cell production leading to severe anemia. The only treatment options are regular transfusions, corticosteroids, or bone marrow transplant, and each has variable effectiveness and considerable morbidity. More effective, safer, and permanent cures for DBA are lacking, but few drugs are in active development. DBA is genetically heterogenous, characterized by loss-of-function mutations in one copy of at least 26 different ribosomal protein (RP) genes that alters the mRNA translation of a select group of genes, most notably the hematopoietic master regulator GATA1. Overexpression of GATA1 in hematopoietic stem and progenitor cells (HSPCs) from patients with DBA rescues the erythroid differentiation defects in vitro, but unregulated GATA1 expression in hematopoietic stem cells (HSCs) promotes premature erythroid differentiation at the expense of long-term (LT-) HSC maintenance. Gene therapy is an attractive strategy to achieve a cure for DBA, but a traditional gene therapy approach that overexpresses a functional copy of a mutated gene is not the ideal option, as it would require the development and validation of dozens of gene therapy vectors, each with a copy of one of the mutated DBA genes. Here, we report a unified gene therapy strategy, using developmentally-regulated and highly restricted expression of GATA1 that would be curative in most, if not all, patients with DBA, regardless of the underlying disease-causing mutation. To be a cure for DBA, a GATA1 gene therapy vector must incorporate into the genome of undifferentiated LT-HSCs but only drive robust GATA1 expression in early erythroid progenitors after lineage commitment. To design a vector with lineage-restricted expression, we identified accessible chromatin regions upstream of GATA1 that only gain accessibility in differentiating erythroid cells, but not in upstream HSPCs. Using these selectively accessible regions concatenated together, we constructed a human GATA1 enhancer (hG1E) element and used it to drive GATA1 and/or GFP expression from a lentiviral backbone. To ensure erythroid-restricted expression of the hG1E-GATA1-GFP vector, we infected CD34+ HSCs from healthy human donors and cultured them in conditions that either allowed HSC maintenance or promoted erythrocyte differentiation. We observed very low GFP expression in bulk HSCs and essentially no expression in the subpopulation of immunophenotypic LT-HSCs. However, more than 80% of committed erythroid cells demonstrated high levels of GFP expression, confirming the fidelity of lineage-restricted expression from the hG1E-GATA1 vector. To examine whether lineage-restricted GATA1 expression confers a functional correction of the erythroid maturation block in DBA, we used CRISPR editing to recapitulate RPS19 haploinsufficiency in human HSPCs. In RPS19 edited samples that were infected with a control GFP virus, we observed a progressive depletion over time of RPS19 edits as the cells underwent erythroid differentiation, consistent with the notion that RPS19 editing leads to impaired erythroid differentiation and are selected against. In contrast, RPS19-edited and hG1E-GATA1 treated samples had a more than 10-fold higher preservation of RPS19 edits in the bulk population and in erythroid colonies derived from single progenitor clones, indicating that regulated GATA1 expression could overcome RPS19 haploinsufficiency. To validate the results from the CRISPR model of DBA, we sought to correct the erythroid maturation block in primary HSCs from a DBA patient with a pathogenic mutation in RPL5. There was skewed differentiation of GFP-infected RPL5+/- HSCs away from the erythroid lineage and toward myeloid and megakaryocyte lineages, but regulated GATA1 expression from the hG1E-GATA1 vector reversed that skewing. There was a 10-fold increase in the ratio of erythroid to non-erythroid cells, and a significant expansion of erythroid cells in the hG1E-GATA1 treated patient cells. Preclinical assessment in additional patient samples both in vitro and in vivo is ongoing. Collectively, our preclinical data demonstrate that lineage-restricted expression of GATA1 is sufficient to rescue the impaired erythroid differentiation in DBA regardless of the underlying genetic lesion. These results will set the stage for the first in-human gene therapy trial for DBA.

489 Whole-transcriptome sequencing-based concomitant detection of viral and human genetic determinants of cutaneous lesions

Severe viral infections of the skin can occur in patients with inborn errors of immunity (IEI). We report an all-in-one whole-transcriptome sequencing-based method by RNA-Seq on a single skin biopsy for concomitant identification of the cutaneous virome and underlying IEI. Skin biopsies were obtained from normal and lesional skin from patients with cutaneous infections suspected to be of viral origin. RNA-Seq was utilized as the first-tier strategy for unbiased human genome-wide rare variant detection. Reads unaligned to the human genome were utilized for the exploration of 926 different viruses in a viral genome catalog. In nine families studied, the patients carried pathogenic variants in six human IEI genes, including IL2RG, WAS, CIB1, STK4, GATA2, and DOCK8. Gene expression profiling also confirmed pathogenicity of the human variants and permitted genome-wide homozygosity mapping which assisted in identification of candidate genes in consanguineous families. This automated, all-in-one computational pipeline, called VirPy, enables simultaneous detection of the viral triggers and the human genetic variants underlying skin lesions in patients with suspicion of IEI and viral dermatosis.

Whole-transcriptome sequencing-based concomitant detection of viral and human genetic determinants of cutaneous lesions.

Severe viral infections of the skin can occur in patients with inborn errors of immunity (IEI). We report an all-in-one whole-transcriptome sequencing–based method by RNA-Seq on a single skin biopsy for concomitantly identifying the cutaneous virome and the underlying IEI. Skin biopsies were obtained from healthy and lesional skin from patients with cutaneous infections suspected to be of viral origin. RNA-Seq was utilized as the first-tier strategy for unbiased human genome-wide rare variant detection. Reads unaligned to the human genome were utilized for the exploration of 926 viruses in a viral genome catalog. In 9 families studied, the patients carried pathogenic variants in 6 human IEI genes, including IL2RG, WAS, CIB1, STK4, GATA2, and DOCK8. Gene expression profiling also confirmed pathogenicity of the human variants and permitted genome-wide homozygosity mapping, which assisted in identification of candidate genes in consanguineous families. This automated, online, all-in-one computational pipeline, called VirPy, enables simultaneous detection of the viral triggers and the human genetic variants underlying skin lesions in patients with suspected IEI and viral dermatosis.

Impairment of the Hypothalamus-Pituitary-Thyroid Axis Caused by Naturally Occurring GATA2 Mutations In Vitro.

The transcription factor GATA2 regulates gene expression in several cells and tissues, including hematopoietic tissues and the central nervous system. Recent studies revealed that loss-of-function mutations in GATA2 are associated with hematological disorders. Our earlier in vitro studies showed that GATA2 plays an essential role in the hypothalamus–pituitary–thyroid axis (HPT axis) by regulating the genes encoding prepro-thyrotropin-releasing hormone (preproTRH) and thyroid-stimulating hormone β (TSHβ). However, the effect of GATA2 mutants on the transcriptional activity of their promoters remains unelucidated. In this study, we created five human GATA2 mutations (R308P, T354M, R396Q, R398W, and S447R) that were reported to be associated with hematological disorders and analyzed their functional properties, including transactivation potential and DNA-binding capacity toward the preproTRH and the TSHβ promoters. Three mutations (T354M, R396Q, and R398W) within the C-terminal zinc-finger domain reduced the basal GATA2 transcriptional activity on both the preproTRH and the TSHβ promoters with a significant loss of DNA binding affinity. Interestingly, only the R398W mutation reduced the GATA2 protein expression. Subsequent analysis demonstrated that the R398W mutation possibly facilitated the GATA2 degradation process. R308P and S447R mutants exhibited decreased transcriptional activity under protein kinase C compared to the wild-type protein. In conclusion, we demonstrated that naturally occurring GATA2 mutations impair the HPT axis through differential functional mechanisms in vitro.

Gata2 heterozygous mutant mice exhibit reduced inflammatory responses and impaired bacterial clearance

SummaryInfectious diseases continually pose global medical challenges. The transcription factor GATA2 establishes gene networks and defines cellular identity in hematopoietic stem/progenitor cells and in progeny committed to specific lineages. GATA2-haploinsufficient patients exhibit a spectrum of immunodeficiencies associated with bacterial, viral, and fungal infections. Despite accumulating clinical knowledge of the consequences of GATA2 haploinsufficiency in humans, it is unclear how GATA2 haploinsufficiency compromises host anti-infectious defenses. To address this issue, we examined Gata2-heterozygous mutant (G2Het) mice as a model for human GATA2 haploinsufficiency. In vivo inflammation imaging and cytokine multiplex analysis demonstrated that G2Het mice had attenuated inflammatory responses with reduced levels of inflammatory cytokines, particularly IFN-γ, IL-12p40, and IL-17A, during lipopolysaccharide-induced acute inflammation. Consequently, bacterial clearance was significantly impaired in G2Het mice after cecal ligation and puncture-induced polymicrobial peritonitis. These results provide direct molecular insights into GATA2-directed host defenses and the pathogenic mechanisms underlying observed immunodeficiencies in GATA2-haploinsufficient patients.

Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli.

Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development. In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers. To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E. coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography. The identity of GATA4 protein was confirmed using western blotting and mass spectrometry. Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices. Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus. Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy.

ELT-2 promotes O-GlcNAc transferase OGT-1 expression to modulate Caenorhabditis elegans lifespan.

O-GlcNAc transferase (OGT) is the enzyme catalyzing protein O-GlcNAcylation by addition of a single O-linked-β-N-acetylglucosamine molecule (O-GlcNAc) to nuclear and cytoplasmic targets, and it uses uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a donor. As UDP-GlcNAc is the final product of the nutrient-sensing hexosamine signaling pathway, overexpression or knockout of ogt in mammals or invertebrate models influences cellular nutrient-response signals and increases susceptibility to chronic diseases of aging. Evidence shows that OGT expression levels decrease in tissues of older mice and rats. However, how OGT expression is modulated in the aging process remains poorly understood. In Caenorhabditis elegans, the exclusive mammalian OGT ortholog OGT-1 is crucial for lifespan control. Here, we observe that worm OGT-1 expression gradually reduces during aging. By combining prediction via the "MATCH" algorithm and luciferase reporter assays, GATA factor ELT-2, the homolog of human GATA4, is identified as a transcriptional factor driving OGT-1 expression. Chromatin immunoprecipitation-quantitative polymerase chain reaction and electrophoretic mobility shift assays show ELT-2 directly binds to and activates the ogt-1 promoter. Knockdown of elt-2 decreases the global O-GlcNAc modification level and reduces the lifespan of wild-type worms. The reduction in lifespan caused by elt-2 RNA interference is abrogated by the loss of ogt-1. These results imply that GATA factors are able to activate OGT expression, which could be beneficial for longevity and the development of therapeutic treatment for aging-related diseases.

GATA2-Dependent Developmental and Regenerative Networks

Coding and regulatory human GATA2 mutations that deregulate protein expression and/or function cause immunodeficiency that often progresses to MDS/AML (McReynolds et al., 2018). In the mouse, decreased GATA2 expression impairs hematopoietic stem/progenitor cell (HSPC) genesis and function (de Pater et al., 2013; Gao et al., 2013; Tsai et al., 1994). While prior studies demonstrated Gata2 +9.5 and -77 enhancers are essential for HSC emergence (+9.5) and/or progenitor cell fate (+9.5 and -77) (Johnson et al., 2012; Johnson et al., 2015; Mehta et al., 2017) and hematopoietic regeneration (+9.5) (Soukup et al., 2019), the mechanisms mediating these processes are not completely established. The -77 enhancer is required for fetal liver progenitors to undergo erythroid, megakaryocytic, granulocytic and monocytic differentiation. By contrast, progenitors with a -77 homozygous deletion (-77-/-) exhibit a predominant monocytic cell fate and generate macrophages ex vivo (Johnson et al., 2015). Using multiomic and single-cell strategies, we asked how this enhancer orchestrates a balance between fate-promoting and -suppressing circuitry in cell populations and single cells. Quantitative proteomics was conducted to discover the -77-regulated protein ensemble conferring multiple fates in a myeloid progenitor population [Common Myeloid Progenitor (CMP) and Granulocyte-Monocyte Progenitor (GMP)] from E14.5 fetal liver of -77+/+ and -77-/- mouse embryos. -77-/- progenitors exhibited decreased levels of GATA2 (4.7-fold) and proteins generated from GATA2 target genes (GATA1: 51-fold; HDC: 52-fold). The 202 proteins upregulated in -77-/- progenitors highlighted immune and inflammatory mechanisms, while the 232 downregulated proteins were linked to erythroid, megakaryocyte and granulocyte biology, indicative of loss of these fate potentials. Innate immune machinery was upregulated in -77-/- vs. -77+/+ progenitors, including interferon (IFN) signaling pathway components such as the IFN-inducible transcription factor and critical monocytic differentiation determinant Interferon Regulatory Factor 8 (IRF8; 2.7 fold higher) (Kurotaki et al., 2013) and diverse pathogen sensors. Expressing GATA2 at physiological levels in -77-/- progenitors normalized the aberrant transcriptome. Since -77 deletion downregulated Gata2 and upregulated Irf8, we tested whether this opposing expression pattern occurs in distinct and/or identical cells in the population using single cell transcriptomics. -77 deletion decreased Gata2 expression, which was anti-correlative with Irf8, and detailed single cell analyses indicated that -77 loss downregulates GATA2, corrupting the transcriptome/proteome, Irf8 expression increases, and IRF8 enables or drives the predominant monocytic differentiation. To determine how GATA2-dependent mechanisms governing progenitor fate relate to those guiding HSPC expansion and differentiation during regeneration, we utilized our +9.5 human disease Ets motif mutation that abrogates myeloablation-dependent GATA2 induction and hematopoietic regeneration in bone marrow (Soukup et al., 2019). While population RNA-seq with Lin-Sca1+c-Kit+ (LSK) cells revealed little to no perturbations in the steady-state, 5-FU treatment of mutants led to altered expression of only 14% of the transcripts regulated in regenerating wild type cells (423/2974). Gene Ontology analysis of differentially expressed genes indicated that genes dysregulated by the Ets motif mutation included those linked to cell cycle regulation and cellular proliferation. As LSKs consist of LT-HSCs, ST-HSCs, and MPPs, we used single-cell transcriptomics to elucidate defective regenerative circuits in individual cells. Analysis of 17000-20000 wild type and mutant LSK cells revealed GATA2-dependent mechanisms distinct from those mediating progenitor cell fate control in the fetal liver. These studies have revealed context-dependent GATA2 mechanisms governing developmental and regenerative hematopoiesis, which will enable the development of strategies to detect, diagnose, and treat GATA2-linked blood diseases. Disclosures No relevant conflicts of interest to declare.

Inhibition of GATA2 restrains cell proliferation and enhances apoptosis and chemotherapy mediated apoptosis in human GATA2 overexpressing AML cells

GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML.

Spiral ganglion cell degeneration-induced deafness as a consequence of reduced GATA factor activity.

Zinc-finger transcription factors GATA2 and GATA3 are both expressed in the developing inner ear, although their overlapping versus distinct activities in adult definitive inner ear are not well understood. We show here that GATA2 and GATA3 are co-expressed in cochlear spiral ganglion cells and redundantly function in the maintenance of spiral ganglion cells and auditory neural circuitry. Notably, Gata2 and Gata3 compound heterozygous mutant mice had a diminished number of spiral ganglion cells due to enhanced apoptosis, which resulted in progressive hearing loss. The decrease in spiral ganglion cellularity was associated with lowered expression of neurotrophin receptor TrkC that is an essential factor for spiral ganglion cell survival. We further show that Gata2 null mutants that additionally bear a Gata2 YAC (yeast artificial chromosome) that counteracts the lethal hematopoietic deficiency due to complete Gata2 loss nonetheless failed to complement the deficiency in neonatal spiral ganglion neurons. Furthermore, cochlea-specific Gata2 deletion mice also had fewer spiral ganglion cells and resultant hearing impairment. These results show that GATA2 and GATA3 redundantly function to maintain spiral ganglion cells and hearing. We propose possible mechanisms underlying hearing loss in human GATA2- or GATA3-related genetic disorders.

Single Cell Transcriptome Analysis of GATA2 Deficiency in Hematopoiesis Modeled with Induced Pluripotent Stem Cells

GATA2 deficiency is a rare, inherited or sporadic genetic disorder characterized by variable onset of a pleomorphic constellation of immune, hematologic and lymphatic abnormalities linked to heterozygous mutations in the Gata2 gene. Patients develop monocyte, B cell, NK cell and dendritic cell deficiencies resulting in vulnerabilities to unusual infections. Patients with GATA2 deficiency also frequently progress to bone marrow failure, myelodysplastic syndrome and/or acute myelogenous leukemia. GATA proteins are transcription factors with central roles in early embryonic development and lineage specification. GATA2 is a master regulator of hematopoiesis, implicated in the initial generation and maintenance of hematopoietic stem cells (HSC).Murine models recapitulate the human phenotype incompletely: GATA2 heterozygous knockout mice do not manifest loss of monocyte, B cells or NK cells; however, serial repopulation assays show decreased engraftment potential. Direct studies of primary HSC from patients with GATA2 deficiency are challenging due to the generally hypocellular marrow. We hypothesized that human pluripotent stem cells, particularly patient-specific iPSC, could be used to study potential developmental defects in GATA2 deficiency, overcoming a lack of primary HSC. In order to gain insights into the impact of human GATA2 deficiency on hematopoietic differentiation, we compared the single cell transcriptomes of HSPC differentiated from (i) iPSCs from a patient with GATA2 deficiency due to a mutation p.R337X (c.1009C>T) (ii) isogenic iPSCs created via homology-directed repair of Gata2 p.R337X, (iii) iPSCs from a healthy control and (iv) isogenic Gata2 heterozygous mutant iPSCs with monoallelic frameshift mutations in the second zinc finger domain.Mesodermal and hematopoietic differentiation was performed under feeder-free, defined media conditions. At day 0, iPSCs were plated in mesodermal induction media containing VEGF, SCF, Activin A and Y27632 in STEMdiff APEL media. Mesodermal induction was continued until day 4, when embryoid bodies were cultured in hematopoietic specification media with SCF, FLT3L, IL3, IL6, G-CSF and BMP4 until day 16, when CD34+CD45+ iPSC-derived hematopoietic stem and progenitor cells (iHSC) were enumerated and sorted by fluorescence-activated cell sorting.Single cell RNA-seq was performed using the 10XGenomics Chromium platform and primary analysis via CellRanger. Scater was used to filter outlier cells. Seurat as used to compute multiple manifold alignment and differential gene expression. Cell classification, pseudotemporal ordering and branch point analysis were performed with monocle. URD was used to calculate confirmatory diffusion maps and pseudotemporal ordering.We analyzed 7,855 iHSPC (2952 from GATA2-deficient patient, 241 isogenic iHSPCs after repair of Gata2 mutation, 2,605 from a healthy volunteer and 2,057 from isogenic heterozygous Gata2 knockout iHSPCs) after filtering of outliers. We computed multiple manifold alignment to mitigate batch effects. Differential gene expression across Gata2 mutation status found that 42 out of 102 (42%) target genes of GATA2 (c.f. TRANSFAC database of curated transcription factor targets) were differentially expressed with adjusted p-values less than 0.05. Semi-supervised classification of cell-types and pseudotemporal ordering via monocle revealed two branch points, consistent with developmental branchings at the level of CLP and CMP multipotent progenitors. The numbers of cells along each branch was found to be statistically different (χ2=30.07, p-value = 3e-7) with the biggest differences noted in the lymphoid branch (state 4). Differential gene expression in this branch revealed a differential up-regulation of Notch1, CD69 and FKBPs and differential down-regulation of CD14.In conclusion, iPSC/iHSPC differentiation models combined with single cell transcriptome analysis may be a valuable tool to identify pathways responsible for impaired hematopoietic/lymphatic development in GATA2 deficiency. DisclosuresDunbar:National Institute of Health: Research Funding. Winkler:National Institute of Health: Research Funding.

Human GATA1 Driven By the Human Μicro LCR/β-Globin Promoter Rescues the Erythroid but Not the Megakaryocytic Phenotype Induced in Mice By the Gata1low Mutation

GATA1 is a member of the GATA family of transcription factors that exerts a concentration/dependent control on the differentiation of erythroid, megakaryocytic (MK), eosinophil and mast cells. Changes of GATA1 content also controls hematopoietic stem/progenitor cell proliferation and commitment. In fact, in addition to X-linked inherited disorders of the erythroid and/or MK lineage, GATA1 mutations are found in inherited and acquired MK leukaemia and myeloproliferative disorders.To study the regulation of the human β-globin locus, the Stamatoyannopoulos laboratory generated transgenic mice carrying human GATA1 driven by the human μLCR coupled with the β-globin promoter (hGATA1) (Li Q et al. PNAS 1997, 18;94:2444). These transgenic mice are born viable with normal phenotype and express hGATA1 at high levels in embryonic and fetal erythroblasts and barely detectable in the adult ones, raising the question whether in adult cells expression of the endogenous Gata1 may suppress the activity of the human μLCR- β-globin promoter.The aim of this study was to determine the effects of Gata1 on the activity of hGATA1 in mice. Since the Gata1null mutation is embryonically lethal, the hypothesis was tested by generating with standard genetic approaches double mutants carrying hGATA1 and the hypomorphic Gata1low mutation, a mutation that strongly reduces expression of Gata1 in adult hematopoietic cells (erythroblasts, MK, hematopoietic progenitors, others). If successful, these experiments would also indirectly assess the extent by which hGATA1 is capable to rescue the complex Gata1low phenotype. Gata1low mice, in fact, are born anemic and thrombocytopenic but recover from their anemia at 1 month by activating extramedullary hematopoiesis in spleen (Vannucchi et al Blood 2001;97:3040). Spleen erythropoiesis is however mostly ineffective since many of the erythroblasts present in this organ are in apoptosis (Tunel+). Gata1low mice remain thrombocytopenic all their life and with age develop myelofibrosis, a phenotype that resemble primary myelofibrosis, the most severe of the Phyladelphia-negative myeloproliferative neoplasms (Vannucchi et al Blood 2002;100:1123). In these mice, hematopoietic stem cells are detected in spleen, and not in bone marrow, and generate bipotent MK/erythroid progenitors (MEP) with a commitment program screwed toward the mast cell lineage and capable to generate in vitro at the single cell basis mast cell lines (Ghinassi et al. Blood 2007;109:1460-71).Double hGATA1Gata1low mutants were easily generated and were not anemic at birth. At 4 months, they expressed normal haematocrit but reduced blood platelet counts (Fig. 1A,B). RT-PCR analyses of prospectively isolated cell populations confermed that the transgene mRNA is barely detectable in cells from single hGATA1 mutants. By contrast, in the double mutants, hGATA1 mRNA was expressed by common myeloid progenitors, its expression increased by 10-fold in MEP, remained at the same levels expressed by MEP in erythroid cells (CD71+/Ter119+) but was barely detectable in MK (CD41+/CD61+). Expression of hGATA1 did not alter the levels of the endogenous Gata1 expressed by these populations that remained low.MEP purified from double hGATA1/Gata1low mice generated in culture more cells (fold increase=250 in Gata1low/hGATA1 cultures vs 50 in single hGATA1 or Gata1low cultures) which were mostly composed by erythroid precursors unable to generate cell lines.By contrast with single Gata1low mutants, by confocal microscopy, GATA1 was readily detectable in erythroid cells from bone marrow and spleen that these cells were not Tunel+ (Fig. 1C), indicating that hGATA1 reduced ineffective erythropoiesis.GATA1 protein remained not detectable in MK from the same tissues (Fig. 1C) and these cells displayed the abnormal morphology characteristic of the retarded maturation of Gata1low MK. Furthermore, double hGATA1/Gata1low mice, as the single Gata1low ones, developed myelofibrosis in bone marrow (by Gomori staining) at 8 months, confirming that myelofibrosis is driven by MK abnormalities.In conclusion, hGATA1 is expressed by adult CMP, MEP and erythroblasts in a genetic Gata1low background rescuing the MEP and erythroid phenotype induced by the hypomorphic mutation. hGATA1 is not expressed and does not rescue the MK phenotype of Gata1low mice, highlighting the specificity of the β-globin promoter for erythroid cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GATA6 Regulates Aortic Valve Remodeling, and Its Haploinsufficiency Leads to Right-Left Type Bicuspid Aortic Valve.

Bicuspid aortic valve (BAV), the most common congenital heart defect affecting 1% to 2% of the population, is a major risk factor for premature aortic valve disease and accounts for the majority of valve replacement. The genetic basis and mechanisms of BAV etiology and pathogenesis remain largely undefined. Cardiac structure and function was assessed in mice lacking a Gata6 allele. Human GATA6 gene variants were analyzed in 452 BAV cases from the BAV consortium and 1849 controls from the Framingham GWAS (Genome Wide Association Study). GATA6 expression was determined in mice and human tissues using quantitative real-time polymerase chain reaction and immunohistochemistry. Mechanistic studies were carried out in cultured cells. Gata6 heterozygous mice have highly penetrant right-left (RL)-type BAV, the most frequent type in humans. GATA6 transcript levels are lower in human BAV compared with normal tricuspid valves. Mechanistically, Gata6 haploinsufficiency disrupts valve remodeling and extracellular matrix composition through dysregulation of important signaling molecules, including matrix metalloproteinase 9. Cell-specific inactivation of Gata6 reveals an essential role for GATA6 in secondary heart field myocytes because loss of 1 Gata6 allele from Isl- 1-positive cells-but not from endothelial or neural crest cells-recapitulates the phenotype of Gata6 heterozygous mice. The data identify a new cellular and molecular mechanism underlying BAV. The availability of an animal model for the most frequent human BAV opens the way for the elucidation of BAV pathogenesis and the development of much needed therapies.

GATA4 Variants in Individuals With a 46,XY Disorder of Sex Development (DSD) May or May Not Be Associated With Cardiac Defects Depending on Second Hits in Other DSD Genes.

Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms.

GATA4 is a transcriptional regulator of R-spondin1 in Japanese flounder (Paralichthys olivaceus)

GATA4 is a well-known transcription factor of the GATA family implicated in regulation of sex determination and gonadal development in mammals. In this study, we cloned the full-length cDNA of Paralichthys olivaceus gata4 (Po-gata4). Phylogenetic, gene structure, and synteny analysis showed that Po-GATA4 is homologous to GATA4 of teleost and tetrapod. Po-gata4 transcripts were detected in Sertoli cells, spermatogonia, oogonia and oocytes, with higher transcript levels overall in the testis than the ovary. The promoter region of P. olivaceus R-spondin1was found to contain a GATA4-binding motif. Results of CBA (cleaved amplified polymorphic sequence-based binding assay) indicated that GATA4 could indeed bind to the promoter sequence of R-spondin1. Moreover, human GATA4 recombinant protein could upregulate R-spondin1 in P. olivaceus ovary cells and FBCs (flounder brain cell line). In FBCs, overexpression of Po-gata4 resulted in elevated transcript levels of R-spondin1. Taken together, our results indicate that Po-GATA4 is involved in gonadal development by regulating R-spondin1 expression.

Integrating Enhancer Mechanisms to Establish a Hierarchical Blood Development Program

GATA-2 levels must be stringently regulated to ensure normal hematopoiesis, and human GATA-2 mutations cause hematologic disorders. GATA-2-regulated enhancers differentially control Gata2 expression in hematopoietic stem/progenitor cells and are essential for hematopoiesis and embryonic development. Mechanisms underlying how the enhancers control Gata2 expression and GATA-2 instigated genetic networks in a cell-specific manner are not completely understood. Targeted deletion of an intronic Gata2 enhancer 9.5 kb downstream of the transcription start site (+9.5) abrogates HSC genesis in the aorta-gonad-mesonephros (AGM) region (Gao et al., JEM, 2013). By contrast, the -77 kb enhancer (-77) activates transcription in myeloid progenitors, and its deletion impairs progenitor differentiation (Johnson et al., Science Advances, 2015). To dissect relationships between the enhancers, we developed a compound heterozygous (CH) mouse model bearing +9.5 and -77 enhancer mutations on different Gata2 alleles. While the CH embryos were alive at E13.5, nearly all died by E14.5 (p = 3.58 x 10-5). Flow cytometric analyses and embryo confocal imaging demonstrated that CH embryos have modestly reduced HSC numbers in the fetal liver (2.9-fold) and the AGM (41%, p = 7.8 x 10-5), which was comparable to +9.5+/- embryos. Thus, -77 does not genetically interact with +9.5 to control HSC emergence. Flow cytometric analysis revealed that Lin-Sca1-Kit+ myelo-erythroid progenitors were 6.6-fold lower in CH vs. WT embryos (p = 1.8 x 10-11), with the difference involving disproportionate losses of GMP (8.6-fold; p = 3.7 x 10-6) and MEP (379-fold; p = 3.2 x 10-9). By contrast, +9.5+/- fetal livers had 2-fold fewer myeloid progenitors, which involved similar reductions of CMP (2.1-fold; p = 1 x 10-6), GMP (2.6-fold; p = 0.0007) and MEP (1.9-fold; p = 0.002). Consistent with the myelo-erythroid progenitor reductions and MEP depletion, CH fetal livers lacked BFU-E (p < 0.001) and CFU-GEMM (p < 0.001) in a colony assay. These results illustrate a genetic interaction between +9.5 and -77 in progenitors, but not HSCs, and a new paradigm in which both enhancers must reside on a single allele to generate MEPs.As erythroid precursor cells express GATA-2, we tested whether the -77 deletion impairs erythroid maturation due to a reduction in myelo-erythroid progenitors or due to a cell-autonomous requirement of the enhancer in erythroid precursors. -77-/- E14.5 fetal livers were pale and smaller than WT counterparts, and -77-/- fetal liver cellularity was reduced 7.2-fold (5.3 x 10-4). When liver size was taken into account, there was little difference in the number of E14.5 R1 cells in -77-/- liver vs. WT littermates (p = 0.31). However, -77-/- R2-R5 cells declined sharply (R2, 8.2-fold, p = 0.004; R3, 14-fold, p < 10-5; R4, 9.7-fold, p = 0.002; R5, 14-fold, p = 0.087). The mutant R1 cells were defective in forming BFU-Es and CFU-Es. Analysis of transcriptomes of purified 77-/- and WT R1 cells from E14.5 fetal livers revealed 2805 and 2519 upregulated and downregulated (p < 0.05) genes, respectively, in -77-/- R1 cells. The -77 enhancer conferred GATA-2 expression, which strongly upregulated GATA-1 and therefore a large GATA-1 target gene cohort. A comparison of WT and -77-/- R1 cell transcriptomes with those of early (Tgbfr3low) and late (Tgbfr3high) BFU-Es (Gao et al., Blood, 2016) revealed a -77-/- R1 signature that correlated with the early BFU-E signature (r = 0.73, p < 10-4) and negatively correlated with the late BFU-E signature (r = -0.42, p = 4 x 10-4) differing from WT cells. In addition to GATA-1 target gene alterations, 253 of the -77-activated genes were not GATA-1-regulated in the G1E-ER-GATA-1 system. These genes included Ryk, which encodes a non-canonical Wnt receptor, and had not been studied in erythroid cells. Two Ryk shRNAs significantly decreased BFU-Es and CFU-GMs in lineage-depleted fetal liver cells. Ongoing studies are integrating Ryk function into signaling circuits that control erythroid maturation and analyzing other -77-regulated targets predicted to constitute new regulators of erythroid cell maturation/function. Thus, loss of the -77 enhancer creates multi-faceted defects in erythroid precursors, involving deficiencies of constituents of signaling and transcriptional circuitry required to enable and drive erythroid maturation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.