GATA2 heterozygosity causes an epigenetic feedback mechanism resulting in myeloid and erythroid dysplasia.

The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.

The role of GATA2 in adult hematopoiesis and cell fate determination.

The correct maintenance and differentiation of hematopoietic stem cells (HSC) in bone marrow is vital for the maintenance and operation of the human blood system. GATA2 plays a critical role in the maintenance of HSCs and the specification of HSCs into the different hematopoietic lineages, highlighted by the various defects observed in patients with heterozygous mutations in GATA2, resulting in cytopenias, bone marrow failure and increased chance of myeloid malignancy, termed GATA2 deficiency syndrome. Despite this, the mechanisms underlying GATA2 deficiency syndrome remain to be elucidated. The detailed description of how GATA2 regulates HSC maintenance and blood lineage determination is crucial to unravel the pathogenesis of GATA2 deficiency syndrome. In this review, we summarize current advances in elucidating the role of GATA2 in hematopoietic cell fate determination and discuss the challenges of modeling GATA2 deficiency syndrome.

GATA2 deficiency syndrome: A decade of discovery.

GATA2 deficiency syndrome (G2DS) is a rare autosomal dominant genetic disease predisposing to a range of symptoms, of which myeloid malignancy and immunodeficiency including recurrent infections are most common. In the last decade since it was first reported, there have been over 480 individuals identified carrying a pathogenic or likely pathogenic germline GATA2 variant with symptoms of G2DS, with 240 of these confirmed to be familial and 24 de novo. For those that develop myeloid malignancy (75% of all carriers with G2DS disease symptoms), the median age of onset is 17 years (range 0-78 years) and myelodysplastic syndrome is the first diagnosis in 75% of these cases with acute myeloid leukemia in a further 9%. All variant types appear to predispose to myeloid malignancy and immunodeficiency. Apart from lymphedema in which haploinsufficiency seems necessary, the mutational requirements of the other less common G2DS phenotypes is still unclear. These predominantly loss-of-function variants impact GATA2 expression and function in numerous ways including perturbations to DNA binding, protein structure, protein:protein interactions, and gene transcription, splicing, and expression. In this review, we provide the first expert-curated ACMG/AMP classification with codes of published variants compatible for use in clinical or diagnostic settings.

Establishment of a Screening System to Identify Novel GATA-2 Transcriptional Regulators.

Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in hematopoietic stem and progenitor cells and is essential for cell proliferation and differentiation. Heterozygous germline GATA2 mutations induce GATA-2 deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia and acute myeloid leukemia, and a profoundly reduced dendritic cell (DC) population, which is associated with increased susceptibility to viral infections. Because patients with GATA-2 deficiency syndrome could retain a wild-type copy of GATA-2, boosting residual wild-type GATA-2 activity may represent a novel therapeutic strategy for the disease. Here, we sought to establish a screening system to identify GATA-2 activators using human U937 monocytic cells as a potential model of the DC progenitor. Enforced GATA-2 expression in U937 cells induces CD205 expression, a marker of DC differentiation, indicating U937 cells as a surrogate of human primary DC progenitors. Transient luciferase reporter assays in U937 cells reveals a high promoter activity of the -0.5 kb GATA-2 hematopoietic-specific promoter (1S promoter) fused with two tandemly connected GATA-2 +9.9 kb intronic enhancers. We thus established U937-derived cell lines stably expressing tandem +9.9 kb/-0.5 kb 1S-luciferase. Importantly, forced GATA-1 expression, a repressor for GATA-2 expression, in the stable clones caused significant decreases in the luciferase activities. In conclusion, our system represents a potential tool for identifying novel regulators of GATA-2, thereby contributing to the development of novel therapeutic approaches.