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. 2025 Jan 30;15(1):7. doi: 10.1038/s41408-025-01213-z

GATA2 mutated allele specific expression is associated with a hyporesponsive state of HSC in GATA2 deficiency syndrome

Laetitia Largeaud 1,2,#, Vincent Fregona 1,#, Laura A Jamrog 1,#, Camille Hamelle 1,2, Stéphanie Dufrechou 1,2, Naïs Prade 1,2, Esmaa Sellam 1, Pauline Enfedaque 1, Manon Bayet 1, Sylvie Hébrard 1, Mathieu Bouttier 1, Christine Didier 1, Bastien Gerby 1, Eric Delabesse 1,2, Marlène Pasquet 1,3,✉, Cyril Broccardo 1,4,✉
PMCID: PMC11782539  PMID: 39885120

Abstract

GATA2 germline mutations lead to a syndrome characterized by immunodeficiency, vascular disorders and myeloid malignancies. To elucidate how these mutations affect hematopoietic homeostasis, we created a knock-in mouse model expressing the recurrent Gata2 R396Q missense mutation. Employing molecular and functional approaches, we investigated the mutation’s impact on hematopoiesis, revealing significant alterations in the hematopoietic stem and progenitor (HSPC) compartment in young age. These include increased LT-HSC numbers, reduced self-renewal potential, and impaired response to acute inflammatory stimuli. The mature HSPC compartment was primarily affected at the CMP sub-population level. In the mutant LT-HSC population, we identified an aberrant subpopulation strongly expressing CD150, resembling aging, but occurring prematurely. This population showed hyporesponsiveness, accumulated over time, and exhibited allele-specific expression (ASE) favoring the mutated Gata2 allele, also observed in GATA2 mutated patients. Our findings reveal the detrimental impact of a Gata2 recurrent missense mutation on the HSC compartment contributing to its functional decline. Defects in the CMP mature compartment, along with the inflammatory molecular signature, explain the loss of heterogeneity in HPC compartment observed in patients. Finally, our study provides a valuable model that recapitulates the ASE-related pathology observed in GATA2 deficiency, shedding light on the mechanisms contributing to the disease’s natural progression.

Subject terms: Haematopoietic stem cells, Haematopoiesis

Introduction

GATA2 is one of the master regulatory transcription factors (TF) of hematopoiesis, characterized by two zinc finger domains (N-ZF and C-ZF), which bind DNA and interact with protein partners to regulate its function [1]. During embryogenesis, GATA2 plays a crucial role in the generation and maintenance of the hematopoietic stem and progenitor cells (HSPCs) [2, 3]. Its deregulation leads to a decrease in the number of HSCs and a loss of their self-renewal capacity in young mice [4, 5].

Over the past decade, germline GATA2 mutations have been extensively identified in patients with a wide range of clinical presentations, including hematological malignancies, immunological defects and vascular disorders [6–12]. The clinical spectrum of germline GATA2 mutations is broad [13]. Overall, frameshift, nonsense and enhancer +9.5 mutations suggest a haploinsufficiency mechanism of pathogenesis, while recurrent missense mutations may have aberrant activity [14, 15]. Moreover, patients with missense mutations are more prone to develop leukemic transformation, suggesting that these mutations may confer additional features [16]. However, their impacts remain poorly studied in vivo due to the very limited number of available models [17, 18]. To investigate the impact of the Gata2 R396Q mutation on hematopoiesis, we established a new knock-in mouse model.

Located at the apex of the hematopoietic hierarchy, long-term HSCs (LT-HSCs) have the ability to self-renew and differentiate to maintain hematopoietic homeostasis throughout the life of an organism [19]. We report here that Gata2R396Q/+ mice, in contrast to the Gata2+/- mice, have an increased number of LT-HSCs from embryogenesis to old age. Challenging assays unveiled functional impairments elucidated at the molecular level by enrichment in pathways associated with exhaustion and hyporesponsiveness.

Moreover, it is now admitted that the HSC population is phenotypically and functionally heterogeneous [20]. Previously, Gata2 has been identified as a gene exhibiting allelic-specific expression (ASE) in non-physiological contexts such as acute myeloid leukemia (AML) [21] or GATA2 deficiency [22]. In this study, we demonstrate that a distinct subset of LT-HSCs predominantly express the mutated allele of Gata2, highlighting a link between the heterogeneity of HSC function and the allele-specific expression (ASE) of the GATA2 gene. Thus, our findings provide insights into the influence of a Gata2 missense mutation on hematopoietic tissue and facilitate a deeper understanding of the pathophysiology in patients with Gata2 germline mutations.

Methods

Mice

C57BL/6 N and CD45.1 B6.SJL mice were sourced from Janvier (Orleans, France) and Jackson Laboratory (Bar Harbor, ME, USA), respectively. Gata2+/- mice, provided by Prof. Stuart Orkin (Harvard Stem Cell Institute, USA), were backcrossed to C57BL/6 N. Gata2R396Q/+ mice were generated in-house by the CIPHE animal facility (Marseille, France). All experiments complied with French law and were approved by the local ethics committee for animal experimentation (Protocol numbers: 19-16U1037-CB-01 and 20-16U1037-CB-02).

Genotyping

DNA for genotyping PCR was extracted from mouse ear tissues (or tails for embryos) using the “Extract-N-Amp PCR” kit (Sigma #XNAT2-1KT). For Gata2R396Q/+ mice, primers were Gata2-forward (5’ AGGCTGTGCAGGCATTTA 3’) and Gata2-reverse (5’ CTGCCAAACCACCCTTGAT 3’). PCR conditions: 94 °C for 3 min; 31 cycles of 94 °C for 30 sec, 58 °C for 40 sec, 72 °C for 30 sec; and 72 °C for 7 min. DNA bands were visualized on a TapeStation (Agilent). Gata2+/- mice were genotyped by amplifying a 650 bp band using Neospecific PCR with Neo504 and Neo505R primers.

Clonogenic Assay

Human GATA2 WT, GATA2 R204X, and GATA2 R396Q cDNAs were subcloned into a MIG plasmid (Addgene #52107) [23] and tagged with 5’ 3xHA to produce retroviruses. Murine lineage-negative cells were transduced with retroviral supernatant supplemented with polybrene, mIL-3, hIL-6, and mSCF (Peprotech). Clonogenic assays were conducted per the manufacturer’s instructions (Stem cell technologies).

Immunofluorescence

BAF/3 cells were transduced with viral supernatants in cytokine-supplemented RPMI by spinoculation and sorted by GFP expression. Cells were fixed, permeabilized, and stained for immunofluorescence analysis using an AXIO microscope and ZEN software (Zeiss). Whole E12.5 fetal livers were fixed, embedded in agarose, sectioned using a vibratome, blocked, permeabilized, and incubated with Endomucin and CD150 antibodies. Sections were stained, mounted, and examined using confocal microscopy.

Flow Cytometry, Cell Sorting, and Data Analyses

Bone marrow (BM) single-cell suspensions were prepared in IMDM with 2% FBS. Immunostaining used antibodies from Pharmingen (BD Biosciences), Invitrogen (Thermo Fisher Scientific), and Miltenyi Biotec. Flow cytometry utilized BD antibodies. Flushed BM cells from Gata2+/+ or Gata2R396Q/+ mice were enriched in the lineage-negative cell fraction using EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (Stem Cell Technology, MethoCult GF M3434). Cell sorting of LSK or LT-HSC, ST-HSC, and MPP3-4 was performed using a BD FACSAria™ III Cell Sorter with DIVA software. Flow cytometry data were analyzed using FlowJo Version 10.8 and normalized using the FlowAI package (V2.1) [24]. Arc sin transformation was used for TriMap dimensional reduction analysis [25].

Transcriptomic Analysis

Total RNA from 60,000 purified LSK Gata2+/+ and Gata2R396Q/+ mice was isolated using the Rneasy Plus Mini Kit (Qiagen, #74134). RNA-seq libraries were prepared with the TruSeq Stranded mRNA Low Sample kit, starting with 300 ng of total RNA. Cluster generation and sequencing were conducted on the Illumina NextSeq 500/550 High Output Kit v2.5 with a read length of 2 × 75 nucleotides. Specific LSK subpopulations were purified by cell sorting, RNA extracted, and Smart-seq v4 libraries prepared. Paired-end sequencing was performed on an Illumina NextSeq 500, and data was processed using fastp, RNA-STAR, and featurecount.

ATAC-Seq

Sorted LSK populations were prepared using the Active-Motif ATAC-Seq kit (#53150). Libraries were assessed on a Tapestation with a DNA High Sensitivity kit and sequenced on a NextSeq 500/550 with an average of 125 million reads per sample. FastQC was used to assess sequence quality. Foreign sequences were removed and trimmed with Sickle. Sequences were mapped to the murine genome with Bowtie2, and various cleaning and filtering steps were performed. GC bias was diagnosed and corrected using deepTools. The pre-processed output was used for peak calling with MACS2 and DiffTF analysis was conducted using the diffTF pipeline.

Functional in Vivo Assays

For BM reconstitution assays, myeloablation was induced by two doses of 5-Fluorouracil (Accord) administered intraperitoneally at days 0 and 11. Acute inflammatory stress was induced using lipopolysaccharide (Sigma #L2630) injected intraperitoneally at 5 mg/kg in 8-week-old mice. Mice were euthanized after 16 or 96 h, and bone marrow cells were characterized by flow cytometry. Transplantations of LSK or LT-HSC cells were performed on myeloablated mice, and cell chimerism quantified by flow cytometry.

WGS and RNA-Seq

Data were generated by France medicine genomic platform (detailed in supplementary methods)

Statistics

Statistical differences were assessed using a two-tailed unpaired Student’s t-test or Mann-Whitney test as appropriate. Error bars denote the standard deviation (SD) or standard error of the mean (SEM) as indicated. Survival variances were evaluated using the log-rank test. A p-value below 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). All analyses were conducted using GraphPad Prism software, version 7.

Results

Disruption of early HSC development by GATA2R396Q mutation

Reported nonsense and frameshift coding mutations occur upstream of the second zinc finger (C-ZF), resulting in haploinsufficiency [14]. GATA2 germline missense mutations cluster on the second C-ZF and are associated with a higher risk of leukemic transformation [16, 26]. Among them, arginine 396 is a mutational hotspot. The GATA2R396Q mutant maintains its nuclear localization, as wild-type GATA2, contrasting with the cytoplasmic localization observed with the truncated variant GATA2R204* lacking the nuclear localization signal (Supplementary Fig. 1A-B).

Previous report indicates that GATA2R396Q exhibits notably reduced DNA-binding and transactivation capabilities [26], suggesting an altered functional phenotype. To further analyze its functional consequences, we generated a Gata2R396Q/+ knock-in mouse (Supplementary Fig. 1C-D). Breeding Gata2R396Q/+ and Gata2+/- mice resulted in the expected genotypes at E12.5 when the yolk sac hematopoiesis ends [27] (Supplementary Fig. 1E). In contrast, Gata2R396Q/- embryos exhibited a translucent fetal liver (Fig. 1A) with an absence of CD150+ hematopoietic stem cells (Fig. 1B). No Gata2R396Q/- pups were present at birth (Supplementary Fig. 1F), similarly to the phenotype observed in Gata2-/- mice [3], demonstrating that Gata2R396Q is ineffective in rescuing the wild-type Gata2 allele.

Fig. 1. Impaired emergence and differentiation of hematopoietic cells due to recurrent Gata2R396Q mutation.

Fig. 1

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A Images of E12.5 embryos resulting from Gata2+/- x Gata2R396Q/+ mating. B Immunostaining of fetal liver sections, with Endomucin (Emcn) in blue and CD150 in purple. Scale bar represents 40 µm. The insert shows the fetal liver in its entirety and the location from which the magnification was performed. C Absolute number of indicated population in E14.5 fetal liver of Gata2+/+ (blue, n = 8) and Gata2R396/+ (red, n = 4) embryos. D Absolute number of LSK, LT-HSC, MPP1, ST-HSC, MPP2 (n = 25), MPP3 and MPP4 (n = 8 Gata2+/+, n = 13 Gata2R396Q/+) cells in 2-month-old Gata2+/+ (blue) and Gata2R396Q/+ (red) mice. E TriMap representation of FACS analyses of LSK subpopulations from 2-month-old mice, showing population density and colored scaled indicated marker intensity. F, G Dot plots showing intracellular Gata1 and Gata2 protein expression in subpopulations of Gata2+/+ and Gata2R396Q/+ LSK and LK cells (n = 3) CD150 marker intensity is color-coded. LSK: Lineage- Sca-1+ Kit+ cells LK : Lineage- Sca-1- Kit+ LT Long term, ST Short term, HSC Hematopoietic stem cell, MPP Multipotent progenitor. Each dot represents an individual mouse. Results are shown as mean ± SD; ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

At E14.5, Gata2R396Q/+ embryos exhibited an increase in fetal liver LSK cell numbers compared to WT embryos, primarily due to a significant increase in Long-Term (LT)-HSCs and Multipotent Progenitors 2 (MPP2) cells while Short-Term (ST)-HSC cells decreased (Fig. 1C, Supplementary Fig. 2A). This substantial impact of the Gata2R396Q mutant on hematopoiesis persisted 2 months after birth, with an additional increase of MPP3 cells (Fig. 1D, Supplementary Fig. 2B). The rise of the number of Gata2R396Q/+ MPP2 cells was associated with increased quiescence, suggesting that their increase is unlikely to be related to cell cycle dysregulation (Supplementary Fig. 2C). Notably, the effect of the Gata2R396Q mutant was significantly different from the loss of one Gata2 allele, as evidenced by the Gata2+/- mouse showing a reduction in both LT-HSCs and ST-HSCs (Supplementary Fig. 2D-E).

In WT mice, Gata2 expression was prominent in LT-HSCs and MPP2, consistent with its role in self-renewal and influencing erythroid and megakaryocyte differentiation, whereas Gata2R396Q/+ mice showed comparatively higher Gata2 levels in all compartments (Fig. 1F, Supplementary Fig. 2G). A specific Gata2high CD150High LT-HSC population is present in the mutant mice, the population also Kit and Sca-1 low (Fig. 1E, F and Supplementary Fig. 2B, F) and absent from the Gata2+/- mice (Supplementary Fig. 2D-F).

We examined the protein expression of Gata2 and one of Gata2’s canonical target genes, Gata1, in the Lin- Kit+ Sca-1- (LK) compartment as Gata1 is poorly expressed in LSK. While Gata2 protein is mainly increased in the GMP subset, Gata1 expression is reduced only in MEP cells of Gata2R396Q/+ mice compared to WT mice (Fig. 1G, Supplementary Fig. 2H-I), suggesting that Gata2R396Q may affect the expression of Gata2 target genes independently of the overall GATA2 protein load in a specific cell subset.

Gata2R396Q affects the mature hematopoietic compartment

We analyzed the more mature hematopoietic compartment in 2-month-old Gata2R396Q/+ mice. LK progenitors in the bone marrow were decreased due to a reduced CMP and MEP cell compartment compared to WT and Gata2+/- mice (Fig. 2A, Supplementary Fig. 3A-D). We observe a decreased hemoglobin and platelet counts in the blood of Gata2R396Q/+ mice (Fig. 2B) likely due to an increased apoptosis rate in mature mutant cells (Fig. 2C, D), despite a similar absolute number of mature cells in the bone marrow including myeloid, B, and T cells (Fig. 2E). Such abnormalities were not detected in Gata2+/- mice (Supplementary Fig. 3D-E), supporting the notion of an ectopic function associated with this recurrent missense mutation.

Fig. 2. Impact of the mutant on hematopoietic differentiated compartment.

Fig. 2

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A FACS analysis statistics of the absolute number of indicated cells in the bone marrow of 2-month-old Gata2+/+ (blue, n = 25) and Gata2R396/+ (red, n = 25) mice. B Blood parameters, including hemoglobin level (Hb) and white blood cells (WBC), in 2-month-old Gata2+/+ (blue, n = 17) and Gata2R396/+ (red, n = 11) mice. C Representative FACS dot plot of the percentage of Annexin V positive cells in B (CD19+) or myeloid (Gr-1+) cells. D FACS analysis statistics of Annexin V positive cells in the myeloid, B, and T compartment. E FACS analysis statistics of the absolute number of total cells, Myeloid cells, B cells, and T cells in the bone marrow of 2-month-old Gata2+/+ (blue, n = 9) and Gata2R396/+ (red, n = 13) mice. Hb: Hemoglobin level, WBC: white blood cells. LK Lineage- Kit+ cells; CMP Common myeloid progenitor, MEP Megakaryocyte–erythroid progenitor, GMP Granulocyte-monocyte progenitor. Each dot represents an individual mouse. Results are shown as mean ± SD (except (E), SEM); ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Hyporesponsiveness and inflammatory molecular signatures are a hallmark of Gata2R396Q

To elucidate the molecular mechanisms underlying the impact of Gata2R396Q on hematopoiesis, we performed comprehensive analyses of gene expression and chromatin accessibility using purified Gata2R396Q/+ and Gata2+/+ LSK cells from 2-month-old mice (Fig. 3A). We assessed TF accessibility [28] and observed a significant reduction in accessibility for Meis [29] and Irf [30] associated motifs along with a decreased expression, suggesting an impact on hematopoietic commitment and interferon response, respectively (Fig. 3B, Supplementary Fig. 4A). Conversely, motifs linked to TF associated with cell quiescence (e.g., Egr) or BMP signaling (e.g., Smad) showed substantial increase in activity and expression (Fig. 3C, Supplementary Fig. 4B). Interestingly, GATA motifs showed only a marginal decrease in accessibility in the mutant cells (Fig. 3B).

Fig. 3. Hyporesponsiveness and exhaustion signatures in mutated LSK cells.

Fig. 3

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A Experimental workflow schematic. B, C Ranked sorted motifs based on differential accessibility: Repressed motifs (B) and enriched motifs (C) in Gata2R396Q/+ LSK cells. D ATAC sequencing signal at the GATA or Erg/Fli1 sites. The color scale represents differential accessibility between Gata2R396Q/+ and Gata2+/+ conditions. The signals are clustered based on their type: more accessible in Gata2R396Q/+, less accessible, or a mix of the two. The top panel shows the footprint of average accessibility at these sites. E Pie charts illustrating the distribution of ATAC-seq consensus peaks relative to gene features for each cluster defined in Fig. 3D. F, G Heatmap of over-represented analysis (ORA) biological processes in Gata2R396Q/+ versus Gata2+/+ cells based on ATAC (F) or RNA (G) sequencing analysis. The color scale represents the enrichment or depletion of each process (-log(p-value)).

Gata2 is a component of a heptad transcription complex critical for defining HSPC identity [31] and among these 7 factors, only Erg and Fli1 showed increased activity without concomitant changes in expression (Supplementary Fig. 4C-D), suggesting a potential disruption of the complex. Subsequent analyses focusing on regions of differential accessibility, identified both enrichments and depletions of Gata motifs across the genome in mutated LSK cells, explaining the absence of widespread repression (Fig. 3D, E). The accessibility of regions containing ETS motifs was predominantly located at promoters (Fig. 3D, E), highlighting the significant role of these motifs in regulating gene transcription (Fig. 3D, E). However, regions containing both Gata2 and ETS motifs were less accessible at key hematopoietic genes such as Meis, Cd34, and Gata2 itself (Supplementary Fig. 4D). In contrast, regions specifically associated with ETS motifs alone, particularly at enhancers and promoters, tended to be more accessible. This increased accessibility correlated with higher expression levels of genes related to hematopoiesis, such as Slamf1 (encoding CD150), Mmp8, and Smad1 (Supplementary Fig. 4D).

The Over-Representation Analysis (ORA) uncovered concurrent changes in cellular processes at both chromatin accessibility and expression levels in Gata2R396Q/+ LSK cells. These cells exhibited enrichment in pathways associated with innate inflammatory response, migration, and extracellular matrix degradation (Fig. 3F, G, Supplementary Fig. 5A-B). Futhermore, the observed depletion of interferon-gamma response pathways (Fig. 3F, G, Supplementary Fig. 5A) was correlated with an observed reduction in Irf expression and accessibility (Supplementary Fig. 4A), suggesting a diminished capacity to respond to stimuli. In conclusion, our analysis of the transcriptional landscape and network of mutated LSK cells revealed specific signatures of inflammation, exhaustion and hyporesponsiveness.

Gata2R396Q expression is associated with a loss of HSCs identity in the mutant mice

We investigated expression profiles by RNA sequencing in sorted LT-, ST-HSC, and MPP3-4 compartments. Notably, Gata2R396Q expression displayed elevated allelic expression levels compared to the wild type in LT- and MPP3-4 (58% and 67% respectively; Fig. 4A), unveiling distinct allele-specific expression (ASE) patterns. Differential gene expression observed in sorted LT-HSCs and MPP3-4 compared to ST-HSCs is consistent with ASE (Fig. 4A, B). Notably, Gata1 and Gfi1b, genes activated by Gata2, showed significantly lower expression in LT-HSCs and MPP3-4, while Cebpa, a gene repressed by Gata2 [32], exhibited increased expression (Fig. 4C). Additionally, key genes for HSPC functions (Epor, Esam, Aqp9, Gpc3) have a lower expression in LT-HSCs and MPP3-4 (Supplementary Fig. 5C). GSEA analysis revealed notable dysregulation of pathways associated with protein synthesis, myeloid differentiation, and TNFα response in all subpopulations, indicating an influence of Gata2R396Q expression level on these processes. Conversely, repression of heme metabolism and interferon-gamma responses showed variability depending on ASE status, suggesting direct involvement of mutant functions. (Fig. 4D).

Fig. 4. Molecular profiling reveals dysregulated pathways and gene expression patterns in Gata2R396Q/+ hematopoietic stem and progenitor cell subpopulations.

Fig. 4

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A Expression of the wild-type Gata2 (plain square) or Gata2R396Q allele (hatched square) in Gata2R396Q/+ (red) versus Gata2+/+ (blue) LT-HSC, ST-HSC, and MPP3-4 subpopulations of LSK cells. Results are shown as mean ± SD; *q-val<0.01. B MA-plots illustrating Log2 fold difference relative to normalized read counts for RNA-sequencing reads, depicting significantly increased (red), significantly decreased (blue), or unchanged (grey) expression in Gata2R396Q/+ versus Gata2+/+ LT-HSC (left panel), ST-HSC (middle panel), and MPP3-4 (right panel) subpopulations of LSK cells. C Expression of indicated genes in Gata2+/+ (blue) or Gata2R396Q/+ (red) LT-, ST-HCS and MPP3 cells. Results are shown as mean ± SD; *q-val<0.01. D Gene set enrichment analysis (GSEA) of enriched or depleted Gene Ontology (GO) terms for LT-, ST-HSC, and MPP3-4 cells, represented by a color scale of normalized enrichment score (NES). False discovery rates (FDR) < 0.05 are indicated by bold-framed squares. E GSEA-enrichment plots showed significant depletion of MolO and NoMO signatures [33] in LT-HSC form Gata2R396Q/+ mice. F, G Expression of indicated genes (from the MolO (F) and NoMO (G) signature), in Gata2+/+ (blue) or Gata2R396Q/+ (red) LT-HSCs. (H) Expression of non-hematopoietic related genes. Results are shown as mean ± SD.

To go deeper, we used molecular signatures associated with the hallmark of HSC properties. Both The MoIO [33] signature, linked with superior HSC function, and the NoMO [33] signature which is associated with less quiescent and functionally inferior HSCs, are overrepresented in wild-type LT-HSCs compared to mutated cells (Fig. 4E). Notably, specific genes within the MolO signature like Neo1, Smtnl1, Cd82 and Vwf exhibited reduced expression in mutated LT-HSCs whereas Ly6a and Procr genes encoding Sca-1 and Epcr respectively showed increased expression (Fig. 4F). Similarly, most of the NoMO signature genes, including Muc13, Itga2b, Gata1 or Aqp9, displayed decreased expression in mutated LT-HSCs while Nkg7 expression increased (Fig. 4C, G). Notably, the expression of Dlk1, a gene associated with increased myeloid potential, quiescent state, and self-renewal capacity [34], was significantly decreased in mutated LT-HSCs (Supplementary Fig. 5D). Furthermore, mutated LT-HSCs expressed genes not typically found in hematopoietic cells, such as Igf1, Sfrp2, S1pr3 or Nsg2 (Fig. 4H and Supplementary Fig. 5E). These findings indicate that mutated LT-HSCs undergo a loss of HSC features, encompassing both dormant and active HSC properties and acquire inappropriate markers.

Gata2R396Q mutation impairs HSCs development and function, mostly at LT-HSC stage

To assess the impact of these molecular features associated with Gata2R396Q expression, we performed clonogenic assays from 2-month-old mice. Gata2R396Q/+ cells exhibited a significant decrease in CFU-GEMM colonies (Fig. 5A). When subjected to serial passages, these cells displayed an increase in colony number (Supplementary Fig. 6A). Morphological and phenotypic analyses revealed a predominance of granular precursors and mature granulocytes in Gata2R396Q/+ cells whereas mast cells were more prevalent in Gata2+/+ condition (Supplementary Fig. 6B-C). Gata2+/- LSK cells have a reduced number of CFU-GM without increased clonogenic activity. As in Gata2+/+, mast cells were the most prominent cells observed in the Gata2+/- condition (Supplementary Fig. 6D-F).

Fig. 5. Functional characterization of hematopoietic stem cells in Gata2R396Q/+ mice under challenging conditions.

Fig. 5

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A Clonogenic assay on 2-month-old Gata2+/+ (blue) and Gata2R396Q/+ (red) Lin-Sca-1+Kit+ (LSK) cells. Colonies were counted after 10 days (n = 3). B Kaplan-Meier survival curve (in days) of Gata2+/+ (blue, n = 8) and Gata2R396Q/+ (red, n = 16) mice after two injections (arrows) of 5-fluorouracil (5-FU) (C) Experimental scheme for transplantation assays. D Representative flow cytometry density plot showing bone marrow engrafted cells (CD45.2+) from Gata2+/+ and Gata2R396Q/+ mice versus host cells (CD45.1+) two months after the first engraftment. E Engraftment quantification of donor CD45.2+ cells six (left panel) and two (left part of the right panel) months after transplantation. The number of engrafted mice ( > 1% of CD45.2+) out of the total number of transplanted mice and the average reconstitution percentage for each group are indicated on top of the graph. The far-right panel shows the percentage of CD45.2+ cells 6 months after a secondary transplant of cells from mice indicated with dark-colored dots. Each blue dot represents an individual Gata2+/+ mouse, and red dots represent Gata2R396Q/+ mice. F Fold expansion between the number of estimated engrafted cells and the number recovered 2 months after engraftment for Gata2+/+ (blue) and Gata2R396Q/+ (red) cells G Experimental scheme of LPS injection assay. H TriMap visualization of bone marrow cells from PBS- or LPS- injected 2-month-old Gata2+/+ (bleu) and Gata2R396Q/+ (red) mice depicting HSPC subpopulations, population density and CD150, Sca-1, CD41 and Ki67 marker intensity of on each population. Intensity of each marker is shown using a colored scale. I Percentage of cells in G0 (Ki67- cells) of total LT-HSC in Gata2+/+ (blue) or CD150high and CD150low LT-HSCs in Gata2R396Q/+ (red) mice injected with PBS (plain box) or LPS (hatched box). CFU Colony forming unit, GEMM Granulocyte, erythroid, macrophage, megakaryocyte, GM Granulocyte, macrophage, M Macrophage, G Granulocytes and BFU-E Burst-forming unit erythrocytes. E erythrocyte, Mk megakaryocyte. LSK Lineage- Sca-1+ Kit+ cells, LT, Long term, ST Short term, HSC Hematopoietic stem cell, MPP Multipotent progenitor. LPS Lipopolysaccharide. Results are shown as mean ± SD; ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To evaluate bone marrow repopulation capacity, myeloablation was induced with 5-FU, revealing that Gata2R396Q/+ mice displayed reduced hematopoietic reconstitution ability, resulting in a significantly higher mortality rate compared to Gata2+/+ mice (Fig. 5B). In a syngeneic transplantation experiment, Gata2R396Q/+ LSK cells exhibited reduce repopulation capacity compared to their Gata2+/+ counterparts after 2 months, with all subpopulations showing reduced expansion. This engraftment deficit worsened after 6 months, indicating a severe impairment of Gata2R396Q/+ LT-HSCs (Fig. 5C-F). Secondary transplantation of total bone marrow from the primary transplanted mice confirmed the loss of long-term engraftment capacity of Gata2R396Q/+ cells (Fig. 5E). Overall, transplantation assays uncovered a long-term exhaustion of Gata2R396Q/+ cells, as evidenced by a decrease in self-renewal capacity.

Furthermore, to assess the functional consequences of the Gata2R396Q mutation on hematopoiesis under challenge conditions, we determined the hematopoietic response after a single dose of lipopolysaccharide (LPS), mimicking an acute bacterial infection (Fig. 5G). LSK cells exhibited an increased proliferation in response to inflammatory stress [35] (Supplementary Fig. 7A) triggering emergency myelopoiesis [36]. After16 hours, we observed an increase in MPP2 and MPP3 numbers in Gata2R396Q/+ mice but to a lesser extent than in Gata2+/+ LSK cells (Supplementary Fig. 7B-C).

Additionally, 96 h after LPS injection, similar patterns were still observed in all cell populations. This persistence suggests that the Gata2R396Q mutation may affect the onset of the response rather than its magnitude (Fig. 5H and Supplementary Fig. 7C). In Gata2R396Q/+ LT-HSCs, we observed two phenotypically distinct populations based on Gata2 and CD150 protein levels (Fig. 1E). Under acute inflammatory stress with LPS, Gata2R396Q/+ mice unveiled an aberrant LT-HSCs with higher CD150 and Sca-1 marker intensities and lower Kit intensity (Fig. 5H, red arrow, Supplementary Fig. 7D). Ki67 labeling revealed that CD150high aberrant LT-HSCs cycled less than CD150low and WT LT-HSCs under steady-state conditions, and the CD150high LT-HSC population exhibited hyporesponsive cycling in response to LPS stimulation (Fig. 5H, I, Supplementary Fig. 7E). Sixteen hours after LPS treatment, the majority of Gata2+/+ LT-HSCs expressed CD41 (Itga2b-encoded platelet marker) compared to a fraction of Gata2R396Q/+ LT-HSCs (Fig. 5H, grey arrow).

Consequently, the Gata2R396Q mutation results in a hematopoietic functional impairment with reduced responsiveness and exhaustion following both short- and long-term inflammatory stress.

Functional decline in Gata2R396Q LT-HSCs induces a specific inflammaging

Given the presence of the CD150high LT-HSC population and the exhaustion and hyporesponsiveness functionality observed in LSK cells from young mutant mice, we examined one-year-old mice, representing a midpoint in their lifespan. Gata2R396Q/+ mice exhibited higher LSK cell numbers, along with increased LT-HSC, MPP2, and MPP3 cells, while MPP1 and ST-HSC were significantly reduced (Fig. 6A, Supplementary Fig. 8A). The increase in LT-HSC numbers was more pronounced at one year than at 2 months (Fig. 6B). Repeated measurements of blood parameters revealed a progressive anemia in Gata2R396Q/+ mice while the platelet count increased rapidly, suggesting a differentiation bias in favor of platelet over erythroid development (Fig. 6C). Because of the cellular phenotypic features resembling a premature aging phenotype, we compared the significant dysregulations observed in mutant LT-HSCs with those found in old versus young WT LT-HSCs [37], revealing similar deregulated pathways, such as an increased enrichment of oxidative phosphorylation and proteasome-related pathways and a decrease of Kit and Cxcr4 pathways (Fig. 6D).

Fig. 6. Functional decline of HSPC in older Gata2R396Q/+ mice.

Fig. 6

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A Absolute number of LSK, LT-HSC, MPP1, ST-HSC, MPP2, MPP3, and MPP4 cells in 12-month-old Gata2+/+ (blue, n = 9) and Gata2R396/+ (red, n = 10) mice. B Comparison of the absolute number of LT-HSC in the bone marrow of 2- and 12-month-old Gata2+/+ (blue, n = 9) and Gata2R396/+ (red, n = 10) mice. Fold increase between 2 and 12 months is indicated. C Time course of hemoglobin level (Hb) and platelet number in the blood of Gata2+/+ (blue) and Gata2R396/+ (red) mice. D Hallmark-term analysis of gene sets enriched in young Gata2+/+ LT-HSCs versus 20-month-old Gata2+/+ LT-HSC cells, with normalized enrichment scores (NES) shown for significant pathways (FDR < 0.05). E GSEA enrichment plots for interferon response and heme metabolism comparing Gata2R396Q/+ to Gata2+/+ LT-HSC (left panels) and old LT-HSC versus young LT-HSC (right panels). F TriMap visualization of FACS analyses of the different LSK subpopulations showing the population density and the intensity of the CD150, CD41 and CD61 markers on each population following a colored scale. G Statistics of absolute number of CD41/CD61 double positive (CD41+/CD61+) or CD41-/CD61-/low LT-HSCs. H Variant allelic frequency at RNA level (VAFRNA) of Gata2 R396Q mutation on sorted CD150low and CD150high LT-HSCs from 2- and 12-month-old mice. I Analysis of CD45.2 cells percentage after 1 month. 500 CD45.2 LT-HSCs from Gata2+/+ mice (2-month-old mice n = 3, blue), Gata2R396Q/+ mice (2-month-old: red circles (CD150high: black box), 12-month-old: red triangle (CD150high: black box)) were transplanted in CD45.1 recipient mice. J Genomic analysis of one GATA2 deficient patient diagnosed with AML. Circos plot with structural variations (left panel). Visualization on IGV viewer of the variant allele frequency (VAF) after Whole Genome Sequencing (WGS) of infiltrated bone marrow (first line) and healthy tissue (third line) or Whole Exome Sequencing of infiltrated bone marrow (second line). VAF at the RNA level is visualized after RNA-Seq analysis (last line). Results are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

While increased interferon signaling and inflammation are typically associated with aging [30], these markers were not elevated in young Gata2R396Q/+ HSPCs, suggesting an unconventional premature aging phenotype (Fig. 6E). Physiological hematopoietic aging is associated with changes in LT-HSCs, including increased expression of the CD150 marker, presence of megakaryocytic surface markers such as CD41 or CD61 [38, 39], and upregulation of Gata2 (Supplementary Fig. 8B) and its target gene Gata1 [40], correlating with a platelet bias. We identified an aberrant CD150high/CD41-/CD61low/- LT-HSCs in middle-aged Gata2R396Q/+ mice, which progressively expanded over time and represented the majority of the LT-HSCs population (Fig. 6F, G). This population exhibited a reduced functionality as evidenced by decreased clonogenic capacity and multipotency with a notable decrease in CFU-GEMM and CFU-Mk colony numbers in clonogenic assay, correlating with the lack of CD41 protein expression (Supplementary Fig. 8C-D).

Allele-specific expression drives functional defects in LT-HSCs and disease progression in GATA2 R396Q model

Based on our identification of an ASE mechanism within HSPC subpopulations, we investigated into the prospect of different allelic imbalances in these two subpopulations. We sorted CD150high and CD150low LT-HSCs from 2- and 12-month-old Gata2R396Q/+ mice and measured the variant allelic frequency (VAF) of the Gata2 mutation at the RNA level. Our results revealed that Gata2R396Q VAFRNA is higher in CD150high compared to CD150low LT-HSCs (73% vs 53% at 2 months and 79% vs 58% at 12 months, respectively), providing evidence for a persistent ASE mechanism in the aberrant CD150high LT-HSCs (Fig. 6H).

In LT-HSC subpopulation transplantation assay, we observed a negative relationship between the level of donor chimerism and Gata2 VAFRNA. Transplantation of CD150high LT-HSC subpopulation resulted in a severe repopulation defect in recipient bone marrow one-month post-injection (Fig. 6I), confirming the hypofunctionality of the CD150high LT-HSCs. On the other hand, transplantation of CD150low underscored a milder defect in BM repopulation capacity compared to Gata2+/+ LT-HSCs.

Interestingly, these functional defects were also detected in old mutant mice at steady state. Phenotypic characterization of 18-month-old Gata2R396Q/+ mice revealed a significantly reduced hemoglobin levels (6 g/dL compared with 10 g/dL for the Gata2+/+ mouse of the same age), enlarged spleen with extramedullary erythropoiesis and a substantial decrease in bone marrow cells (Supplementary Fig. 8E-F). The LSK compartment is predominantly composed of CD150high LT-HSC cells (Supplementary Fig. 8G) suggesting bone marrow hypoplasia could be due to the exhaustion of the most functional LT-HSCs (CD150low). Our RNA-Seq analysis of a patient with the germline missense GATA2R362G (c.1084 C > G) mutation [16, 41], confirmed the presence of ASE with higher relative expression of the mutated allele at leukemic transformation (Fig. 6J). These findings highlight the clinical relevance of the ASE mechanism in human GATA2 deficiency syndrome and its potential implications for disease development and progression.

Discussion

Our work comprehensively illustrates the impact of the GATA2R396Q germline mutation on HSPCs, generating a Gata2R396Q/+ knock-in mouse model. This model shows increased LT-HSC numbers, diminished self-renewal potential, and impaired responsiveness to inflammatory stimuli.

While the missense GATA2R396Q mutant maintains nuclear localization unlike truncated mutants [15, 42], it doesn’t rescue the wild-type allele, indicating a global loss-of-function mutation.

Interestingly, three GATA2 missense mutations—Gata2R396Q, Gata2R398W, and Gata2L359V (our study; [17, 18])—have been introduced into mouse models, each with distinct molecular impact. Structurally, leucine 359 interacts with DNA in the major groove, while arginine 361 and R396 are essential for recognizing the WGATAR motif and interacting with thymine and adenine residues. R362 and R398 enhance DNA binding by stabilizing interactions. The L359V mutation swaps one hydrophobic residue for another, whereas R396Q and R398W reduce positive charge, affecting DNA binding differently. Homology modeling suggests arginine mutations disrupt DNA interactions but leave the second zinc finger structure intact [26, 43].

Despite reduced DNA-binding activity [26, 42], Gata2R396Q/- embryos survive longer than Gata2-/- embryos [3], suggesting potential ectopic functions. Unlike models with lower Gata2 expression, such as Gata2+/- or Gata2+9.5+/- models [5, 44], our Gata2R396Q/+ model, like Gata2R398W/+, does not exhibit a decrease in overall Gata2 levels within the LSK compartment [17]. Instead, we observed a notable increase in global Gata2 protein levels, particularly in LT-HSCs, MPP2, and MPP3, with no alterations in ST-HSCs. These changes mirror the abnormal LSK subpopulation distribution from embryonic stages throughout their lifespan. This distribution pattern is distinct from Gata2+/- and Gata2R398W/+ models in which a reduction or no difference in LT-HSC proportions is observed, respectively [5, 17, 45]. Furthermore, GATA2R396Q mutant affects the more mature HSPC compartment by reducing the CMP and MEP compartments inducing anemia over time while platelet number increase rapidly, indicating differentiation bias in favor of platelet over erythroid development.

Despite higher LT-HSC counts mutant mice, repopulation and serial transplantation assays unveiled impaired LSK functionality. Chromatin accessibility and gene expression profiles in young mice showed reduced accessibility for motifs linked to commitment and interferon response, compromised infection responses to mycobacteria for example in patients [43]. In addition, GATA2 deficient patients bone marrow with spectrum 1 showed a higher proportion of MEP and a lower proportion of GMP than aplastic anaemia patients [41, 46]. Under chronic inflammation, this differentiation bias is due to exhaustion of CMP and GMP populations, rather than MEP proliferation.

Gata2 is a constituent of a crucial heptad of TF defining stemness [31]. Our findings demonstrate that the Gata2R396Q mutation selectively impairs DNA binding at the heptamer binding site, but does not affect other TF expression. Furthermore, we observe increased accessibility specifically at Fli1/Erg motifs, resulting in higher expression of BMP pathway genes like the Smad family. This supports previous research showing a crosstalk between Smad signaling and Gata2 [47, 48] and may explain Gata2 levels similar to WT cells [32]. There is also a correlation between CD150 and Gata2 expression levels in WT HSCs [49]. In mutated HSPCs, Fli1 sites at Slamf1 (encoding CD150) transcriptional regulatory elements show heightened accessibility. This suggests a compensatory response to the diminished Gata2 activity.

Hence, we identify a mutant specific CD150high-expressing LT-HSCs population in young Gata2R396Q/+ mice compared to WT mice, showing similarities to certain features of an aging. However, some pathways were not elevated in young Gata2R396Q/+ HSPCs compared to aged wild-type HSPCs, indicating an unconventional premature aging phenotype [37].

Mutated LT-HSCs exhibited a notable reduction in the expression of Neo1, a component of the MolO signature. In HSPC from Neo1-mutant mice, the NoMO signature was enriched along with increased Gata1 and Itga2b expressions [50]. In Gata2R396Q/+ mouse model, the physiological upregulation of Gata1was hindered leading to the absence of enrichment of the NoMO signature in mutated LT-HSCs. Additionally, mutated LT-HSCs In addition, mutated LT-HSC express genes from cells in the microenvironment. This is in line with findings from De Pater’s group [51], which demonstrated impaired suppression of endothelial identity in Gata2+/- HSPCs during embryonic maturation due to reduced Gfi1b activity, as observed in Gata2R396Q/+ LT-HSCs. These observations suggest a loss of molecular stemness identity in mutated HSCs.

LPS challenge uncovered functional diversity within the LT-HSC compartment, notably linked to CD150 expression levels. While CD150low mutant LT-HSCs behaved similarly to WT cells, CD150high cells displayed pronounced hyporesponsiveness. The CD150high LT-HSC population exhibited the highest expression levels of the mutant allele, suggesting an allele-specific expression mechanism at play. This ASE mechanism has previously being reported in the context of GATA2 [21]. Interestingly, Al Seraihi and colleagues have reported that differential allele expression can influence the clinical phenotypes in a GATA2 deficient patient [22]. Our findings showed that ASE is present in mice as early as 2 months old and might persist even in the context of a hematologic progression in patients. This raises questions about potential secondary events that could favor the selection of cells demonstrating ASE. Furthermore despite the existence of some genotype-phenotype correlations [41], questions remain regarding the wide range of symptoms observed even within a same family. The investigation of ASE mechanism could provide valuable insights into understanding these divergences.

In conclusion, our study provides compelling evidence for the substantial impact of a missense Gata2 mutation on the HSPC compartment during hematopoietic development. The alterations at phenotypic, functional, and molecular levels highlight the crucial role of Gata2 in maintaining HSPC homeostasis. The disruption of critical molecular pathways in mutated HSPCs hampers their ability to respond appropriately to environmental signals and may contribute to their exhaustion. These insights significantly advance our understanding of initiation of bone marrow failure in GATA2 deficiency. Therefore, our findings hold promising implications for improving clinical follow-up for patients with GATA2-related conditions, providing valuable insights into the mechanisms involved in maintaining HSPC homeostasis.

Supplementary information

Supplementary material (12.4MB, pdf)

Acknowledgements

We greatly thank Pr. Stuart Orkin for the Gata2+/- mice and Pr. Emery Bresnick for the Gata2 antibody. We are grateful to Manon Farcé from the cytometry and cell-sorting facility of the Pole Technologique of the CRCT (INSERM U1037) for technical assistance. We thank the Anexplo/Genotoul platforms for technical assistance (UMS006). L.J. was supported by la Société Française des Cancers de l’Enfant (SFCE, association Capucine, R17094BB) and Région Occitanie grants (RPH17006BBA). This specific project was founded mainly by INCa (R17164BB and R21196BB), association « Laurette Fugain » (R20110BB), Fédération leucémie espoir (R2004BB), association « Constance la petite guerrière astronaute » (R19043BB) and association « les 111 des arts » (R18062BB). The team is « Equipe labélisée Ligue Contre le Cancer » and was also supported by la Ligue Nationale Contre le Cancer (R19015BB), Société Française des Cancers de l’Enfant (R17094BB, association Capucine), la Région Occitanie (R16038BB), Association « Cassandra » (R18041BB). The team is a part of Institut Carnot « Opale ». We thank the Auragen platform of the “Plan France Médecine Génomique” for contributing to the generation of the genomic data of the patient presented in this study.

Author contributions

L.L., L.J., V.F., C.H., E.S., P.E., M.B., S.H. and C.D. performed and analyzed in vivo and in vitro studies. N.P. and S.D. performed library preparation. L.L., L.J. and V.F analyzed the data from RNA-Seq and ATAC-Seq. L.L., V.F., B.G., C.D., E.D. and C.B. conceived and designed the experiments. E.D., M.P and C.B. acquire the funding. L.L., L.J., V.F. and C.B. wrote the manuscript with feedback from all authors.

Funding

This work was supported by la Société Française des Cancers de l’Enfant (SFCE), Région Occitanie grants, Institut National du Cancer (INCa), association « Laurette Fugain », Fédération leucémie espoir, association « Constance la petite guerrière astronaute », association « les 111 des arts ». The team is « Equipe labélisée Ligue Contre le Cancer » and was also supported by la Ligue Nationale Contre le Cancer, Société Française des Cancers de l’Enfant (association Capucine), la Région Occitanie, Association Cassandra. The team is a part of Institut Carnot « Opale ».

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Laetitia Largeaud, Vincent Fregona, Laura A. Jamrog.

These authors jointly supervised this work: Marlène Pasquet, Cyril Broccardo.

Contributor Information

Marlène Pasquet, Email: pasquet.m@chu-toulouse.fr.

Cyril Broccardo, Email: cyril.broccardo@inserm.fr.

Supplementary information

The online version contains supplementary material available at 10.1038/s41408-025-01213-z.

References

  • 1.Crispino JD, Horwitz MS. GATA factor mutations in hematologic disease. Blood. 2017;129:2103–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.de Pater E, Kaimakis P, Vink CS, Yokomizo T, Yamada-Inagawa T, van der Linden R, et al. Gata2 is required for HSC generation and survival. J Exp Med. 2013;210:2843–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Tsai F-Y, Keller G, Kuo FC, Weiss M, Chen J, Rosenblatt M, et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature. 1994;371:221–6. [DOI] [PubMed] [Google Scholar]
  • 4.Soukup AA, Zheng Y, Mehta C, Wu J, Liu P, Cao M, et al. Single-nucleotide human disease mutation inactivates a blood-regenerative GATA2 enhancer. J Clin Invest. 2019;129:1180–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Rodrigues NP. Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis. Blood. 2005;106:477–84. [DOI] [PubMed] [Google Scholar]
  • 6.Hahn CN, Chong C-E, Carmichael CL, Wilkins EJ, Brautigan PJ, Li X-C, et al. Heritable GATA2 Mutations Associated with Familial Myelodysplastic Syndrome and Acute Myeloid Leukemia. Nat Genet. 2011;43:1012–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Dickinson RE, Milne P, Jardine L, Zandi S, Swierczek SI, McGovern N, et al. The evolution of cellular deficiency in GATA2 mutation. Blood. 2014;123:863–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Wlodarski MW, Hirabayashi S, Pastor V, Starý J, Hasle H, Masetti R, et al. Prevalence, clinical characteristics, and prognosis of GATA2-related myelodysplastic syndromes in children and adolescents. Blood. 2016;127:1387–97. [DOI] [PubMed] [Google Scholar]
  • 9.Pasquet M, Bellanné-Chantelot C, Tavitian S, Prade N, Beaupain B, Larochelle O, et al. High frequency of GATA2 mutations in patients with mild chronic neutropenia evolving to MonoMac syndrome, myelodysplasia, and acute myeloid leukemia. Blood. 2013;121:822–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Hsu AP, Sampaio EP, Khan J, Calvo KR, Lemieux JE, Patel SY, et al. Mutations in GATA2 are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (MonoMAC) syndrome. Blood. 2011;118:2653–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bigley V, Haniffa M, Doulatov S, Wang X-N, Dickinson R, McGovern N, et al. The human syndrome of dendritic cell, monocyte, B and NK lymphoid deficiency. J Exp Med. 2011;208:227–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ostergaard P, Simpson MA, Connell FC, Steward CG, Brice G, Woollard WJ, et al. Mutations in GATA2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (Emberger syndrome). Nat Genet. 2011;43:929–31. [DOI] [PubMed] [Google Scholar]
  • 13.Homan CC, Venugopal P, Arts P, Shahrin NH, Feurstein S, Rawlings L, et al. GATA2 deficiency syndrome: A decade of discovery. Hum Mutat. 2021;42:1399–421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Bresnick EH, Jung MM, Katsumura KR. Human GATA2 mutations and hematologic disease: how many paths to pathogenesis? Blood Adv. 2020;4:4584–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Katsumura KR, Mehta C, Hewitt KJ, Soukup AA, Fraga de Andrade I, Ranheim EA, et al. Human leukemia mutations corrupt but do not abrogate GATA-2 function. Proc Natl Acad Sci. 2018;115:E10109–E10118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Donadieu J, Lamant M, Fieschi C, Fontbrune FS, de Caye A, Ouachee M, et al. Natural history of GATA2 deficiency in a survey of 79 French and Belgian patients. Haematologica. 2018;103:1278–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hasegawa A, Hayasaka Y, Morita M, Takenaka Y, Hosaka Y, Hirano I, et al. Heterozygous variants in GATA2 contribute to DCML deficiency in mice by disrupting tandem protein binding. Commun Biol. 2022;5:1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Fu Y-K, Tan Y, Wu B, Dai Y-T, Xu X-G, Pan M-M, et al. Gata2-L359V impairs primitive and definitive hematopoiesis and blocks cell differentiation in murine chronic myelogenous leukemia model. Cell Death Dis. 2021;12:568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M, et al. Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair. Cell. 2008;135:1118–29. [DOI] [PubMed] [Google Scholar]
  • 20.Haas S, Trumpp A, Milsom MD. Causes and Consequences of Hematopoietic Stem Cell Heterogeneity. Cell Stem Cell. 2018;22:627–38. [DOI] [PubMed] [Google Scholar]
  • 21.Mulet-Lazaro R, van Herk S, Erpelinck C, Bindels E, Sanders MA, Vermeulen C, et al. Allele-specific expression of GATA2 due to epigenetic dysregulation in CEBPA double-mutant AML. Blood. 2021;138:160–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Al Seraihi AF, Rio-Machin A, Tawana K, Bödör C, Wang J, Nagano A, et al. GATA2 monoallelic expression underlies reduced penetrance in inherited GATA2-mutated MDS/AML. Leukemia. 2018;32:2502–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Holst J, Szymczak-Workman AL, Vignali KM, Burton AR, Workman CJ, Vignali DA. Generation of T-cell receptor retrogenic mice. Nat Protoc. 2006;1:406–17. [DOI] [PubMed] [Google Scholar]
  • 24.Monaco G, Chen H, Poidinger M, Chen J, de Magalhães JP, Larbi A. flowAI: automatic and interactive anomaly discerning tools for flow cytometry data. Bioinforma Oxf Engl. 2016;32:2473–80. [DOI] [PubMed] [Google Scholar]
  • 25.Amid E, Warmuth MK TriMap: Large-scale Dimensionality Reduction Using Triplets. 2022. 10.48550/arXiv.1910.00204.
  • 26.Chong C-E, Venugopal P, Stokes PH, Lee YK, Brautigan PJ, Yeung DTO, et al. Differential effects on gene transcription and hematopoietic differentiation correlate with GATA2 mutant disease phenotypes. Leukemia. 2018;32:194–202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.McGrath KE, Frame JM, Fegan KH, Bowen JR, Conway SJ, Catherman SC, et al. Distinct sources of hematopoietic progenitors emerge before HSCs and provide functional blood cells in the mammalian embryo. Cell Rep. 2015;11:1892–904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Li Z, Schulz MH, Look T, Begemann M, Zenke M, Costa IG. Identification of transcription factor binding sites using ATAC-seq. Genome Biol. 2019;20:45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ariki R, Morikawa S, Mabuchi Y, Suzuki S, Nakatake M, Yoshioka K, et al. Homeodomain Transcription Factor Meis1 Is a Critical Regulator of Adult Bone Marrow Hematopoiesis. PLoS ONE. 2014;9:e87646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pietras EM, Lakshminarasimhan R, Techner J-M, Fong S, Flach J, Binnewies M, et al. Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons. J Exp Med. 2014;211:245–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Wilson NK, Foster SD, Wang X, Knezevic K, Schütte J, Kaimakis P, et al. Combinatorial Transcriptional Control In Blood Stem/Progenitor Cells: Genome-wide Analysis of Ten Major Transcriptional Regulators. Cell Stem Cell. 2010;7:532–44. [DOI] [PubMed] [Google Scholar]
  • 32.Guo G, Luc S, Marco E, Lin T-W, Peng C, Kerenyi MA, et al. Mapping cellular hierarchy by single cell analysis of the cell surface repertoire. Cell Stem Cell 2013; 13. 10.1016/j.stem.2013.07.017. [DOI] [PMC free article] [PubMed]
  • 33.Wilson NK, Kent DG, Buettner F, Shehata M, Macaulay IC, Calero-Nieto FJ, et al. Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations. Cell Stem Cell. 2015;16:712–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Rosales-Alvarez RE, Rettkowski J, Herman JS, Dumbović G, Cabezas-Wallscheid N, Grün D Gene expression noise dynamics unveil functional heterogeneity of ageing hematopoietic stem cells. Systems Biology, 2022 10.1101/2022.08.04.502776. [DOI] [PMC free article] [PubMed]
  • 35.King KY, Goodell MA. Inflammatory modulation of HSCs: viewing the HSC as a foundation for the immune response. Nat Rev Immunol. 2011;11:685–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Haas S, Hansson J, Klimmeck D, Loeffler D, Velten L, Uckelmann H, et al. Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors. Cell Stem Cell. 2015;17:422–34. [DOI] [PubMed] [Google Scholar]
  • 37.Maryanovich M, Zahalka AH, Pierce H, Pinho S, Nakahara F, Asada N, et al. Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche. Nat Med. 2018;24:782–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Bernitz JM, Kim HS, MacArthur B, Sieburg H, Moore K. Hematopoietic Stem Cells Count and Remember Self-Renewal Divisions. Cell. 2016;167:1296–309.e10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Mann M, Mehta A, de Boer CG, Kowalczyk MS, Lee K, Haldeman P, et al. Heterogeneous Responses of Hematopoietic Stem Cells to Inflammatory Stimuli Are Altered with Age. Cell Rep. 2018;25:2992–3005.e5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gekas C, Graf T. CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age. Blood. 2013;121:4463–72. [DOI] [PubMed] [Google Scholar]
  • 41.Largeaud L, Collin M, Monselet N, Vergez F, Fregona V, Larcher L, et al. Somatic genetic alterations predict haematological progression in GATA2 deficiency. Haematologica 2023. 10.3324/haematol.2022.282250. [DOI] [PMC free article] [PubMed]
  • 42.Cortés-Lavaud X, Landecho MF, Maicas M, Urquiza L, Merino J, Moreno-Miralles I, et al. GATA2 Germline Mutations Impair GATA2 Transcription, Causing Haploinsufficiency: Functional Analysis of the p.Arg396Gln Mutation. J Immunol. 2015;194:2190–8. [DOI] [PubMed] [Google Scholar]
  • 43.Oleaga-Quintas C. Inherited GATA2 Deficiency Is Dominant by Haploinsufficiency and Displays Incomplete Clinical Penetrance. J Clin Immunol. 2021;41:639–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.You X, Zhou Y, Chang Y-I, Kong G, Ranheim EA, Johnson KD, et al. Gata2 +9.5 enhancer regulates adult hematopoietic stem cell self-renewal and T-cell development. Blood Adv. 2022;6:1095–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Abdelfattah A, Hughes-Davies A, Clayfield L, Menendez-Gonzalez JB, Almotiri A, Alotaibi B, et al. Gata2 haploinsufficiency promotes proliferation and functional decline of hematopoietic stem cells with myeloid bias during aging. Blood Adv. 2021;5:4285–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Ganapathi KA, Townsley DM, Hsu AP, Arthur DC, Zerbe CS, Cuellar-Rodriguez J, et al. GATA2 deficiency-associated bone marrow disorder differs from idiopathic aplastic anemia. Blood. 2015;125:56–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Marks-Bluth J, Khanna A, Chandrakanthan V, Thoms J, Bee T, Eich C, et al. SMAD1 and SMAD5 Expression Is Coordinately Regulated by FLI1 and GATA2 during Endothelial Development. Mol Cell Biol. 2015;35:2165–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Trompouki E, Bowman TV, Lawton LN, Fan ZP, Wu D-C, DiBiase A, et al. Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration. Cell. 2011;147:577–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Guo G Mapping cellular hierarchy by single cell analysis of the cell surface repertoire. Cell Stem Cell 2013;13. [DOI] [PMC free article] [PubMed]
  • 50.Renders S, Svendsen AF, Panten J, Rama N, Maryanovich M, Sommerkamp P, et al. Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1. Nat Commun. 2021;12:608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Koyunlar C, Gioacchino E, Vadgama D, de Looper HWJ, Zink J, ter Borg M, et al. Gata2-regulated Gfi1b expression controls endothelial programming during endothelial-to-hematopoietic transition. Blood Adv 2023; bloodadvances.2022008019. [DOI] [PMC free article] [PubMed]

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Supplementary Materials

Supplementary material (12.4MB, pdf)

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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