BACKGROUND: Haemophagocytic lymphohistiocytosis (HLH) is a syndrome that occurs in patients with severe systemic hyperinflammation. GATA binding protein 2 (GATA2) is a transcription factor and key component in haematopoiesis and stem cell biology.
METHODS: Three patients with HLH, one with Mycobacterium avium infection, one with Epstein-Barr virus (EBV) infection, and one with Mycobacterium kansasii infection, were all subsequently found to have a defect in the GATA2 gene through genetic testing.
CONCLUSIONS: GATA2 deficiency syndrome should be considered in patients with myelodysplastic syndrome, nontuberculous mycobacterium infection and HLH. In addition, the GATA2 gene variant may be a genetic defect that could be the cause of the primary HLH. However, further studies are needed to confirm the role of GATA2 pathogenic variants in the pathogenesis of HLH.

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP‑like cell line HEL western blotting, RT‑qPCR, lentivirus‑mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation‑specific (ETS) transcription factor friend leukemia integration factor 1 (Fli‑1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1‑mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation‑specific‑related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA‑mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.

Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are \"open\" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin \"pioneering factor\" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

UNASSIGNED: Long noncoding RNAs (lncRNAs) are extensively expressed in eukaryotic cells and have been revealed to be important for regulating cell differentiation. Many lncRNAs have been found to regulate erythroid differentiation in the mouse. However, given the low sequence conservation of lncRNAs between mouse and human, our understanding of lncRNAs in human erythroid differentiation remains incomplete. lncRNAs are often transcribed opposite to protein coding genes and regulate their expression. Here, we characterized a human erythrocyte-expressed lncRNA, GATA2AS, which is transcribed opposite to erythroid transcription regulator GATA2. GATA2AS is a 2080-bp long, primarily nucleus-localized noncoding RNA that is expressed in erythroid progenitor cells and decreases during differentiation. Knockout of GATA2AS in human HUDEP2 erythroid progenitor cells using CRISPR-Cas9 genome editing to remove the transcription start site accelerated erythroid differentiation and dysregulated erythroblast gene expression. We identified GATA2AS as a novel GATA2 and HBG activator. Chromatin isolation by RNA purification showed that GATA2AS binds to thousands of genomic sites and colocalizes at a subset of sites with erythroid transcription factors including LRF and KLF1. RNA pulldown and RNA immunoprecipitation confirmed interaction between GATA2AS and LRF and KLF1. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that knockout of GATA2AS reduces binding of these transcription factors genome wide. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and H3K27ac ChIP-seq showed that GATA2AS is essential to maintain the chromatin regulatory landscape during erythroid differentiation. Knockdown of GATA2AS in human primary CD34+ cells mimicked results in HUDEP2 cells. Overall, our results implicate human-specific lncRNA GATA2AS as a regulator of erythroid differentiation by influencing erythroid transcription factor binding and the chromatin regulatory landscape.

Mutations in genes encoding transcription factors inactivate or generate ectopic activities to instigate pathogenesis. By disrupting hematopoietic stem/progenitor cells, GATA2 germline variants create a bone marrow failure and leukemia predisposition, GATA2 deficiency syndrome, yet mechanisms underlying the complex phenotypic constellation are unresolved. We used a GATA2-deficient progenitor rescue system to analyze how genetic variation influences GATA2 functions. Pathogenic variants impaired, without abrogating, GATA2-dependent transcriptional regulation. Variants promoted eosinophil and repressed monocytic differentiation without regulating mast cell and erythroid differentiation. While GATA2 and T354M required the DNA-binding C-terminal zinc finger, T354M disproportionately required the N-terminal finger and N terminus. GATA2 and T354M activated a CCAAT/Enhancer Binding Protein-ε (C/EBPε) enhancer, creating a feedforward loop operating with the T-cell Acute Lymphocyte Leukemia-1 (TAL1) transcription factor. Elevating C/EBPε partially normalized hematopoietic defects of GATA2-deficient progenitors. Thus, pathogenic germline variation discriminatively spares or compromises transcription factor attributes, and retaining an obligate enhancer mechanism distorts a multilineage differentiation program.

Drug resistance in breast cancer (BC) is a clinical challenge. Exploring the mechanism and identifying a precise predictive biomarker for the drug resistance in BC is critical. Three first-line drug (paclitaxel, doxorubicin and tamoxifen) resistance datasets in BC from GEO were merged to obtain 1,461 differentially expressed genes for weighted correlation network analysis, resulting in identifying ATRX as the hub gene. ATRX is a chromatin remodelling protein, therefore, ATRX-associated transcription factors were explored, thereby identifying the network of AR, GLI3 and GATA2. GO and KEGG analyses revealed immunity, transcriptional regulation and endocrinotherapy/chemotherapy resistance were enriched. Moreover, CIBERSORT revealed immunity regulation was inhibited in the resistance group. ssGSEA showed a significantly lower immune status in the ATRX-Low group compared to the ATRX-High group. Furthermore, the peaks of H3K9me3 ChIP-seq on the four genes were higher in normal tissues than in BC tissues. Notably, the frequency of ATRX mutation was higher than BRCA in BC. Moreover, depressed ATRX revealed worse overall survival and disease-free survival in the human epidermal growth factor receptor 2 (HER2)-/hormone receptor (HR)+ BC. Additionally, depressed ATRX predicted poor results for patients who underwent endocrinotherapy or chemotherapy in the HER2-/HR+ BC subgroup. A nomogram based on ATRX, TILs and ER exhibited a significantly accurate survival prediction ability. Importantly, overexpression of ATRX significantly inhibited the IC50 of the three first-line drugs on MCF-7 cell. Thus, ATRX is an efficient predictive biomarker for endocrinotherapy and chemotherapy resistance in HER2-/HR+ BC and acts by suppressing the AR, GLI3 and GATA2 transcriptional network.

GATA binding protein 2 (GATA2) is a conserved zinc finger transcription factor that regulates the emergence and maintenance of complex genetic programs driving development and function of hematopoietic stem and progenitor cells (HSPCs). Patients born with monoallelic GATA2 mutations develop myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML), whereas acquired GATA2 mutations are reported in 3% to 5% of sporadic AML cases. The mechanisms by which aberrant GATA2 activity promotes MDS and AML are incompletely understood. Efforts to understand GATA2 in basic biology and disease will be facilitated by the development of broadly efficacious antibodies recognizing physiologic levels of GATA2 in diverse tissue types and assays. Here, we purified a polyclonal anti-GATA2 antibody and generated multiple highly specific anti-GATA2 monoclonal antibodies, optimized them for immunohistochemistry on patient bone marrow bioosy samples, and analyzed GATA2 expression in adults with healthy bone marrow, MDS, and acute leukemia. In healthy bone marrow, GATA2 was detected in mast cells, subsets of CD34+ HSPCs, E-cadherin-positive erythroid progenitors, and megakaryocytes. In MDS, GATA2 expression correlates with bone marrow blast percentage, positively correlates with myeloid dysplasia and complex cytogenetics, and is a nonindependent negative predictor of overall survival. In acute leukemia, the percent of GATA2+ blasts closely associates with myeloid lineage, whereas a subset of lymphoblastic and undifferentiated leukemias with myeloid features also express GATA2. However, the percent of GATA2+ blasts in AML is highly variable. Elevated GATA2 expression in AML blasts correlates with peripheral neutropenia and complex AML cytogenetics but, unlike in MDS, does not predict survival.

Previous studies of the murine Ly49 and human KIR gene clusters implicated competing sense and antisense promoters in the control of variegated gene expression. In the current study, an examination of transcription factor genes defines an abundance of convergent and divergent sense/antisense promoter pairs, suggesting that competing promoters may control cell fate determination. Differentiation of CD34+ hematopoietic progenitors in vitro shows that cells with GATA1 antisense transcription have enhanced GATA2 transcription and a mast cell phenotype, whereas cells with GATA2 antisense transcription have increased GATA1 transcripts and an erythroblast phenotype. Detailed analyses of the AHR and RORC genes demonstrate the ability of competing promoters to act as binary switches and the association of antisense transcription with an immature/progenitor cell phenotype. These data indicate that alternative cell fates generated by promoter competition in lineage-determining transcription factors contribute to the programming of cell differentiation.

OBJECTIVE: Hepatitis B virus (HBV) spliced variants are associated with viral persistence or pathogenicity. Hepatitis B doubly spliced protein (HBDSP), which has been previously reported as a pleiotropic transactivator protein, can potentially serve as an HBV virulence factor. However, the underlying mechanisms of HBDSP in HBV-associated liver diseases remain to be elucidated. In this study, we revealed that HBDSP promotes cellular apoptosis and induces wt-p53-dependent apoptotic signaling pathway in wt-p53 hepatocellular cells by transactivating p53 transcription, and increases the release of HBV progeny. Therefore, HBDSP may promote the HBV particles release through wt-p53-dependent hepatocellular apoptosis. Our findings suggest that blocking HBDSP-induced wt-p53-dependent apoptosis might have therapeutic values for chronic hepatitis B.