We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.

Generalized verrucosis is a characteristic of several genetic and immunodeficiency disorders including epidermodysplasia verruciformis; warts, hypogammaglobulinaemia, infections and myelokathexis (WHIM) syndrome; warts, immunodeficiency, lymphoedema and anogenital dysplasia (WILD) syndrome; severe combined immune deficiency and HIV, among others. In recent years, it has been consistently recognized in patients with GATA2 deficiency, a novel immunodeficiency syndrome characterized by monocytopenia, B-cell and natural killer-cell lymphopenia, and a tendency to develop myeloid leukaemias and disseminated mycobacterial, human papillomavirus (HPV) and opportunistic fungal infections. Mutations in GATA2 cause haploinsufficiency and track in families as an autosomal dominant immunodeficiency. GATA2 is a transcription factor involved in early haematopoietic differentiation and lymphatic and vascular development. We describe a case of generalized verrucosis with HPV type 57 presenting in a young man with GATA2 deficiency. GATA2 deficiency is a novel dominant immunodeficiency that is often recognized later in life and should be considered in the differential diagnosis of patients with generalized verrucosis.

Fetal stem cells isolated from umbilical cord blood (UCB) possess a great capacity for proliferation and differentiation and serve as a valuable model system to study gene regulation. Expanded knowledge of the molecular control of hemoglobin synthesis will provide a basis for rational design of therapies for β-hemoglobinopathies. Transcriptome data are available for erythroid progenitors derived from adult stem cells, however studies to define molecular mechanisms controlling globin gene regulation during fetal erythropoiesis are limited. Here, we utilize UCB-CD34+ stem cells induced to undergo erythroid differentiation to characterize the transcriptome and transcription factor networks (TFNs) associated with the γ/β-globin switch during fetal erythropoiesis. UCB-CD34+ stem cells grown in the one-phase liquid culture system displayed a higher proliferative capacity than adult CD34+ stem cells. The γ/β-globin switch was observed after day 42 during fetal erythropoiesis in contrast to adult progenitors where the switch occurred around day 21. To gain insights into transcription factors involved in globin gene regulation, microarray analysis was performed on RNA isolated from UCB-CD34+ cell-derived erythroid progenitors harvested on day 21, 42, 49 and 56 using the HumanHT-12 Expression BeadChip. After data normalization, Gene Set Enrichment Analysis identified transcription factors (TFs) with significant changes in expression during the γ/β-globin switch. Forty-five TFs were silenced by day 56 (Profile-1) and 30 TFs were activated by day 56 (Profile-2). Both GSEA datasets were analyzed using the MIMI Cytoscape platform, which discovered TFNs centered on KLF4 and GATA2 (Profile-1) and KLF1 and GATA1 for Profile-2 genes. Subsequent shRNA studies in KU812 leukemia cells and human erythroid progenitors generated from UCB-CD34+ cells supported a negative role of MAFB in γ-globin regulation. The characteristics of erythroblasts derived from UCB-CD34+ stem cells including prolonged γ-globin expression combined with unique TFNs support novel mechanisms controlling the γ/β-globin switch during UCB-derived erythropoiesis.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment option for myelodysplastic syndromes (MDS). Little is known about the prognostic impact of mutations, for example, in TP53 specifically after allo-HSCT. We here describe the prognostic impact of mutations in a panel of 19 genes analyzed by amplicon-based next-generation-sequencing in a uniformly treated patient cohort. Sixty-two patients with a median age of 61 yr suffered from MDS with 0-20% bone marrow blasts. International Prognostic Score was intermediate 1 (15%) and higher (79%). Conditioning uniformly was performed using a sequential approach in which FLAMSA chemotherapy was followed by Busulfan-based conditioning. Patients mostly were transplanted from an unrelated donor (77%), and 36% of patients received a graft from a mismatched donor. Median number of mutations was 2 (range 0-6). RUNX1, GATA2, TET2, and CEBPA were the genes most frequently found mutated. TP53, a factor previously reported to confer adverse prognostic impact after allogeneic stem cell transplantation, was mutated in samples from eight patients, one of which showed a silent mutation. With an estimated 5-yr overall/disease-free survival of 48 ± 7%/41 ± 7%, none of the mutations analyzed showed a prognostic impact in this analysis of the largest uniformly treated cohort thus far. This especially holds true for patients with a mutation in TP53.

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.

Although thyroid hormone is a known stimulator of erythropoietic differentiation, severe anemia is sometimes observed in patients with hyperthyroidism and this mechanism is not fully understood. The aim of this study was to investigate the effect of triiodothyronine (T3) on hemin-induced erythropoiesis. Human erythroleukemia K562 cells were used as an erythroid differentiation model. Cell differentiation was induced by hemin and the effect of pre-incubation with T3 (0.1 to 100 nM) was analyzed by measuring the benzidine-positive rate, hemoglobin content, CD71 expression (transferrin receptor), and mRNA expression for transcription factors related to erythropoiesis and thyroid hormone receptors (TRs). Hemin, a promoter of erythroid differentiation, increased the levels of mRNAs for TRα, TRβ, and retinoid X receptor α (RXRα), as well as those for nuclear factor-erythroid 2 (NFE2), GATA-binding protein 1 (GATA1) and GATA-binding protein 2 (GATA2). Lower concentrations of T3 had a stimulatory effect on hemin-induced hemoglobin production (1 and 10 nM), CD71 expression (0.1 nM), and α-globin mRNA expression (1 nM), while a higher concentration of T3 (100 nM) abrogated the stimulatory effect on these parameters. T3 at 100 nM did not affect cell viability and proliferation, suggesting that the abrogation of erythropoiesis enhancement was not due to toxicity. T3 at 100 nM also significantly inhibited expression of GATA2 and RXRα mRNA, compared to 1 nM T3. We conclude that a high concentration of T3 attenuates the classical stimulatory effect on erythropoiesis exerted by a low concentration of T3 in hemin-induced K562 cells.

Background & Aims Autoimmune hepatitis (AIH), an immune-mediated liver disease, originates as a consequence of interacting genetic and environmental risk factors. Treatment remains non-specific and prone to side effects. Deficiencies in regulatory T cell (Treg) function are hypothesized to contribute to the pathogenesis of AIH. Methods We describe an adult patient who presented with AIH in the context of monocytopenia. The patient was characterized by GATA2 gene sequencing, flow cytometry of peripheral blood for leucocyte subsets, ELISA for serum Flt-3 ligand, and immunohistochemistry of liver biopsy tissue. Results Sequencing confirmed a GATA2 mutation. Peripheral Treg were absent in the context of a preserved total T cell count. Immunostaining for the Treg transcription factor FOXP3 was reduced in liver tissue as compared to a control AIH specimen. There were marked deficiencies in multiple antigen-presenting cell subsets and Flt-3 ligand was elevated. These findings are consistent with previous reports of GATA2 dysfunction. Conclusions The association of a GATA2 mutation with AIH is previously unrecognized. GATA2 encodes a hematopoietic cell transcription factor, and mutations may manifest as monocytopenia, dendritic and B cell deficiencies, myelodysplasia, and immunodeficiency. Tregs may be depleted as in this case. Our findings provide support for the role of Tregs in AIH, complement reports of other deficiencies in T cell regulation causing AIH-like syndromes, and support the rationale of attempting to modulate the Treg axis for the therapeutic benefit of AIH patients.

GATA binding protein 2 (GATA2) deficiency is a rare disorder of hematopoiesis, lymphatics, and immunity caused by spontaneous or autosomal dominant mutations in the GATA2 gene. Clinical manifestations range from neutropenia, lymphedema, deafness, to severe viral and mycobacterial infections, bone marrow failure, and acute myeloid leukemia. Patients also present with monocytopenia, dendritic cell, B- and natural killer (NK)-cell deficiency. We studied the T-cell and NK-cell compartments of four GATA2-deficient patients to assess if changes in these lymphocyte populations could be correlated with clinical phenotype. Patients with more severe clinical complications demonstrated a senescent T-cell phenotype whereas patients with lower clinical score had undetectable changes relative to controls. In contrast, patients' NK-cells demonstrated an immature/activated phenotype that did not correlate with clinical score, suggesting an intrinsic NK-cell defect. These studies will help us to determine the contribution of T- and NK-cell dysregulation to the clinical phenotype of GATA2 patients, and may help to establish the most accurate therapeutic options for these patients. Asymptomatic patients may be taken into consideration for hematopoietic stem cell transplantation when dysregulation of T-cell and NK-cell compartment is present.

GATA1 is a critical regulator of erythropoiesis. While the mechanisms underlying the high-level expression of GATA1 in maturing erythroid cells have been studied extensively, the initial activation of the Gata1 gene in early hematopoietic progenitors remains to be elucidated. We previously identified a hematopoietic stem and progenitor cell (HSPC)-specific silencer element (the Gata1 methylation-determining region [G1MDR]) that recruits DNA methyltransferase 1 (Dnmt1) and provokes methylation of the Gata1 gene enhancer. In the present study, we hypothesized that removal of the G1MDR-mediated silencing machinery is the molecular basis of the initial activation of the Gata1 gene and erythropoiesis. To address this hypothesis, we generated transgenic mouse lines harboring a Gata1 bacterial artificial chromosome in which the G1MDR was deleted. The mice exhibited abundant GATA1 expression in HSPCs, in a GATA2-dependent manner. The ectopic GATA1 expression repressed Gata2 transcription and induced erythropoiesis and apoptosis of HSPCs. Furthermore, genetic deletion of Dnmt1 in HSPCs activated Gata1 expression and depleted HSPCs, thus recapitulating the HSC phenotype associated with GATA1 gain of function. These results demonstrate that the G1MDR holds the key to HSPC maintenance and suggest that release from this suppressive mechanism is a fundamental requirement for subsequent initiation of erythroid differentiation.

The hematopoietic GATA factors GATA-1 and GATA-2, which have distinct and overlapping roles to regulate blood cell development, are reciprocally expressed during erythropoiesis. GATA-1 directly represses Gata2 transcription, and reduced GATA-2 synthesis promotes red blood cell development. Gata2 repression involves "GATA switches" in which GATA-1 displaces GATA-2 from Gata2 regulatory regions. We show that extragenic GATA switch sites occupied by GATA-2 associate with as much RNA polymerase II (Pol II) and basal transcription factors as present at the active Gata2 promoters. Pol II bound to GATA switch sites in the active locus was phosphorylated on serine 5 of the carboxyl-terminal domain, indicative of elongation competence. GATA-1-mediated displacement of GATA-2 from GATA switch sites reduced Pol II recruitment to all sites except the far upstream -77-kb region. Surprisingly, TFIIB occupancy persisted at most sites upon repression. These results indicate that GATA-2-bound extragenic regulatory elements recruit Pol II, GATA-1 binding expels Pol II, and despite the persistent TFIIB-chromatin complexes, Pol II recruitment is blocked.

The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self-renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34+ cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway-PD0325901 (PD)-significantly reduces the expansion of CD34+ and CD34+ CD38- cells, while there is no change in the expression of stemness-related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34+ cells. Notably, when ERK pathway is blocked in UCB-MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst-forming unit-erythroid colony (BFU-E) as well as enhancement of erythroid glycophorin-A marker. These results are in total conformity with up-regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down-regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self-renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB-haematopoietic progenitor cells.

We had analyzed 10 exome sequencing data and single nucleotide polymorphism chips for blood cancer provided by the PGM21 (The National Project for Personalized Genomic Medicine) Award program. We had removed sample G06 because the pair is not correct and G10 because of possible contamination. In-house software somatic copy-number and heterozygosity alteration estimation (SCHALE) was used to detect one loss of heterozygosity region in G05. We had discovered 27 functionally important mutations. Network and pathway analyses gave us clues that NPM1, GATA2, and CEBPA were major driver genes. By comparing with previous somatic mutation profiles, we had concluded that the provided data originated from acute myeloid leukemia. Protein structure modeling showed that somatic mutations in IDH2, RASGEF1B, and MSH4 can affect protein structures.

Various genetic disorders are known to be associated with cancer predisposition. For example, children with Down syndrome are predisposed to developing acute myeloid leukemia, and those with RASopathies, such as Noonan syndrome, are predisposed to juvenile myelomonocytic leukemia. To date, more than 250 diseases or syndromes have been reported to be associated with the development of pediatric cancers. Recently, the advent of the massive parallel sequencing technique revealed several germline mutations, including RUNX1, CEBPA, GATA2, SRP72, ETV6, and DDX41, which are associated with familial myeloid malignancies. A significant number of children with myeloid malignancies may harbor pathognomonic germline variants. It is strongly recommended that precise diagnosis, genetic counseling, familial screening, and follow-up programs be provided for patients with such a predisposition to cancer. To identify genetic disorders associated with predispositions to pediatric myeloid malignancies, the development of an efficient screening system with the massive parallel sequencer for germline and somatic mutations, which would also be useful for familial genetic studies and prediction of tumor progression, is needed.

Primitive hematopoiesis generates red blood cells that deliver oxygen to the developing embryo. Mesodermal cells commit to a primitive blood cell fate during gastrulation and, in order to do so the mesoderm must receive non-cell autonomous signals transmitted from other germ layers. In Xenopus, the transcription factor Gata2 functions in ectodermal cells to generate or transmit the non-cell autonomous signals. Here we have identified Breast Cancer Antiestrogen Resistance 3 (bcar3) as a gene that is induced in ectodermal cells downstream of Gata2. Bcar3 and its binding partner Bcar1 function to transduce integrin signaling, leading to changes in cellular morphology, motility and adhesion. We show that gata2, bcar3 and bcar1 are co-expressed in ventral ectoderm from early gastrula to early tailbud stages. At later stages of development, bcar3 and bcar1 are co-expressed in the spinal cord, notochord, fin mesenchyme and pronephros but each shows additional unique sites of expression. These co-expression and unique expression patterns suggest that Bcar3 and Bcar1 may function together but also independently during Xenopus development.

Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily; dysregulated expression of NGAL has been observed in several benign and malignant diseases. In the present study, differentially expressed genes, in comparison with those of control cells, in the mRNA expression profile of EC109 esophageal squamous cell carcinoma (ESCC) cells following NGAL overexpression were analyzed by multiple bioinformatic tools for a comprehensive understanding. A total of 29 gene ontology (GO) terms associated with immune function, chromatin structure and gene transcription were identified among the differentially expressed genes (DEGs) in NGAL overexpressing cells. In addition to the detected GO categories, the results from the functional annotation chart revealed that the differentially expressed genes were also associated with 101 functional annotation category terms. A total of 59 subpathways associated locally with the differentially expressed genes were identified by subpathway analysis, a markedly greater total that detected by traditional pathway enrichment analysis only. Promoter analysis indicated that the potential transcription factors Snail, deltaEF1, Mycn, Arnt, MNB1A, PBF, E74A, Ubx, SPI1 and GATA2 were unique to the downregulated DEG promoters, while bZIP910, ZNF42 and SOX9 were unique for the upregulated DEG promoters. In conclusion, the understanding of the role of NGAL overexpression in ESCC has been improved through the present bioinformatic analysis.

GATA2 deficiency is a genetic disorder caused by inherited or sporadic haploinsufficient mutations in the GATA2 gene. Patients have abnormalities in hematopoiesis, lymphangiogenesis and immunity; encompassing a broad range of clinical syndromes, mainly characterized by monocytopenia, B and NK cell cytopenia, severe or recurrent infections, and a high risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). We report a case of an Argentinean woman who presented with multiple opportunistic infections as her first manifestation of GATA2 deficiency.

Tartrate-resistant acid phosphatase (TRAP) is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. However, factors regulating human TRAP gene expression have not been clearly defined. Therefore, we isolated a genomic clone (CL-9) for TRAP containing a 14-kb insert. A restriction map was generated for this insert, and a 2.6-kb ApaI fragment containing the 5'-flanking region was subcloned. Sequence analysis of this fragment revealed the presence of candidate transcription factor-binding sequences for H-APF-1, SP1, GATA2, and the c-Myc proto-oncogene. PCR analysis of RNA isolated from human osteoclastomas and pagetic bone revealed a 276-bp intron at -1 bp to -276 bp relative to the ATG and a transcript originating from this intron. Rapid amplification of the 5' end of the human TRAP mRNA by PCR indicated the presence of a 93-bp untranslated region 5' from the intron. Promoter activity was detected in the DNA fragment from +1 bp to -1903 bp relative to the ATG initiation codon, which drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. Comparison of the human TRAP 5'-flanking region with mouse TRAP and uteroferrin revealed 41% and 47% homology, respectively. This suggests that regulation of human TRAP gene expression may differ from that for the murine TRAP gene.

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult β-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene.

BACKGROUND

Leukemic and mesenchymal stem cells interact in the leukemic microenvironment and affect each other differently. This interplay has also important implications for the hematopoietic stem cell (HSC) biology and function. This study evaluated human HSC self-renewal potential and quiescence in an in vitro leukemic niche without leukemic cells.

METHODS

A leukemic niche was established by co-culturing mesenchymal stem cells with a fresh conditioned medium obtained from a leukemic (REH) cell line. After 3 days, the REH-conditioned medium was removed and freshly isolated CD34+ at a density of up to 100,000 cells/ml were added to the leukemic niche. CD34+ cell evaluations (cell cycle, self-renewal gene expression and migration capacity) were performed after 3 further days of co-culture. Additionally, we preliminary investigated the soluble factors present in the leukemic niche and their effect on the mesenchymal stem cells. Statistical significance was assessed by Student's t test or the nonparametric test Kolmogorov-Smirnov.

RESULTS

By co-culturing normal mesenchymal stem cells with the REH-conditioned medium we showed that hematopoietic stem cells, normally in a quiescent state, enter cell cycle and proliferate. This loss of quiescence was accompanied by an increased expression of Ki-67 and c-Myc, two well-known cell proliferation-associated markers. Two central regulators of quiescence GATA2 and p53 were also down regulated. Importantly, two genes involved in HSC self-renewal, Klf4 and the histone-lysine N-methyltransferase enzyme Ezh2, were severely affected. On the contrary, c-Kit expression, the stem cell factor receptor, was upregulated in hematopoietic stem cells when compared to the normal niche. Interestingly, mesenchymal stem cells incubated with the REH-conditioned medium stopped growing, showed a flattened morphology with the appearance of small vacuoles, and importantly, became positive for the senescence-associated beta-galactosidase activity. Evaluation of the leukemic-conditioned medium showed increased IL-6 and IL-8, suggesting that these cytokines could be responsible for the observed changes.

CONCLUSIONS

Our results showed that quiescence and self-renewal are severely affected in this leukemic niche. This in vitro leukemic niche, established without leukemic cells, will facilitate HSC gene expression evaluation and the development of therapeutic agents aimed to neutralize soluble factors and the cell signaling pathways involved in HSC alterations.

Pulmonary alveolar proteinosis (PAP) is categorized into hereditary, secondary and autoimmune PAP (aPAP) types. The common pathogenesis is the ability of the alveolar macrophages to catabolize phagocytized surfactant is affected. Hereditary PAP is caused by mutations involving the GM-CSF signaling, particularly in genes for the GM-CSF receptor and sometimes by GATA2 mutations. Secondary PAP occurs in hematologic malignancies, other hematologic disorders, miscellaneous malignancies, fume and dust inhalation, drugs, autoimmune disorders and immunodeficiencies. aPAP is related to the production of GM-CSF autoantibodies. PAP is characterized morphologically by the inappropriate and progressive 'occupation' of the alveolar spaces by an excessive amount of unprocessed surfactant, limiting gas exchange and gradually exhausting the respiratory reserve. Myeloid cells' immunity deteriorates, increasing the risk of infections. Treatment of PAP is based on its etiology. In aPAP, recent therapeutic advances might shift the treatment option from the whole lung lavage procedure under general anesthesia to the inhalation of GM-CSF 'as needed'.

BACKGROUND

Acute myeloid leukemia (AML) 1-Evi-1 is a chimeric gene generated by the t (3; 21) (q26; q22) translocation, which leads into malignant transformation of hematopoietic stem cells by unclear mechanisms. This in vivo study aimed to establish a stable line of zebrafish expressing the human RUNX1-Evi-1 fusion gene under the control of a heat stress-inducible bidirectional promoter, and investigate its roles in hematopoiesis and hematologic malignancies.

METHODS

We introduced human RUNX1-Evi-1 fusion gene into embryonic zebrafish through a heat-shock promoter to establish Tg(RE:HSE:EGFP) zebrafish. Two males and one female mosaic F0 zebrafish embryos (2.1%) were identified as stable positive germline transgenic zebrafish.

RESULTS

The population of immature myeloid cells and hematopoietic blast cells were accumulated in peripheral blood and single cell suspension from kidney of adult Tg(RE:HSE:EGFP) zebrafish. RUNX1-Evi-1 presented an intensive influence on hematopoietic regulatory factors. Consequently, primitive hematopoiesis was enhanced by upregulation of gata2 and scl, while erythropoiesis was downregulated due to the suppression of gata1. Early stage of myelopoiesis was flourishing with the high expression of pu.1, but it was inhibited along with the low expression of mpo. Microarray analysis demonstrated that RUNX1-Evi-1 not only upregulated proteasome, cell cycle, glycolysis/gluconeogenesis, tyrosine metabolism, drug metabolism, and PPAR pathway, but also suppressed transforming growth factor β, Jak-STAT, DNA replication, mismatch repair, p53 pathway, JNK signaling pathway, and nucleotide excision repair. Interestingly, histone deacetylase 4 was significantly up-regulated. Factors in cell proliferation were obviously suppressed after 3-day treatment with histone deacetylase inhibitor, valproic acid. Accordingly, higher proportion of G1 arrest and apoptosis were manifested by the propidium iodide staining.

CONCLUSIONS

RUNX1-Evi-1 may promote proliferation and apoptosis resistance of primitive hematopoietic cell, and inhibit the differentiation of myeloid cells with the synergy of different pathways and factors. VPA may be a promising choice in the molecular targeting therapy of RUNX1-Evi-1-related leukemia.

The dynamic and reversed expression of GATA1 and GATA2 are essential for proper erythroid differentiation. Our previous work demonstrates that LSD1, a histone H3K4 demethylase, represses GATA2 expression at late stage of erythroid differentiation. K562 and MEL cells were used and cultured in Roswell Park Memorial Institute-1640 medium (RPMI) and Dulbecco's modified Eagle's medium (DMEM), respectively. Western blot assay was used to examine the GATA1, GATA2, TAL1, HDAC1, HDAC2, CoREST and β-actin protein. The immunoprecipitation assay and GST pull-down assay were employed to detect the precipitated protein complexes and investigate the interaction between the proteins. The small interfering RNA (siRNA) and nonspecific control siRNA were synthesized to silence the target genes. Double fluorescence immunostaining was used to observe the association of LSD1 with GATA2 in K562 cells. The results indicated that knockdown of LSD1 in K562 cell causes increased H3K4 di-methylation at GATA1 locus and activates GATA1 expression, demonstrating that LSD1 represses GATA1 expression through LSD1-mediated histone demethylation. Upon induced erythroid differentiation of K562 cells, the interaction between GATA2 and LSD1 is decreased, consistent with a de-repression of GATA1 expression. Meanwhile, the interaction between TAL1 and LSD1 is increased, which forms a complex that efficiently suppresses GATA2 expression. In conclusion, these observations reveal an elegant mechanism to modulate GATA1 and GATA2 expression during erythroid differentiation. While LSD1 mainly forms complex with GATA2 to repress GATA1 expression in hematopoietic progenitor cells, it mostly forms complex with TAL1 to repress GATA2 expression in differentiated cells.

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.

We performed allogeneic hematopoietic stem cell transplantation in a patient with GATA2 deficiency and an Epstein-Barr virus (EBV)-related spindle cell tumor involving the liver and possibly bone. He received a matched-related donor transplant with donor peripheral blood stem cells following a myeloablative conditioning regimen. He achieved rapid and high levels of donor engraftment and had complete reversal of the clinical and immunologic phenotype of MonoMAC/GATA2 deficiency and eradication of the EBV tumors after 3 years of follow-up. Thus, allogeneic hematopoietic stem cell transplant results in reconstitution of immunologic function and cure of EBV-associated malignancy in MonoMAC/GATA2 deficiency.