GATA2 +9.5 enhancer: from principles of hematopoiesis to genetic diagnosis in precision medicine.

PURPOSE OF REVIEW: By establishing mechanisms that deliver oxygen to sustain cells and tissues, fight life-threatening pathogens and harness the immune system to eradicate cancer cells, hematopoietic stem and progenitor cells (HSPCs) are vital in health and disease. The cell biological framework for HSPC generation has been rigorously developed, yet recent single-cell transcriptomic analyses have unveiled permutations of the hematopoietic hierarchy that differ considerably from the traditional roadmap. Deploying mutants that disrupt specific steps in hematopoiesis constitutes a powerful strategy for deconvoluting the complex cell biology. It is striking that a single transcription factor, GATA2, is so crucial for HSPC generation and function, and therefore it is instructive to consider mechanisms governing GATA2 expression and activity. The present review focuses on an essential GATA2 enhancer (+9.5) and how +9.5 mutants inform basic and clinical/translational science. RECENT FINDINGS: +9.5 is essential for HSPC generation and function during development and hematopoietic regeneration. Human +9.5 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. Qualitatively and quantitatively distinct contributions of +9.5 cis-regulatory elements confer context-dependent enhancer activity. The discovery of +9.5 and its mutant alleles spawned fundamental insights into hematopoiesis, and given its role to suppress blood disease emergence, clinical centers test for mutations in this sequence to diagnose the cause of enigmatic cytopenias. SUMMARY: Multidisciplinary approaches to discover and understand cis-regulatory elements governing expression of key regulators of hematopoiesis unveil biological and mechanistic insights that provide the logic for innovating clinical applications.

The Transcription Factor Ehf Is Involved in TGF-ß-Induced Suppression of FcεRI and c-Kit Expression and FcεRI-Mediated Activation in Mast Cells.

FcεRI, which is composed of α, ß, and γ subunits, plays an important role in IgE-mediated allergic responses. TGF-ß1 has been reported to suppress FcεRI and stem cell factor receptor c-Kit expression on mast cell surfaces and to suppress mast cell activation induced by cross-linking of FcεRI. However, the molecular mechanism by which these expressions and activation are suppressed by TGF-ß1 remains unclear. In this study, we found that the expression of Ets homologous factor (Ehf), a member of the Ets family transcriptional factors, is upregulated by TGF-ß/Smad signaling in mouse bone marrow-derived mast cells (BMMCs). Forced expression of Ehf in BMMCs repressed the transcription of genes encoding FcεRIα, FcεRIß, and c-Kit, resulting in a reduction in cell surface FcεRI and c-Kit expression. Additionally, forced expression of Ehf suppressed FcεRI-mediated degranulation and cytokine production. Ehf inhibited the promoter activity of genes encoding FcεRIα, FcεRIß, and c-Kit by binding to these gene promoters. Furthermore, the mRNA levels of Gata1, Gata2, and Stat5b were lower in BMMCs stably expressing Ehf compared with control cells. Because GATA-1 and GATA-2 are positive regulators of FcεRI and c-Kit expression, decreased expression of GATAs may be also involved in the reduction of FcεRI and c-Kit expression. Decreased expression of Stat5 may contribute to the suppression of cytokine production by BMMCs. In part, mast cell response to TGF-ß1 was mimicked by forced expression of Ehf, suggesting that TGF-ß1 suppresses FcεRI and c-Kit expression and suppresses FcεRI-mediated activation through upregulation of Ehf.

Protective vaccination and blood-stage malaria modify DNA methylation of gene promoters in the liver of Balb/c mice.

Epigenetic mechanisms such as DNA methylation are increasingly recognized to be critical for vaccination efficacy and outcome of different infectious diseases, but corresponding information is scarcely available for host defense against malaria. In the experimental blood-stage malaria Plasmodium chabaudi, we investigate the possible effects of a blood-stage vaccine on DNA methylation of gene promoters in the liver, known as effector against blood-stage malaria, using DNA methylation microarrays. Naturally susceptible Balb/c mice acquire, by protective vaccination, the potency to survive P. chabaudi malaria and, concomitantly, modifications of constitutive DNA methylation of promoters of numerous genes in the liver; specifically, promoters of 256 genes are hyper(=up)- and 345 genes are hypo(=down)-methylated (p < 0.05). Protective vaccination also leads to changes in promoter DNA methylation upon challenge with P. chabaudi at peak parasitemia on day 8 post infection (p.i.), when 571 and 1013 gene promoters are up- and down-methylated, respectively, in relation to constitutive DNA methylation (p < 0.05). Gene set enrichment analyses reveal that both vaccination and P. chabaudi infections mainly modify promoters of those genes which are most statistically enriched with functions relating to regulation of transcription. Genes with down-methylated promoters encompass those encoding CX3CL1, GP130, and GATA2, known to be involved in monocyte recruitment, IL-6 trans-signaling, and onset of erythropoiesis, respectively. Our data suggest that vaccination may epigenetically improve parts of several effector functions of the liver against blood-stage malaria, as, e.g., recruitment of monocyte/macrophage to the liver accelerated liver regeneration and extramedullary hepatic erythropoiesis, thus leading to self-healing of otherwise lethal P. chabaudi blood-stage malaria.

Molecular basis of crosstalk between oncogenic Ras and the master regulator of hematopoiesis GATA-2.

Disease mutations provide unique opportunities to decipher protein and cell function. Mutations in the master regulator of hematopoiesis GATA-2 underlie an immunodeficiency associated with myelodysplastic syndrome and leukemia. We discovered that a GATA-2 disease mutant (T354M) defective in chromatin binding was hyperphosphorylated by p38 mitogen-activated protein kinase. p38 also induced multisite phosphorylation of wild-type GATA-2, which required a single phosphorylated residue (S192). Phosphorylation of GATA-2, but not T354M, stimulated target gene expression. While crosstalk between oncogenic Ras and GATA-2 has been implicated as an important axis in cancer biology, its mechanistic underpinnings are unclear. Oncogenic Ras enhanced S192-dependent GATA-2 phosphorylation, nuclear foci localization, and transcriptional activation. These studies define a mechanism that controls a key regulator of hematopoiesis and a dual mode of impairing GATA-2-dependent genetic networks: mutational disruption of chromatin occupancy yielding insufficient GATA-2, and oncogenic Ras-mediated amplification of GATA-2 activity.

Hyperosmolarity-Induced Down-Regulation of Claudin-2 Mediated by Decrease in PKCß-Dependent GATA-2 in MDCK Cells.

Hyperosmolarity decreases claudin-2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity-induced decrease in claudin-2 expression is inhibited by Go6983, a non-selective protein kinase C (PKC) inhibitor, and PKCß specific inhibitor in Madin-Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca(2+) concentration and phosphorylated PKCß level, which were inhibited by RN-1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12-myristate 13-acetate, a PKC activator, decreased claudin-2 expression. These results indicate hyperosmolarity decreases claudin-2 expression mediated by the activation of RN-1734-sensitive channel and PKCß. Hyperosmolarity decreased promoter activity of claudin-2, which was inhibited by Go6983 and PKCß inhibitor similar to those in real-time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of -469/-6, a deletion mutant. Claudin-2 has hyperosmolarity-sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA-2, which was inhibited by Go6983 and PKCß inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity-induced decrease in reporter activity and claudin-2 expression were rescued by over-expression of wild type GATA-2. Chromatin immunoprecipitation assay showed that GATA-2 bound to promoter region of claudin-2. These results suggest that hyperosmolarity decreases the expression level of claudin-2 via a decrease in PKCß-dependent GATA-2 transcriptional activity in renal tubular epithelial cells.

Differential distribution of competence for panplacodal and neural crest induction to non-neural and neural ectoderm.

It is still controversial whether cranial placodes and neural crest cells arise from a common precursor at the neural plate border or whether placodes arise from non-neural ectoderm and neural crest from neural ectoderm. Using tissue grafting in embryos of Xenopus laevis, we show here that the competence for induction of neural plate, neural plate border and neural crest markers is confined to neural ectoderm, whereas competence for induction of panplacodal markers is confined to non-neural ectoderm. This differential distribution of competence is established during gastrulation paralleling the dorsal restriction of neural competence. We further show that Dlx3 and GATA2 are required cell-autonomously for panplacodal and epidermal marker expression in the non-neural ectoderm, while ectopic expression of Dlx3 or GATA2 in the neural plate suppresses neural plate, border and crest markers. Overexpression of Dlx3 (but not GATA2) in the neural plate is sufficient to induce different non-neural markers in a signaling-dependent manner, with epidermal markers being induced in the presence, and panplacodal markers in the absence, of BMP signaling. Taken together, these findings demonstrate a non-neural versus neural origin of placodes and neural crest, respectively, strongly implicate Dlx3 in the regulation of non-neural competence, and show that GATA2 contributes to non-neural competence but is not sufficient to promote it ectopically.

Transcriptional regulation of lineage commitment--a stochastic model of cell fate decisions.

Molecular mechanisms employed by individual multipotent cells at the point of lineage commitment remain largely uncharacterized. Current paradigms span from instructive to noise-driven mechanisms. Of considerable interest is also whether commitment involves a limited set of genes or the entire transcriptional program, and to what extent gene expression configures multiple trajectories into commitment. Importantly, the transient nature of the commitment transition confounds the experimental capture of committing cells. We develop a computational framework that simulates stochastic commitment events, and affords mechanistic exploration of the fate transition. We use a combined modeling approach guided by gene expression classifier methods that infers a time-series of stochastic commitment events from experimental growth characteristics and gene expression profiling of individual hematopoietic cells captured immediately before and after commitment. We define putative regulators of commitment and probabilistic rules of transition through machine learning methods, and employ clustering and correlation analyses to interrogate gene regulatory interactions in multipotent cells. Against this background, we develop a Monte Carlo time-series stochastic model of transcription where the parameters governing promoter status, mRNA production and mRNA decay in multipotent cells are fitted to experimental static gene expression distributions. Monte Carlo time is converted to physical time using cell culture kinetic data. Probability of commitment in time is a function of gene expression as defined by a logistic regression model obtained from experimental single-cell expression data. Our approach should be applicable to similar differentiating systems where single cell data is available. Within our system, we identify robust model solutions for the multipotent population within physiologically reasonable values and explore model predictions with regard to molecular scenarios of entry into commitment. The model suggests distinct dependencies of different commitment-associated genes on mRNA dynamics and promoter activity, which globally influence the probability of lineage commitment.

MicroRNA-24 regulates vascularity after myocardial infarction.

BACKGROUND: Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. METHODS AND RESULTS: Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. CONCLUSIONS: Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.

Spontaneous autologous graft-versus-host disease in plasma cell myeloma autograft recipients: flow cytometric analysis of hematopoietic progenitor cell grafts.

Nine plasma cell myeloma patients spontaneously developed histologically proven autologous graft-versus-host disease (GVHD) limited predominantly to the gastrointestinal tract within 1 month of initial autologous hematopoietic cell transplantation (AHCT) using high-dose melphalan conditioning. All recipients responded promptly to systemic and nonabsorbable oral corticosteroid therapy. All patients previously received systemic therapy with thalidomide, lenalidomide, or bortezomib before AHCT. Using enzymatic amplification staining-enhanced flow cytometry, we evaluated expression of selected transcription regulators, pathway molecules, and surface receptors on samples of the infused hematopoietic cell grafts. We demonstrated significantly enhanced expression of GATA-2, CD130, and CXCR4 on CD34(+) hematopoietic progenitor cells of affected patients compared with 42 unaffected AHCT controls. These 3 overexpressed markers have not been previously implicated in autologous GVHD. Although we did not specifically evaluate T cells, we postulate that exposure over time to the various immunomodulating therapies used for induction treatment affected not only the CD34(+) cells but also T cells or relevant T cell subpopulations capable of mediating GVHD. After infusion, the affected hematopoietic progenitor cells then encounter a host that has been further altered by the high-dose melphalan preparative regimen; such a situation leads to the syndrome. These surface markers could be used to develop a model to predict development of this syndrome. Autologous GVHD potentially is a serious complication of AHCT and should be considered in plasma cell myeloma patients with otherwise unexplained gastrointestinal symptoms in the immediate post-AHCT period. Prompt recognition of this condition and protracted treatment with nonabsorbable or systemic corticosteroids or the combination may lead to resolution.

High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle.

Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G(0) residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34(+)CD38(-)Hoechst(lo)Pyronin Y(lo)) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2-conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21(CIP1) and p27(KIP1) do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2(hi)) failed to contribute to hematopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2(lo) cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.

Regulation of trophoblast-specific factors by GATA2 and GATA3 in bovine trophoblast CT-1 cells.

Numerous transcription factors that regulate trophoblast developmental processes have been identified; however, the regulation of trophoblast-specific gene expression has not been definitively characterized. While a new role of Gata3 in trophoblast development was being demonstrated in mice, we examined effects of GATA transcription factors on conceptus interferon tau (IFNT), a major trophectoderm factor in ruminants. In this study, expression patterns of trophoblast ASCL2, CDX2, CSH1, ELF5, HAND1, IFNT, and TKDP1 mRNAs were initially examined, from which ASCL2, CDX2, IFNT, and TKDP1 mRNAs were found to be similar to those of GATA2 and GATA3 in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses. A chromatin immunoprecipitation (ChIP) assay revealed that endogenous GATA2 and GATA3 occupied GATA binding sites on the upstream regions of CSH1, IFNT, and TKDP1 genes and on the intron 1 region of CDX2 gene in bovine trophoblast CT-1 cells. In transient transfection analyses of the upstream region of bovine CSH1, and IFNT or the intron 1 region of CDX2 gene, over-expression of GATA2 induced transactivation of these trophoblast-specific genes in bovine non-trophoblast ear fibroblast EF cells, but over-expression of GATA3 did not substantially affect their transactivation. In CT-1 cells, endogenous CDX2 and IFNT mRNAs were down-regulated by GATA2 siRNA, while endogenous ASCL2 and CDX2 mRNAs were down-regulated by GATA3 siRNA. Our results indicate that in addition to trophectoderm lineage specification, GATA2 and/or GATA3 are involved in the regulation of trophoblast-specific gene transcription in bovine trophoblast CT-1 cells.

The transcription factor NFE2 enhances expression of the hematopoietic master regulators SCL/TAL1 and GATA2.

Activity of the transcription factor NFE2 is elevated in the majority of patients with myeloproliferative neoplasms (MPNs), either by overexpression of the wild-type alleles or by the presence of an activating mutation. In murine models, enhanced NFE2 activity causes an MPN phenotype with spontaneous transformation to acute leukemia. However, little is known about the downstream target genes activated by augmented NFE2 levels. Here, we describe that NFE2 regulates expression of the hematopoietic master regulators GATA2 and SCL/TAL1, which are in turn overexpressed in primary MPN cells, suggesting that concomitant aberrant activation of several transcription factors coordinately contributes to the cellular expansion characteristic of these disorders.

Circulating GATA2 mRNA is decreased among women destined to develop preeclampsia and may be of endothelial origin.

Preeclampsia is a pregnancy complication associated with elevated placental secretion of anti-angiogenic factors, maternal endothelial dysfunction and organ injury. GATA2 is a transcription factor expressed in the endothelium which regulates vascular homeostasis by controlling transcription of genes and microRNAs, including endothelial miR126. We assessed GATA2 and miR126 in preeclampsia. Whole blood circulating GATA2 mRNA and miR126 expression were significantly decreased in women with established early-onset preeclampsia compared to gestation-matched controls (p = 0.002, p < 0.0001, respectively). Using case-control groups selected from a large prospective cohort, whole blood circulating GATA2 mRNA at both 28 and 36 weeks' gestation was significantly reduced prior to the clinical diagnosis of preeclampsia (p = 0.012, p = 0.015 respectively). There were no differences in GATA2 mRNA or protein expression in preeclamptic placentas compared to controls, suggesting the placenta is an unlikely source. Inducing endothelial dysfunction in vitro by administering either tumour necrosis factor-α or placenta-conditioned media to endothelial cells, significantly reduced GATA2 mRNA expression (p < 0.0001), suggesting the reduced levels of circulating GATA2 mRNA may be of endothelial origin. Circulating GATA2 mRNA is decreased in women with established preeclampsia and decreased up to 12 weeks preceding onset of disease. Circulating mRNAs of endothelial origin may be a novel source of biomarker discovery for preeclampsia.

Clenbuterol increases muscle fiber size and GATA-2 protein in rat skeletal muscle in utero.

Certain beta(2)-adrenoceptor agonists, such as clenbuterol, are known to elicit a muscle-specific anabolism or hypertrophy in both normal and catabolic muscle in a wide variety of species. However, the underlying mechanism(s) of the beta(2)-agonist-induced anabolism remains unclear. This study aimed to determine the effects of clenbuterol administration in utero on skeletal muscle and to examine the underlying molecular mechanisms. Pregnant rats were fed clenbuterol (2 mg/kg diet) from Day 4 of gestation (4 dg) until weanling and fetal samples were taken from 13.5, 15.5, 17.5, and 19.5 dg and from 1d neonatal pups. Muscles were analyzed for total DNA, RNA and protein and sections examined morphologically for changes in muscle development. Western and immunohistochemical analyses were performed to identify changes in known myogenic signaling proteins. Clenbuterol increased the size of both fast and slow fibers in utero which was associated with a decreased DNA:protein ratio (28%) and an increased RNA:DNA ratio (36%). Additionally, drug treatment in utero induced a decrease in the fast:slow fiber ratio (38%). These myogenic changes were correlated with an increase in the GATA-2 hypertrophic transcription factor at both 17.5 dg (by 250%) and 19.5 dg (by 40%) in fetuses from clenbuterol treated dams. In addition, drug treatment resulted in increased membrane association of PKC-micro at 17.5 dg (325%) and increased PKC-alpha cytosolic abundance (40%) and PKC-theta membrane abundance at 19.5 dg (250%). These results are the first demonstration that beta(2)-agonists such as clenbuterol may act through upregulating the GATA-2 transcription factor and implicate certain PKC isoforms in the drug-induced regulation of skeletal muscle development.

A comparison of inflammatory mediators released by basophils of asthmatic and control subjects in response to high-affinity IgE receptor aggregation.

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to secretion of inflammatory mediators and cytokine production. Basophils are known to play an important role in the pathogenesis of asthma but there has been no comprehensive examination of the effectors these cells produce. Here a study of the transcription and release of a selection of chemokines and cytokines from basophils was undertaken. METHODS: A Cartesian antibody array provided an effective method of assaying for multiple cytokines and chemokines simultaneously. Results were verified by RT-PCR and ELISA assays. This allowed the comparison of freshly prepared peripheral blood basophil responses to cross-linking of the high-affinity IgE receptor, with and without preincubation with IL-3. RESULTS: Evidence that human blood basophils produce the chemokines MIP-5, eotaxin and GM-CSF was provided by antibody array and RT-PCR analyses. Preincubation with IL-3 enhanced the expression and release of IL-13, IL-8 and mRNA transcripts encoding MIP-5 and GATA2 in basophils from both asthmatic and control subjects. Leptin mRNA transcription, storage and release in basophils are described for the first time. CONCLUSIONS: Surveying cytokine and chemokines stored and released by peripheral blood basophils shows that asthmatic and control subjects share similar profiles even when their degranulation responses are distinct. Evidence is provided for the production of leptin, GM-CSF, eotaxin and MIP-5 by peripheral blood basophils. IL-3 preincubation enhances the production and release of IL-8 upon IgE receptor cross-linking.

Delta-like homolog1/GATA binding protein 2 axis mediates leptin inhibition of PPARγ2 expression in hepatic stellate cells in vitro.

AIMS: Peroxisome-proliferator activated receptor γ (PPARγ) plays a pivotal role in inhibition of hepatic stellate cell (HSC) activation, a key step for liver fibrogenesis. Adipocyte-derived hormone leptin has been shown to promote liver fibrosis in murine and human. PPARγ includes two subtypes, PPARγ1 and PPARγ2. Our previous study indicated that leptin down-regulated PPARγ1 expression in HSCs. The aim of this study was to investigate the effect of leptin on PPARγ2 expression and the underlying mechanisms in HSCs. MAIN METHODS: Real-time PCR and western blot analyses were used to examine gene expression. The promoter activities were detected by luciferase assay. KEY FINDINGS: Leptin reduced PPARγ2 expressions at promoter level, mRNA level, and protein level in HSCs, which required ß-catenin, p38 mitogen-activated protein kinase, and delta-like homolog1 (DLK1) signaling pathways. Leptin induced GATA binding protein 2 (GATA2) expression through DLK1 pathway and GATA2 reduced PPARγ2 expression. Ectopic expression of PPARγ2 reduced the protein levels of α-smooth muscle actin and α1(I)collagen in HSCs. SIGNIFICANCE: Since obese patients, often accompanied by hyperleptinemia, are more prone to liver fibrosis, the data from this study might have potential implications for clarifying the mechanisms for liver fibrogenesis in obese patients with hyperleptinemia.

Alterations of specific chromatin conformation affect ATRA-induced leukemia cell differentiation.

Chromatin conformation plays a key role in regulating gene expression and controlling cell differentiation. However, the whole-genome chromatin conformation changes that occur during leukemia cell differentiation are poorly understood. Here, we characterized the changes in chromatin conformation, histone states, chromatin accessibility, and gene expression using an all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation model. The results showed that the boundaries of topological associated domains (TADs) were stable during differentiation; however, the chromatin conformations within several specific TADs were obviously changed. By combining H3K4me3, H3K27ac, and Hi-C signals, we annotated the differential gene-regulatory chromatin interactions upon ATRA induction. The gains and losses of the gene-regulatory chromatin interactions are significantly correlated with gene expression and chromatin accessibility. Finally, we found that the loss of GATA2 expression and DNA binding are crucial for the differentiation process, and changes in the chromatin structure around the GATA2 regulate its expression upon ATRA induction. This study provided both statistical insights and experimental details regarding the relationship between chromatin conformation changes and transcription regulation during leukemia cell differentiation, and the results suggested that the chromatin conformation is a new type of potential drug target for cancer therapy.

Induction of erythroid-specific genes by overexpression of GATA-2 in K562 cells.

GATA transcription factors have been shown to play important roles in hematopoiesis. GATA-2 is expressed in stem and progenitor cells, and has been speculated to control the proliferation and maintain the immaturity of these cells. To examine whether the function of GATA-2 is changeable according to the differentiation stage, we established GATA-2 overexpressing subclones of K562, which is a leukemic cell line committed to the erythroid lineage. Via an increase in the GATA-2 expression level, the expression levels of erythroid-specific genes including alpha-, beta-, and gamma-globin were increased compared to control cells, while the expression level of GATA-1 was unchanged. Expression of the transferrin receptor was also increased in GATA-2 overexpressing K562 cells when examined by flow cytometry. In addition, the heme content of GATA-2 overexpressing K562 cells was more than 2 times higher than control cells. Chromatin immunoprecipitation analysis showed that GATA-2 protein binding to the GATA element in alpha-globin LCR was increased in GATA-2 overexpressing K562 cells. These findings suggest that GATA-2 could induce erythroid-specific genes without competition with GATA-1 when expressed in erythroid-committed cells, and thus further suggest that temporal and spatial regulation may be important for displaying specific functions of GATA-2.

GATA binding protein 2 overexpression is associated with poor prognosis in KRAS mutant colorectal cancer.

Colorectal cancer (CRC) is one of the most lethal cancers worldwide. Mutations in KRAS occur with the frequency of 30-50% in CRC leading to decreased therapeutic response to anti-epidermal growth factor receptor (EGFR) agents. Recently GATA2 was proven to be essential in the survival of KRAS mutant non-small cell lung cancer (NSCLC) cells. However, the association between KRAS mutation and GATA2 expression in CRC remains largely unknown. In the present study, dideoxy sequencing and immunohistochemistry were used to determine KRAS mutation and GATA2 expression, respectively, in a cohort of 236 patients. Cox proportional hazard regression and Kaplan-Meier survival analysis were performed to study the association between KRAS mutation or GATA2 expression and clinical outcomes. Kaplan-Meier analysis revealed that KRAS mutant patients with high expression of GATA2 had significantly worse long-term clinical outcomes than those with low expression of GATA2 (P<0.001). Further analysis showed that patients with both KRAS mutation and high GATA2 expression experienced significantly more unfavorable 5-year outcomes than patients with wild- type KRAS and low GATA2 expression (P=0.001). Univariate and multivariate Cox proportional hazard regression demonstrated the GATA2 expression level was an independent risk factor for overall survival of CRC patients (HR 1.645; 95% CI 1.004-2.696; P=0.048). In conclusion, the results of this study demonstrated that high expression of GATA2 is correlated with worse survival outcomes in KRAS mutant CRC patients, suggesting that GATA2 may serve as a novel biomarker for the survival of CRC patients harboring KRAS mutation.