G9a is an epigenetic regulator that methylates H3K9, generally causing repression of gene expression, and participates in diverse cellular functions. G9a is genetically deregulated in a variety of tumor types and can silence tumor suppressor genes and, therefore, is important for carcinogenesis. Although hypoxia is recognized to be an adverse factor in tumor growth and metastasis, the role of G9a in regulating gene expression in hypoxia has not been described extensively. Here, we show that G9a protein stability is increased in hypoxia via reduced proline hydroxylation and, hence, inefficient degradation by the proteasome. This inefficiency leads to an increase in H3K9me2 at its target promoters. Blocking the methyltransferase activity of G9a inhibited cellular proliferation and migration in vitro and tumor growth in vivo. Furthermore, an increased level of G9a is a crucial factor in mediating the hypoxic response by down-regulating the expression of specific genes, including ARNTL, CEACAM7, GATA2, HHEX, KLRG1, and OGN This down-regulation can be rescued by a small molecule inhibitor of G9a. Based on the hypothesis that the changes in gene expression would influence patient outcomes, we have developed a prognostic G9a-suppressed gene signature that can stratify breast cancer patients. Together, our findings provide an insight into the role G9a plays as an epigenetic mediator of hypoxic response, which can be used as a diagnostic marker, and proposes G9a as a therapeutic target for solid cancers.

GATA-binding protein 2 (GATA2) and LIM domain only 2 (Lmo2) form common transcription complexes during hematopoietic differentiation. Here we show that these two transcription factors also play a key role in endothelial cells (EC) and lymphatic EC (LEC) function. Primary EC and tumor-associated blood vessels expressed GATA2 and Lmo2. VEGF-induced sprouting angiogenesis in both differentiating embryonic stem cells (embryoid bodies) and primary EC increased GATA2 and Lmo2 levels. Conversely, silencing of GATA2 and Lmo2 expression in primary EC inhibited VEGF-induced angiogenic activity, including EC migration and sprouting in vitro, two key steps of angiogenesis in vivo. This inhibition of EC function was associated with downregulated expression of neuropilin-2 (NRP2), a co-receptor of VEGFRs for VEGF, at the protein, mRNA and promoter levels. NRP2 overexpression partially rescued the impaired angiogenic sprouting in the GATA2/Lmo2 knockdown EC, confirming that GATA2 and Lmo2 mediated EC function, at least in part, by directly regulating NRP2 gene expression. Furthermore, it was found that primary LEC expressed GATA2 and Lmo2 as well. Silencing of GATA2 and Lmo2 expression in LEC inhibited VEGF-induced LEC sprouting, also in a NRP2-dependent manner. In conclusion, our results demonstrate that GATA2 and Lmo2 cooperatively regulate VEGF-induced angiogenesis and lymphangiogenesis via NRP2.

Cranial placodes contribute to many sensory organs and ganglia of the vertebrate head. The olfactory, otic, and lateral line placodes form the sensory receptor cells and neurons of the nose, ear, and lateral line system; the lens placode develops into the lens of the eye; epibranchial, profundal, and trigeminal placodes contribute sensory neurons to cranial nerve ganglia; and the adenohypophyseal placode gives rise to the anterior pituitary, a major endocrine control organ. Despite these differences in fate, all placodes are now known to originate from a common precursor, the preplacodal ectoderm (PPE). The latter is a horseshoe-shaped domain of ectoderm surrounding the anterior neural plate and neural crest and is defined by expression of transcription factor Six1, its cofactor Eya1, and other members of the Six and Eya families. Studies in zebrafish, Xenopus, and chick reveal that the PPE is specified together with other ectodermal territories (epidermis, neural crest, and neural plate) during early embryogenesis. During gastrulation, domains of ventrally (e.g., Dlx3/Dlx5, GATA2/GATA3, AP2, Msx1, FoxI1, and Vent1/Vent2) and dorsally (e.g., Zic1, Sox3, and Geminin) restricted transcription factors are established in response to a gradient of BMP and help to define non-neural and neural competence territories, respectively. At neural plate stages, the PPE is then induced in the non-neural competence territory by signals from the adjacent neural plate and mesoderm including FGF, BMP inhibitors, and Wnt inhibitors. Subsequently, signals from more localized signaling centers induce restricted expression domains of various transcription factors within the PPE, which specify multiplacodal areas and ultimately individual placodes. For further resources related to this article, please visit the WIREs website.

BACKGROUND

The author has declared no conflicts of interest for this article.

The bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of bone marrow mesenchymal stem cell differentiation is poorly understood. The transcription factor GATA2 is indispensable for hematopoietic stem cell function as well as other hematopoietic lineages, suggesting that it may maintain bone marrow mesenchymal stem cells in an immature state and also contribute to their differentiation. To explore this possibility, we established bone marrow mesenchymal stem cells from GATA2 conditional knockout mice. Differentiation of GATA2-deficient bone marrow mesenchymal stem cells into adipocytes induced accelerated oil-drop formation. Further, GATA2 loss- and gain-of-function analyses based on human bone marrow mesenchymal stem cells confirmed that decreased and increased GATA2 expression accelerated and suppressed bone marrow mesenchymal stem cell differentiation to adipocytes, respectively. Microarray analysis of GATA2 knockdowned human bone marrow mesenchymal stem cells revealed that 90 and 189 genes were upregulated or downregulated by a factor of 2, respectively. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell cycle regulation, and the number of G1/G0 cells increased after GATA2 knockdown. Concomitantly, cell proliferation was decreased by GATA2 knockdown. When GATA2 knockdowned bone marrow mesenchymal stem cells as well as adipocytes were cocultured with CD34-positive cells, hematopoietic stem cell frequency and colony formation decreased. We confirmed the existence of pathological signals that decrease and increase hematopoietic cell and adipocyte numbers, respectively, characteristic of aplastic anemia, and that suppress GATA2 expression in hematopoietic stem cells and bone marrow mesenchymal stem 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 molecular mechanism involved in the liganded thyroid hormone receptor suppression of the TSHbeta (thyroid-stimulating hormone beta, or thyrotropin beta) gene transcription is undetermined. One of the main reasons is the limitation of useful cell lines for the experiments. We have developed an assay system using non-pituitary CV1 cells and studied the negative regulation of the TSHbeta gene. In CV1 cells, the TSHbeta-CAT (chloramphenicol acetyltransferase) reporter was stimulated by Pit1 and GATA2 and suppressed by T3 (3,3',5-tri-iodothyronine)-bound thyroid hormone receptor. The suppression was dependent on the amounts of T3 and the receptor. Unliganded receptor did not stimulate TSHbeta activity, suggesting that the receptor itself is not an activator. Analyses using various receptor mutants revealed that the intact DNA-binding domain is crucial to the TSHbeta gene suppression. Co-activators and co-repressors are not necessarily essential, but are required for the full suppression of the TSHbeta gene. Among the three receptor isoforms, beta2 exhibited the strongest inhibition and its protein level was the most predominant in a thyrotroph cell line, TalphaT1, in Western blotting. The dominant-negative effects of various receptor mutants measured on the TSHbeta-CAT reporter were not simple mirror images of those in the positive regulation under physiological T3 concentration.

The fetal liver kinase 1 (FLK-1)(+) hemangioblast can generate hematopoietic, endothelial, and smooth muscle cells (SMCs). ER71/ETV2, GATA2, and SCL form a core transcriptional network in hemangioblast development. Transient coexpression of these three factors during mesoderm formation stage in mouse embryonic stem cells (ESCs) robustly enhanced hemangioblast generation by activating bone morphogenetic protein (BMP) and FLK-1 signaling while inhibiting phosphatidylinositol 3-kinase, WNT signaling, and cardiac output. Moreover, etsrp, gata2, and scl inhibition converted hematopoietic field of the zebrafish anterior lateral plate mesoderm to cardiac. FLK-1(+) hemangioblasts generated by transient coexpression of the three factors (ER71-GATA2-SCL [EGS]-induced FLK-1(+)) effectively produced hematopoietic, endothelial, and SMCs in culture and in vivo. Importantly, EGS-induced FLK-1(+) hemangioblasts, when codelivered with mesenchymal stem cells as spheroids, were protected from apoptosis and generated functional endothelial cells and SMCs in ischemic mouse hindlimbs, resulting in improved blood perfusion and limb salvage. ESC-derived, EGS-induced FLK-1(+) hemangioblasts could provide an attractive cell source for future hematopoietic and vascular repair and regeneration.

A 50-year-old woman was diagnosed with acute myeloid leukemia (AML). She has history of thrombocytopenia for 25 years and a significant family history of thrombocytopenia, affecting her mother, siblings and their children, as well as her own children. Both her mother and maternal aunt died from myelodysplastic syndrome (MDS). Additional genetic analysis was performed and identified two heterozygous missence mutations in the second zinc finger domain of GATA2 gene (p.Thr358Lys, and p.Leu359Val), occurring in cis on the same allele. Given the patient's family history and clinical manifestation, this was interpreted as an acute myeloid leukemia with heritable GATA2 mutations associated with familial AML-MDS. Germline GATA2 mutations are involved in a group of complex syndromes with overlapping clinical features of immune deficiency, lymphedema and propensity to acute myeloid leukemia or myelodysplastic syndrome (AML-MDS). Here we reported a case of familial AML-MDS with two novel GATA2 mutations. This case illustrates the importance of recognizing the clinical features for this rare category of AML-MDS and performing the appropriate molecular testing. The diagnosis of heritable gene mutations associated familial AML-MDS has significant clinical implication for the patients and affected families. Clinical trials are available to further investigate the role of allogeneic hematopoietic stem cell transplant in managing these patients.

GATA2 transcription factor and LIM homeodomain proteins Islet1 (ISL1) and LIM homeobox 3 (LHX3) are suspected to be involved in gonadotrope cell fate and maintenance. The GnRH receptor gene (Gnrhr), crucial for gonadotrope function, is expressed in the pituitary gland from embryonic day 13.5 onward, well before LH and FSH β-subunits. This expression pattern together with the presence of WGATAR and TAAT motifs in Gnrhr promoter sequences suggests the involvement of early transcription factors in promoter activation. In this study, using a well-characterized transgenic mouse model, GATA2 was found colocalized with Gnrhr promoter activity in the pituitary. Transient transfection of Gnrhr promoter luciferase fusion constructs together with either GATA2 expression vectors or small interfering RNA in gonadotrope cell lines indicated that GATA2, which typically acts as a trans-activator, unexpectedly repressed Gnrhr promoter activity. Using DNA chromatography affinity and EMSA, we demonstrated that GATA2 operates via a response element containing a peculiar palindromic GATA motif that overlaps a critical TAAT motif involved in LHX3/ISL1 trans-activation. Indeed, despite the inhibitory action of GATA2, this element displayed a clear-cut enhancer activity in gonadotrope cells. Chromatin immunoprecipitation assays indicated that GATA2, LHX3, and ISL1 interact with a Gnrhr promoter fragment encompassing this element. The trans-repressive action of GATA2 on Gnrhr promoter activity is likely balanced or even hindered by trans-activating effects of LIM homeodomain proteins via this novel bifunctional LIM/GATA response element. Such a hierarchical interplay may contribute to finely adjust Gnrhr gene expression in gonadotrope cell lineage during pituitary development as well as in the adult animal.

Embryonic stem cells can differentiate in vitro into hematopoietic cells through two intermediate stages; the first being FLK1(+) E-cadherin- proximal lateral mesoderm and the second being CD45(-) VE-cadherin+ endothelial cells. To further dissect the CD45(-) VE-cadherin+ cells, we have examined distribution of alpha4-integrin on this cell population, because alpha4-integrin is the molecule expressed on hematopoietic stem cells. During culture of FLK1(+) E-cadherin- cells, CD45(-) VE-cadherin+ alpha4-integrin- cells differentiate first, followed by alpha4-integrin+ cells appearing in both CD45(-) VE-cadherin+ and CD45(-) VE-cadherin- cell populations. In the CD45(-) VE-cadherin+ cell population, alpha4-integrin+ subset but not alpha4-integrin- subset had the potential to differentiate to hematopoietic lineage cells, whereas endothelial cell progenitors were present in both subsets. The CD45(-) VE-cadherin- alpha4-integrin+ cells also showed hematopoietic potential. Reverse transcription-polymerase chain reaction analyses showed that differential expression of the Gata2 and Myb genes correlated with the potential of the alpha4-integrin+ cells to give rise to hematopoietic cell differentiation. Hematopoietic CD45(-) VE-cadherin+ alpha4-integrin+ cells were also present in the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our results suggest that the expression of alpha4-integrin is a marker of the earliest precursor of hematopoietic cell lineage that was diverged from endothelial progenitors.

BACKGROUND

Senescence marker protein-30 (SMP30), whose expression declines during aging in rat liver, has been proposed as an important aging marker. Besides apoptosis, SMP30 also protects cells against various other injuries by enhancement of membrane calcium-pump activity. The mechanism of this differential gene expression mechanism is not known. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed to elucidate the mechanism of transcriptional regulation of SMP30 gene.

RESULTS

We have characterized up to -2750 bp of the promoter by DNA-protein interactions studies. Twenty eight transcription factor binding sites have been identified by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Transient transfection of 5' and 3' -deleted promoter-reporter constructs and luciferase assay illustrated the region between -128/+157 bp is sufficient to drive promoter activity. We have mapped an essential regulatory region between -513 to -352 bp which causes a drastic decline of reporter activity. This region contains CdxA, GATA2 and SRY transcription factor binding sites. Individual mutation of these three sites showed increase in reporter activity. Mutation in SRY site (-403/-368) showed maximum increase in reporter activity among these three sites. Therefore, we suggest that SRY like protein may be acting as a strong repressor of SMP30 gene along with CdxA and GATA-2. We also report that mutation of both Sp1 (172/-148 bp) and a C/EBPbeta (-190/-177 bp) transcription binding site located adjacent to each other on SMP30 gene promoter, causes a significant enhancement in reporter activity than individual mutation, thus may be causing the repression of SMP30 promoter activity.

CONCLUSIONS

These studies provide novel insights into the mechanism that regulate SMP30 gene expression.

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.

Here we present a simple two-step in vitro model of vascularized trophoblastic tissue derived from human embryonic stem (hES) cells. The first step is the formation of cystic embryoid bodies (EBs) in suspension in a semisolid methyl cellulose medium, within which an endothelial platelet/endothelial cell adhesion molecule-1 (PECAM-1(+)) cell network develops. In a second step, deposition of these EBs on the bottom of nontreated, polystyrene tissue culture plates, leads by centrifugal outgrowth of the EB to the emergence of an adherent cell layer, with which a PECAM-1(+) network is associated. Cells of this adherent layer expressed VE-cadherin (CD144), PECAM-1 (CD31), and alpha-fetoprotein (alpha-FP). Trophoblastic differentiation was strongly suggested by the secretion of beta-human chorionic gonadotropin (beta-hCG) and by the presence of the cytotrophoblast and syncytiotrophoblast marker GB25. The INSL4 gene, a cyto and syncytio-trophoblast marker, was also highly expressed in the adherent layer, as well as other trophoblast genes such as CGA, CDX1, CDX2, and HAND1, compared to hES cell gene expression taken as reference. In contrast, expression of self-renewal genes, such as TERT, POU5F1, ZFP42, GDF3, and NODAL were decreased. No ectodermal or endodermal genes were expressed, but the mesodermal genes PECAM-1 and GATA2 were. The possibility of removing the EBs during the second step would permit analysis of their relative contribution to angiogenesis or possible hemangioblast formation, compared to that of the trophoblastic adherent layer. This primitive vascularized trophoblastic model could also provide a tool to study early steps of normal and pathological placental development.

GATA transcription factors have been implicated in controlling adipogenesis in Drosophila and in mammals. In mammals, both GATA2 and GATA3 have been shown to be present in preadipocytes, and their silencing allows the onset of adipogenesis. Overexpression of GATA proteins blocks adipogenesis in cellular assays. GATA factors have been found to operate through recruiting cofactors of the Friend of GATA (FOG) family. FOG proteins, in turn, recruit co-regulators, including C-terminal binding proteins (CTBPs). We have investigated whether FOGs and CTBPs influence adipogenesis. We found that both FOG1 and FOG2 are expressed in cells prior to adipogenesis but are down-regulated as adipogenesis proceeds. Overexpression of FOG1 or FOG2 interferes with adipogenesis. Mutant versions of FOG2 unable to bind CTBP or GATA proteins are impaired in their inability to inhibit adipogenesis. Finally, a mutant version of GATA2, unable to associate with FOGs, also displays abnormal activity and causes enhanced cell proliferation. These results implicate FOGs and CTBPs as partners of GATA proteins in the control of adipocyte proliferation and differentiation.

Androgen receptor (AR), a ligand-dependent transcription factor, plays a critical role in prostate cancer onset and progression, and its transcriptional function is mediated largely by distinct nuclear receptor co-regulators. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1) functions as an AR co-activator. CCAR1 interacted with and enhanced the transcriptional activity of AR. Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes. We further showed that CCAR1 is required for recruitment of AR, MED1 and RNA polymerase II to the enhancers of AR target genes and for androgen-induced long-range prostate specific antigen enhancer-promoter interaction. The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites. CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers. Furthermore, CCAR1 depletion inhibited the growth, migration, invasion of prostate cancer cells and reduced the tumorigenicity of prostate cancer cells in vivo. Our results firmly established CCAR1 as an AR co-activator that plays a key role in AR transcription complex assembly and has an important physiological role in androgen signaling and prostate tumorigenesis.

Alzheimer's disease (AD) is a dynamic degeneration of the brain with progressive dementia. Considering the uncertainties in its molecular mechanism, in the present study, we employed network-based integrative analyses, and aimed to explore the key molecules and their associations with small drugs to identify potential biomarkers and therapeutic agents for the AD. First of all, we studied a transcriptome dataset and identified 1521 differentially expressed genes (DEGs). Integration of transcriptome data with protein-protein and transcriptional regulatory interactions resulted with central (hub) proteins (UBA52, RAC1, CREBBP, AR, RPS11, SMAD3, RPS6, RPL12, RPL15, and UBC), regulatory transcription factors (FOXC1, GATA2, YY1, FOXL1, NFIC, E2F1, USF2, SRF, PPARG, and JUN) and microRNAs (mir-335-5p, mir-26b-5p, mir-93-5p, mir-124-3p, mir-17-5p, mir-16-5p, mir-20a-5p, mir-92a-3p, mir-106b-5p, and mir-192-5p) as key signaling and regulatory molecules associated with transcriptional changes for the AD. Considering these key molecules as potential therapeutic targets and Connectivity Map (CMap) architecture, candidate small molecular agents (such as STOCK1N-35696) were identified as novel potential therapeutics for the AD. This study presents molecular signatures at RNA and protein levels which might be useful in increasing discernment of the molecular mechanisms, and potential drug targets and therapeutics to design effective treatment strategies for the AD.

The IL1RL1/ST2 gene encodes a receptor for IL-33. Signaling from IL1RL1/ST2 induced by IL-33 binding was recently identified as a modulator of the Th2 response. The target cells for IL-33 are restricted in some hematopoietic lineages, including mast cells, basophils, eosinophils, Th2 cells, natural killer cells, and dendritic cells. To clarify the molecular mechanisms of cell type-specific IL1RL1/ST2 expression in mast cells and basophils, transcriptional regulation of the human IL1RL1/ST2 promoter was investigated using the mast cell line LAD2 and the basophilic cell line KU812. Reporter assays suggested that two GATA motifs just upstream of the transcription start site in the ST2 promoter are critical for transcriptional activity. These two GATA motifs possess the capacity to bind GATA1 and GATA2 in EMSA. ChIP assay showed that GATA2, but not GATA1, bound to the ST2 promoter in LAD2 cells and that histone H3 at the ST2 promoter was acetylated in LAD2 cells, whereas binding of GATA1 and GATA2 to the ST2 promoter was detected in KU812 cells. Knockdown of GATA2 mRNA by siRNA reduced ST2 mRNA levels in KU812 and LAD2 cells and ST2 protein levels in LAD2 cells; in contrast, GATA1 siRNA transfection up-regulated ST2 mRNA levels in KU812 cells. The ST2 promoter was transactivated by GATA2 and repressed by GATA1 in coexpression analysis. When these siRNAs were introduced into human peripheral blood basophils, GATA2 siRNA reduced ST2 mRNA, whereas GATA1 siRNA up-regulated ST2 mRNA. These results indicate that GATA2 and GATA1 positively and negatively control human ST2 gene transcription, respectively.

Synthetic lethal interaction provides a conceptual framework for the development of wiser cancer therapeutics. In this study, we exploited a therapeutic strategy based on the interaction between GATA binding protein 2 (GATA2) downregulation and the KRAS mutation status by delivering small interfering RNA targeting GATA2 (siGATA2) with cationic lipid-assisted polymeric nanoparticles for treatment of non-small-cell lung carcinoma (NSCLC) harboring oncogenic KRAS mutations. Nanoparticles carrying siGATA2 (NPsiGATA2) were effectively taken up by NSCLC cells and resulted in targeted gene suppression. NPsiGATA2 selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells. However, this intervention was harmless to normal KRAS wild-type NSCLC cells and HL7702 hepatocytes, confirming the advantage of synthetic lethality-based therapy. Moreover, systemic delivery of NPsiGATA2 significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model, suggesting the therapeutic promise of NPsiGATA2 delivery in KRAS mutant NSCLC therapy.

The gata2 gene encodes a transcription factor implicated in regulating early patterning of ectoderm and mesoderm, and later in numerous cell-specific gene expression programs. Activation of the gata2 gene during embryogenesis is dependent on the bone morphogenetic protein (BMP) signaling pathway, but the mechanism for how signaling controls gene activity has not been defined. We developed an assay in Xenopus embryos to analyze regulatory sequences of the zebrafish gata2 promoter that are necessary to mediate the response to BMP signaling during embryogenesis. We show that activation is Smad dependent, since it is blocked by expression of the inhibitory Smad6. Deletion analysis identified an octamer binding site that is necessary for BMP-mediated induction, and that interacts with the POU homeodomain protein Oct-1. However, this element is not sufficient to transfer a BMP response to a heterologous promoter, requiring an additional more proximal cooperating element. Based on recent studies with other BMP-dependent promoters (Drosophila vestigial and Xenopus Xvent-2), our studies of the gata2 gene suggest that POU-domain proteins comprise a common component of the BMP signaling pathway, cooperating with Smad proteins and other transcriptional activators.

Gata2 is an essential hematopoietic transcriptional factor that is also expressed prominently in the nervous system. The early lethality of knockout mice due to severe anemia has largely precluded studies of gata2 neural regulation and function. In this report, we describe the identification of zebrafish Pur alpha and Sp8 orthologs as two factors that function to regulate neuronal expression of gata2. During embryogenesis, Pur alpha is expressed widely, whereas Sp8 has an overlapping pattern of expression with gata2 in the nervous system. Knockdown and ectopic expressions of Pur alpha and Sp8 indicate that these factors function, respectively, as a repressor and an activator of gata2 gene expression in the nervous system. With consideration given to the previously established roles for these factors, we propose a model for how the transcriptional regulation of neural gata2 expression may be involved in controlling cellular proliferation in the nervous system.

Ecotropic viral integration site 1 (EVI1) is an important transcription factor for leukemogenesis. EVI1 is a member of a group of transcription factors with C-terminal binding protein (CtBP)-binding motifs that act as transcriptional co-repressors; however, we recently found that EVI1 directly activates GATA2 transcription, which is an important gene for the maintenance of hematopoietic stem cells. We show here that EVI1-activated GATA2 transcripts derive from exon 1S of GATA2, which is specifically activated in neural and hematopoietic cells. EVI1 was acetylated by the histone acetyltransferase p300/CBP association factor (P/CAF) in myeloid leukemia cells and hematopoietic progenitor cells. Acetylation at Lys(564), which is adjacent to the CtBP-binding consensus sequence of EVI1, was found to be important for transcriptional activation of GATA2. Mutation of Lys(564) to alanine (K564A) markedly reduced the ability of EVI1 to bind DNA and activate transcription of GATA2. Furthermore, we confirmed that Lys(564) in EVI1 was specifically acetylated in leukemia and primary hematopoietic cells by using an antibody directed against an acetylated Lys(564) EVI1 peptide. Moreover, co-transfection of P/CAF with EVI1 overcame the suppressive effect of the CtBP co-repressor and resulted in GATA2 transcriptional activation; nonetheless, CtBP2 was still included in the protein complex with EVI1 and P/CAF on the EVI1-binding site in the GATA2 promoter region. Thus, acetylation of EVI1 at Lys(564) by P/CAF enhances the DNA binding capacity of EVI1 and thereby contributes to the activation of GATA2.

Increased local immune and inflammatory responses in obese individuals with periodontitis may explain the aggressive clinical presentation and altered treatment response when compared to that of normal weight subjects. Our goal was to identify any differences in microRNA (miRNA) expression profiles of gingival tissue in periodontitis when obesity is present, which may suggest novel molecular pathways that this miRNA network may affect. Total RNA was extracted from gingival tissue biopsies collected from normal weight and obese individuals with periodontitis; miRNA expression profiling was performed with Affymetrix GeneChip miRNA 3.0 arrays; and results were validated with quantitative reverse transcription polymerase chain reaction (qRT-PCR). In silico identification of previously confirmed miRNA gene targets was conducted through miRTarBase and miRWalk databases, and pathway enrichment analysis identified enriched miRNA gene sets. Expression of selected genes in the same biopsy samples was tested with qRT-PCR. The gingival tissue miRNA profile of obese patients, compared to that of normal weight patients, showed 13 upregulated and 22 downregulated miRNAs, among which miR-200b was validated by qRT-PCR to be significantly increased in obesity. Functional analysis of 51 experimentally validated miR-200b gene targets identified enrichment of genes involved in cell motility, differentiation, DNA binding, response to stimulus, and vasculature development pathways not previously identified in the obesity-specific disease profile. Furthermore, the expression of the miR-200b gene targets ZEB1/2, GATA2, and KDR was confirmed by qRT-PCR as being lower in obese patients with periodontitis versus normal weight patients, suggesting a role of miR-200b in regulation of a set of gene targets and biological pathways relevant to wound healing and angiogenesis. Functional studies to explore the role of miR-200b in the above processes may offer new insights on putative therapeutic targets for this group of patients.

Cis-element encyclopedias provide information on phenotypic diversity and disease mechanisms. Although cis-element polymorphisms and mutations are instructive, deciphering function remains challenging. Mutation of an intronic GATA motif (+9.5) in GATA2, encoding a master regulator of hematopoiesis, underlies an immunodeficiency associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas an inversion relocalizes another GATA2 cis-element (-77) to the proto-oncogene EVI1, inducing EVI1 expression and AML, whether this reflects ectopic or physiological activity is unknown. We describe a mouse strain that decouples -77 function from proto-oncogene deregulation. The -77(-/-) mice exhibited a novel phenotypic constellation including late embryonic lethality and anemia. The -77 established a vital sector of the myeloid progenitor transcriptome, conferring multipotentiality. Unlike the +9.5(-/-) embryos, hematopoietic stem cell genesis was unaffected in -77(-/-) embryos. These results illustrate a paradigm in which cis-elements in a locus differentially control stem and progenitor cell transitions, and therefore the individual cis-element alterations cause unique and overlapping disease phenotypes.