OBJECTIVE: To analyze the clinical and molecular biological characteristics of a neonate with myeloid proliferation related to Down syndrome (DS). METHODS: The neonate, who was suspected for Down syndrome, was analyzed in terms of clinical feature, peripheral blood cell morphology, fluorescence in situ hybridization (FISH), immunological classification and other laboratory tests. On hundred and fourteen leukemia-related genes were subjected to next-generation sequencing (NGS). RESULTS: Laboratory test revealed obvious abnormal liver function and coagulation function, anemia, and extreme leukocytosis. Cell smear indicated significantly increased progenitor cells, which conformed to proliferation of megakaryocytes. FISH showed trisomy 21. By NGS, c.220+dupT, a novel mutation, was identified in exon 2 of the GATA1 gene, which encodes a N-terminal activation domain and has a frequency of 95.8%. No mutation was identified among the remaining 113 genes. CONCLUSION The neonate had DS and GATA1 gene mutation. High percentage of circulating blasts should be considered as transient myelodysplasia but not congenital leukemia.
Down Syndrome ; genetics ; GATA1 Transcription Factor ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Infant, Newborn ; Mutation ; Trisomy

OBJECTIVETo find out how GATA-1 and GATA-2 behave in the bone marrow of patients with Monge's disease.

METHODSThe levels of mRNA in mononuclear cells (MNC) and proteins of GATA-1 and GATA-2 in the bone marrow of patients with Monge's disease and controls were determined by RT-PCR and immune cytolysis chemical method.

RESULTS(1) All patients and controls expressed GATA-1 mRNA (Monge's disease 1.033 +/- 0.146, Control 0.458 +/- 0.076) and GATA-2 mRNA (Monge's disease 0.451 +/- 0.073, Control 0.185 +/- 0.074). All patients expressed both GATA-1 (positive cell counts 77.3 +/- 33.3, positive score 135.4 +/- 75.4) and GATA-2 ( positive cell counts 29.4 +/- 11.4, positive score 48.4 +/- 19.7). All the controls expressed GATA-1 (positive cell counts 18.1 +/- 11.3, positive score 24.2 +/- 13.4) while 12 of 20 controls expressed GATA-2 ( positive cell counts 5.4 +/- 3.0, positive score 7.3 +/- 4.2). The expression of mRNA and proteins of GATA-1 and GATA-2 in Monge's disease were higher than in controls (P < 0.01). (2) There was a positive correlation between GATA-1 and Hb (P < 0.01), as did between mRNA and proteins of GATA-1 and GATA-2. (3) Both the proteins of GATA-1 and GATA-2 located only in the cytoplasm but not the nucleus.

CONCLUSIONSTwo of inherent genes, GATA-1 and GATA-2 which were expressed at higher levels in patients with Monge's disease than in controls might play significant roles in the pathogenesis of Monge's disease.

Adult ; Altitude Sickness ; metabolism ; GATA1 Transcription Factor ; metabolism ; GATA2 Transcription Factor ; metabolism ; Humans ; Male ; Polycythemia ; metabolism ; RNA, Messenger ; metabolism

OBJECTIVE: To investigate the changes of GATA-1 protein expression during erythroid differentiation of K562 cells under hypoxia and how GATA-1 can regulate erythroid differentiation by up-regulating the expression of miR-451a and inhibiting the expression of 14-3-3ζ. METHODS: K562 cells were divided into 2 groups: the normoxia group and the hypoxia group, after the induction of hemin for 96 h, the positive cells rate of the benzidine staining, the mRNA expression of γ-globin and the expression of CD235a were detected, and the success of the model was verified. The changes of GATA-1 and miR-451a expression in the above-mentioned 2 groups, the changes of miR-451a expression after over-expressed GATA-1 were detected by Western blot and qRT-PCR. The cells in normoxic group and hypoxia group were divided into negative control group (NC group) and miR-451a over-expression group respectively, and the degree of erythroid differentiation in the four groups was judged according to the corresponding erythroid differentiation indexes, and the expression of 14-3-3ζ was detected by Western blot after over-expressed miR-451a. RESULTS: The positive cell rate of benzidine staining, mRNA expression of γ-globin and the expression of CD235a after 96 h induction by K562 cells under hypoxia were significantly higher than 0 h, suggesting that the erythroid differentiation model of K562 cells under hypoxia was replicated successfully. The expression levels of GATA-1 protein and miR-451a in the hypoxic group were significantly higher than that in the normoxic group (P<0.05). The expression level of miR-451a in hypoxia group was significantly higher than that in NC group after overexpressed GATA-1 (P<0.05). After over-expressed of miR-451a under hypoxia, the positive cell rate of benzidine staining, the mRNA expression level of γ-globin and the expression of CD235a were significantly higher than those in NC group (P<0.05). The expression level of 14-3-3ζ protein in miR-451a over-expressed group was lower than that in NC group under hypoxia (P<0.05). CONCLUSION Hypoxia can significantly increase the expression of GATA-1 protein, and the increase of GATA-1 expression can up-regulate the expression of miR-451a, thereby inhibiting the expression of 14-3-3ζ protein, which hinders the cell proliferation in erythroid differentiation model of K562 cells and plays an important role in promoting erythroid differentiation.
14-3-3 Proteins ; Cell Differentiation ; Erythroid Cells/metabolism* ; GATA1 Transcription Factor/metabolism* ; Humans ; Hypoxia ; K562 Cells ; MicroRNAs/genetics*

Transient myeloproliferative disorder (TMD) is similar to congenital leukemia, with leukocytosis, increased peripheral blast cells, and hepatomegaly in the neonatal period. Although TMD occurs only in patients with Down syndrome and trisomy 21 mosaicism, there have been reports of congenital leukemia with trisomy 21 limited to hematopoietic cells showing spontaneous resolution. We identified trisomy 21 in the leukemic cells in a patient with congenital leukemia. As there was no other gene abnormality, we stopped chemotherapy, and the disease resolved spontaneously. We reviewed the cases of clonal trisomy 21 TMD and found that their clinical features were similar to those of TDM associated with Down syndrome. In conclusion, in a phenotypically normal patient with suspected congenital leukemia, it is necessary to confirm the presence of 21 trisomy. If the neonate has only trisomy 21 without other gene abnormalities, intensive chemotherapy may not be required.
Chromosomes, Human, Pair 21 ; Down Syndrome* ; Drug Therapy ; GATA1 Transcription Factor ; Hepatomegaly ; Humans ; Infant ; Infant, Newborn* ; Leukemia ; Leukocytosis ; Mosaicism ; Myeloproliferative Disorders* ; Trisomy*

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1-TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.
Animals ; Astrocytes/metabolism* ; DNA Demethylation ; Epigenesis, Genetic ; GATA1 Transcription Factor/metabolism* ; Inflammation/metabolism* ; Oligodeoxyribonucleotides/metabolism* ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X7/metabolism* ; Visceral Pain/metabolism*

Children with Down syndrome (DS) have a higher risk of developing leukemia than do healthy children, and they especially have a higher risk for developing transient myeloproliferative disorder (TMD) or acute megakaryocytic leukemia (AMKL). In recent studies, it has been reported that most of these patients have acquired mutation of the GATA1 gene, which encodes the erythroid/megakaryocytic transcription factor GATA1. GATA1 mutations have not been found in AMKL patients who did not have DS and other hematologic malignancies in DS. Most of the GATA1 mutations in DS-TMD/AMKL are nonsense mutations that are mainly located in exon 2. We observed a nonsense mutation in exon 2 of GATA1 [c.189_190delCA (Tyr63X)] in one case of DS-TMD. The GATA1 mutation has been thought to be an early event in the leukemogenesis of DS-TMD/AMKL and it could be used as a stable molecular marker to assess the treatment response or to monitor for the recurrence of DS-TMD/AMKL.
Child ; Codon, Nonsense ; Down Syndrome ; Exons ; GATA1 Transcription Factor ; Hematologic Neoplasms ; Humans ; Leukemia ; Leukemia, Megakaryoblastic, Acute ; Myeloproliferative Disorders ; Organothiophosphorus Compounds ; Recurrence

OBJECTIVETo investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2.

METHODSWe established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45.

RESULTSWe found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45.

CONCLUSIONNF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.

Base Sequence ; Blood Proteins ; genetics ; DNA Primers ; GATA1 Transcription Factor ; physiology ; Gene Expression Regulation ; physiology ; Gene Silencing ; HeLa Cells ; Humans ; Methylation ; Molecular Chaperones ; genetics ; NF-E2 Transcription Factor, p45 Subunit ; physiology ; Promoter Regions, Genetic ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effect of simulated microgravity on erythroid differentiation of K562 cells and explore the possible mechanism.

METHODSThe fourth generation rotating cell culture system was used to generate the simulated microgravity environment. Benzidine staining was used to evaluate the cell inhibition rate, and real-time quantitative PCR (qRT-PCR) was used to detect GATA-1, GATA-2, Ets-1, F-actin, β-Tubulin and vimentin mRNA expressions. The changes of cytoskeleton were observed by fluorescence microscopy, and Western blotting was employed to assay F-actin, β-tubulin and vimentin protein expression levels.

RESULTSBenzidine staining showed that simulated microgravity inhibited erythroid differentiation of K562 cells. K562 cells treated with Hemin presented with increased mRNA expression of GATA-1 and reduced GATA-2 and Ets-1 mRNA expressions. Simulated microgravity treatment of the cells resulted in down-regulated GATA-1, F-actin, β-tubulin and vimentin mRNA expressions and up-regulated mRNA expressions of GATA-2 and Ets-1, and reduced F-actin, β-tubulin and vimentin protein expressions. Exposure to simulated microgravity caused decreased fluorescence intensities of cytoskeletal filament F-actin, β-tubulin and vimentin in the cells.

CONCLUSIONSimulated microgravity inhibits erythroid differentiation of K562 cells possibly by causing cytoskeleton damages to result in down-regulation of GATA-1 and up-regulation of GATA-2 and Ets-1 expressions.

Actins ; metabolism ; Cell Differentiation ; Down-Regulation ; GATA1 Transcription Factor ; metabolism ; GATA2 Transcription Factor ; metabolism ; Hemin ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Protein c-ets-1 ; metabolism ; Tubulin ; metabolism ; Up-Regulation ; Vimentin ; metabolism ; Weightlessness Simulation

OBJECTIVETo identify the interaction partners of a new splicing product of LMO2 gene (LMO2-C), and study its function in K562 cells.

METHODSMaltose binding protein (MBP) pull down and mammalian two-hybrid assay (MTHA) were used to identify the interaction partners of LMO2-C in K562 cells. Semiquantitative RT-PCR was used to detect the expression of hematopoietic specific gene glycoprotein (GPA) in K562 cells.

RESULTSMBP-LMO2-C fusion protein was expressed and purified in soluble form successfully. Endogenous GATA1 and LDB1 proteins were confirmed to bind to LMO2-C by MBP pull down analysis. The MTHA also showed that LMO2-C had comparable binding affinities to LDB1 with LMO2-L, and over expression of LMO2-C prevented LMO2-L from binding to LDB1, the inhibition rate being (81.13 +/- 0.68)%. RT-PCR results showed that the expression level of GPA was reduced [(51.00 +/- 1.58)%] in K562 cells while LMO2-C overexpressed.

CONCLUSIONLMO2-C can bind endogenous GATA1 and LDB1 protein in K562 cells and down regulates the expression of GPA.

Adaptor Proteins, Signal Transducing ; DNA-Binding Proteins ; genetics ; metabolism ; GATA1 Transcription Factor ; metabolism ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; genetics ; metabolism ; Periplasmic Binding Proteins ; Proto-Oncogene Proteins ; RNA Splicing ; Transcription Factors ; metabolism ; Two-Hybrid System Techniques

The role of cytokines and transcription factors on the regulation of megakaryocy topoiesis and platelet production are reviewed in this article. Megakaryocytopoiesis involves the proliferation and differentiation of megakaryocytic pro genitor cells into immature megakaryocytes, and the differentiation of immature megakaryocytes to mature megakaryocytes which produce platelets. The former is regulated mainly by thrombopoietin (TPO) and to a lesser degree by other cytokines such as interleukin-1 (IL-1), IL-3 and platelet-derived growth factor (PDGF), the later by TPO and probably IL-6 and IL-11. A number of transcription factors have been implicated in the control of megakaryocyte differentiation. GATA-1, FOG-1 and Fli-1 are essential regulators in early- and mid-stages of megakaryocytopoiesis. NF-E2 regulates late-stage of megakaryocytopoiesis and platelet production. However, the platelet release mechanism is poorly understood. Nitric oxide (NO) may act in the stage of platelet release through induction of apoptosis in megakaryocytes.
Animals ; Cytokines ; physiology ; DNA-Binding Proteins ; physiology ; Erythroid-Specific DNA-Binding Factors ; GATA1 Transcription Factor ; Hematopoiesis ; Humans ; Interleukins ; physiology ; Megakaryocytes ; physiology ; NF-E2 Transcription Factor ; NF-E2 Transcription Factor, p45 Subunit ; Platelet-Derived Growth Factor ; physiology ; Thrombopoiesis ; physiology ; Thrombopoietin ; physiology ; Transcription Factors ; physiology