GATA-1 Gene Expression Research Articles

Changes of hemoglobin resulted by distant hybridization contributed to increased hypoxia tolerance of allotriploid crucian carp

Hypoxia tolerance, one of the important goals of fish breeding, is beneficial for aquaculture and the transporting of fish. Based on the bisexual fertile allotetraploid crucian carp hybrid lineage obtained by distant hybridization, we successfully developed the allotriploid crucian carp via interploidy crossing with diploid red crucian carp. This allotriploid fish exhibited obvious hypoxia tolerance with lower values of dissolved oxygen at Pcrit (critical oxygen press) and 50 % ASR (aquatic surface respiration) than its original parents. Hemoglobin is the main vector of oxygen in vivo. This study examined the changes in amino acid sequences and natural protein conformation of hemoglobin, and found that the Hbα sequences of allotriploid crucian carp displayed obvious hybridity, including recombination and variation, when compared with its original parents. Moreover, the expression of hbα gene is higher in the liver and blood of allotriploid crucian carp than in its original parents. After acute anoxia treatment, the expression of gata1 gene, which served as the general switch for erythroid development, decreased in the blood of all the three kinds of fish. However, only in the allotriploid crucian carp hbα gene expression significantly decreased, coupled with the erythrocytes displaying an increased ratio of nuclei/cytoplasm. Taken together, this study suggested that changes in hemoglobin coupled with the erythrocyte morphological alterations under hypoxia increased the hypoxia tolerance ability of allotriploid crucian carp, which would provide a new perspective to guide fish breeding to obtain high-quality fish species with increased hypoxia tolerance.

Growth hormone is involved in GATA1 gene expression via STAT5B in human erythroleukemia and monocytic cell lines

GATAs are a family of transcription factors consisting of six members. Particularly, GATA1 and GATA2 have been reported to promote the development of erythrocytes, megakaryocytes, eosinophils, and mast cells. However, little information is available on the extracellular ligands that promote GATA1 expression. We evaluated whether growth hormone (GH) is an extracellular stimulator that participates in the signal transduction of GATAs, focusing on GATA1 expression in hematopoietic cell lineages. We used a reporter assay, RT-PCR, real-time quantitative PCR, and western blotting to evaluate GH-induced expression of GATA1 and GATA2 in the human erythroleukemic cell line K562 and the non-erythroid cell line U937. GATA1 expression in these hematopoietic cell lines increased at the transcriptional and protein levels in the presence of GH, and was inhibited by a STAT5 specific inhibitor. Cells transfected with activated STAT5B showed increased expression of GATA1. We identified functional STAT5B consensus sequences as binding site-158 bp from the transcription starting site in the GATA1 promoter region. These results suggest that GH directly induces GATA1 expression via GHR/JAK/STAT5 and is related to hematopoietic cell proliferation.

GATA1 and GATA2 Sense/Antisense Transcriptional Switching in Early Hematopoietic Development

The GATA1 and GATA2 genes play essential roles in hematopoietic development. GATA1 is the master regulator of erythropoiesis and GATA2 is required for the expansion and survival of hematopoietic progenitor and stem cells, as well as the development of mast cells. A switch in GATA expression occurs during erythropoiesis, with silencing of the GATA2 gene and increased expression of GATA1. A GATA2 antisense transcript ( GATA2-AS1) has been associated with silencing of GATA2 expression. We have identified a novel GATA1 antisense promoter located in the first intron that opposes the GATA1 promoter and generates a spliced antisense transcript complementary to the SUV39H1 H3K9 methyltransferase gene. The competition between opposing sense and antisense promotersin the GATA1 and GATA2 genes may play a central role in the switching of GATA expression that occurs during hematopoiesis. To address this hypothesis, gene expression analysis of cells expressing sense versus antisense transcripts from the GATA1 or GATA2 genes was performed using 5'scRNAseq data from in vitro differentiated CD34 cells. Cells expressing only sense or only antisense GATA1 transcriptswere readily detected and comprised the majority (90%) of cells with GATA1 transcripts. Differential gene expression analysis of cells with GATA1 sense-only versus cells with GATA1 antisense-only showed association of sense transcription with erythrocyte genes, whereas antisense expression was associated with a mast cell phenotype and upregulation of GATA2 transcript levels. Conversely, GATA2 antisense expressing cells had increased GATA1 transcription and an erythrocyte phenotype, and GATA2 sense expressing cells demonstrated mast cell-specific gene expression. Single cell RNAseq analysis of HL60 cells induced to differentiate in vitro identified significant changes in GATA2 gene expression. Induction of granulocyte differentiation in HL60 cells treated with DMSO resulted in increased GATA2 antisense transcription, whereas PMA induction of monocyte differentiation was associated with increased GATA2 sense transcription. Taken together these results indicate a central role for sense/antisense promoter switching in hematopoiesis.

Recent progress in analyses of GATA1 in hematopoietic disorders: a mini-review

GATA1 is an essential master regulator of erythropoiesis and megakaryopoiesis. Accumulating lines of evidence have shown that dynamic changes in GATA1 gene expression levels during erythropoiesis are crucial for proper erythroid differentiation. Since GATA1 is an X-chromosome gene, GATA1 knockout leads to embryonic lethal dyserythropoiesis in male mice, while heterozygous female mice can survive. In the past decade, it has become clear that germline GATA1 gene mutations leading to structural changes in the GATA1 protein are involved in congenital dyserythropoiesis in males. In contrast, decreased GATA1 expression levels, which cause embryonic lethal dyserythropoiesis in male mice, increase the risk of erythroleukemia development in female mice, while female GATA1-knockout mice do not show substantial phenotypic alterations in erythroid or megakaryocyte lineages. In this review, we summarize the recent progress in elucidating the roles of GATA1 in normal and pathogenetic erythropoiesis and discuss the possible mechanisms of pathogenesis of dyserythropoiesis and erythroleukemia.

Evaluating the effect of nurses' supportive and educational care on GATA2 gene expression and quality of life in patients with endometriosis

Endometriosis is one of the women's aggressive but benign diseases, formed by the presence of endometrial glands and stroma outside the uterine cavity. Various genes, including the GATA2 gene, are involved in the pathogenesis of endometriosis. Since this disease affects patients' quality of life, this study was conducted to investigate the effect of nurses' supportive and educational care on the quality of life of endometriosis patients and its role in GATA2 gene expression. For this purpose, 45 patients with endometriosis participated in this research, a semi-experimental before-and-after study. The instrument used was demographic information and quality of life questionnaires affiliated with Beckman Institute, which were completed in two stages before and after implementing patient training and support sessions. The real-time PCR technique was also used to evaluate the expression level of the GATA2 gene after obtaining endometrial tissue from patients before and after the intervention. Finally, the received information was analyzed using SPSS software and statistical tests. Based on the obtained results, the average quality of life score before the intervention was 51.73±13.91, and after the intervention was 60.46±13.80 (P<0.001). Also, in all four dimensions of quality of life, patients' average scores increased after the intervention compared to before the intervention. Still, this difference was significant only in the two dimensions of physical and mental health (P<0.001). GATA2 gene expression before intervention was 0.35 ± 0.13 among endometriosis patients. After the intervention, its amount reached about three times, i.e., 0.96 ± 0.32, which showed a significant difference between the two groups at the 5% probability level. In general, the results of this research confirmed the positive effect of educational and support programs in improving the quality of life of breast cancer patients. Therefore, it is suggested to design and implement such programs in a broader manner and based on patients' educational and support needs.

The upregulation of Gata transcription factors family and FOG-1 in expanded and differentiated cord blood-derived CD34+ hematopoietic stem cells to megakaryocyte lineage during co-culture with cord blood mesenchymal stem cells

BackgroundUmbilical cord blood (UCB) has improved into an attractive and alternative source of allogeneic hematopoietic stem cells (all-HSCs) in clinics and, research for three decades. Recently, it has been shown that the limited cell dose of, this valuable source can be enhanced by the ex vivo expansion of cells in many, ways. We evaluated the expression of the Gata transcription factors family and FOG-1, in expanded and differentiated cord blood-derived CD34 + hematopoietic stem cells to, megakaryocytes lineage., Methods: Separated mononuclear cells were cultured in DMEM complete medium., Harvested cells as a mesenchymal stem cell at 85 % confluency were cultured with, trypsin/EDTA and in 24-well plates. The characteristic analyses of isolated UCB- MSCs, were done by flow cytometry and adipogenic, chondrogenic, and osteogenic, differentiation assays. MACS purified UCB-CD34 + hematopoietic cells cultivated and, differentiated to megakaryocyte progenitor cells in the presence of cytokine cocktail, with UCB-MSCs. Then, the GATA1, GATA2, GATA3, and FOG-1 genes expression, after differentiation to megakaryocyte progenitor cells were performed by quantitative, real-time polymerase chain reaction (PCR)., Results: In this study, the results of real-time-PCR showed that the fold change, expression of GATA-1, FOG-1, and GATA-2 genes after co-culturing with UCB-MSCs, significantly increased to 7.3, 4.7, and 3.3-fold in comparison with control groups;respectively., Conclusion: UCB-MSCs can increase the expansion and differentiation of UCBCD34 + , to megakaryocyte progenitor cells through upregulation of GATA-1, GATA-2, and FOG-1 gene expression.

GATA-1 mutation alters the spermatogonial phase and steroidogenesis in adult mouse testis

GATA-1 is a transcription factor from the GATA family, which features zinc fingers for DNA binding. This protein was initially identified as a crucial regulator of blood cell differentiation, but it is currently known that the Gata-1 gene expression is not limited to this system. Although the testis is also a site of significant GATA-1 expression, its role in testicular cells remains considerably unexplored. In the present study, we evaluated the testicular morphophysiology of adult ΔdblGATA mice with a mutation in the GATA-1 protein. Regarding testicular histology, GATA-1 mutant mice exhibited few changes in the seminiferous tubules, particularly in germ cells. A high proportion of differentiated spermatogonia, an increased number of apoptotic pre-leptotene spermatocytes (Caspase-3-positive), and a high frequency of sperm head defects were observed in ΔdblGATA mice. The main differences were observed in the intertubular compartment, as ΔdblGATA mice showed several morphofunctional changes in Leydig cells. Reduced volume, increased number and down-regulation of steroidogenic enzymes were observed in ΔdblGATA Leydig cells. Moreover, the mutant animal showed lower serum testosterone concentration and high LH levels. These results are consistent with the phenotypic and biometric data of mutant mice, i.e., shorter anogenital index and reduced accessory sexual gland weight. In conclusion, our findings suggest that GATA-1 protein is an important factor for germ cell differentiation as well as for the steroidogenic activity in the testis.

Cloning and expression analysis of GATA1 gene in Carassius auratus red var

BackgroundGATA1 is a key transcription factor in the GATA family, and promotes the differentiation and maturation of red blood cell, which is essential for normal hematopoiesis.ResultsOur results showed that the cDNA sequence of GATA1 was 2730 bp long encoding 443 amino acids. qRT-PCR analysis demonstrated that GATA1 had the highest expression in testis (T), followed by pituitary (P) and spleen (S). GATA1 gene expression in C. auratus red var. embryo from the neuroblast stage (N) to the embryo hatching (H) changes continuously; and the gene expression levels of nonylphenol (NP)-treated and those of control embryos were significantly different. Moreover, Methylation levels of GATA1 gene in NP-treated embryos were higher than those in control embryos, indicating that NP affected GATA1 methylation.ConclusionsOur study provides cues for further studying the roles of GATA1 gene in fish development, and suggested a potential molecular mechanism by which NP leads to abnormal development of fish embryos.

Astragaloside IV enhances GATA-4 mediated myocardial protection effect in hypoxia/reoxygenation injured H9c2 cells

Background and aimThe transcription factor GATA-4 plays an important role in myocardial protection. Astragaloside IV (Ast-IV) was reported with the effects on improving cardiac function after ischemia. In this study, we explored how Ast-IV interacts with GATA-4 to protect myocardial cells H9c2 against Hypoxia/Reoxygenation (H/R) stress. Methods and resultsH9c2 cells were cultured under the H/R condition. Various cell activity and morphology assays were used to assess the rates of apoptosis and autophagy. In these H/R injured H9c2 cells, increased apoptosis (P < 0.01) and autophagosome number (P < 0.01) were observed, and the addition of Ast-IV ameliorated this tendency. Mechanistically, we used the RT-qPCR and Western blot to evaluate the expressions of various molecules. The results showed that Ast-IV treatment upregulated gene expression of GATA-4 (P < 0.01) and the survival factors (Bcl-2, P < 0.05; p62, P < 0.01), but suppressed apoptosis and autophagy related genes (PARP, Caspase-3, Beclin-1, and LC3-II; All P < 0.01). Furthermore, overexpressing of GATA-4 by its agonist phenylephrine can also protect H/R injured H9c2 cells, and the addition of Ast-IV further enhanced this protection of GATA-4. In contrast, silencing GATA-4 expression abolished the H/R protection of Ast-IV, which demonstrated that the myocardial protection of Ast-IV is mediated by GATA-4. Lastly, along with GATA overexpression, enhanced interactions between Bcl-2 and Beclin-1 were detected by Chromatin immunoprecipitation (P < 0.01). ConclusionAst-IV rescued the H/R injury induced apoptosis and autophagy in H9c2 cells. Ast-IV treatment can stimulate the overexpression of GATA-4, and further enhanced the myocardial protection effect of GATA-4.

Hematopoiesis in aged female mice devoid of thyroid hormone receptors.

Hypothyroidism is often associated with anemia and immunological disorders. Similar defects are found in patients and in mice with a mutated dominant-negative thyroid hormone receptor α (TRα) and in knockout mice devoid of this receptor, suggesting that this isoform is responsible for the effects of the thyroid hormones in hematopoiesis. However, the hematological phenotype of mice lacking also TRβ has not yet been examined. We show here that TRα1/TRβ-knockout female mice, lacking all known thyroid hormone receptors with capacity to bind thyroid hormones, do not have overt anemia and in contrast with hypothyroid mice do not present reduced Gata1 or Hif1 gene expression. Similar to that found in hypothyroidism or TRα deficiency during the juvenile period, the B-cell population is reduced in the spleen and bone marrow of ageing TRα1/TRβ-knockout mice, suggesting that TRβ does not play a major role in B-cell development. However, splenic hypotrophy is more marked in hypothyroid mice than in TRα1/TRβ-knockout mice and the splenic population of T-lymphocytes is not significantly impaired in these mice in contrast with the reduction found in hypothyroidism. Our results show that the overall hematopoietic phenotype of the TRα1/TRβ-knockout mice is milder than that found in the absence of hormone. Although other mechanism/s cannot be ruled out, our results suggest that the unoccupied TRs could have a negative effect on hematopoiesis, likely secondary to repression of hematopoietic gene expression.

The Effect of Swimming Training plan and Vitamin B6 Intake on GATA2 Gene Expression in Endometriosis Rat Model

The Effect of Swimming Training plan and Vitamin B6 Intake on GATA2 Gene Expression in Endometriosis Rat Model

Single-cell analyses demonstrate that a heme–GATA1 feedback loop regulates red cell differentiation

AbstractErythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell's surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

Oral consumption of cinnamon enhances the expression of immunity and lipid absorption genes in the small intestinal epithelium and alters the gut microbiota in normal mice

Because cinnamon is orally ingested, its biological activity is likely to influence the small intestinal epithelium and the microbiota along the intestinal tract. We investigated small intestinal epithelial gene expression associated with immunity and lipid metabolism and measured IgA level in the small intestines and sera of mice fed cinnamon. We also analyzed microbial changes in the small and large intestines and feces using pyrosequencing of the 16 s rRNA gene. Cinnamon increased the gene expression of Muc2, RegIIIγ, and Pigr, which are necessary for defense against gut bacteria. It also increased the gene expression of GATA4 and Slc27a2, which are implicated in lipid absorption. Cinnamon increased luminal IgA level but not serum IgA and decreased the class Gammaproteobacteria (classified within the phylum Proteobacteria), particularly the genus Pseudomonas, in the large intestine. Cinnamon thus affected immunity and lipid absorption in the small intestinal epithelium and microbial composition in the intestines.

Therapy with mesenchymal stromal cells or conditioned medium reverse cardiac alterations in a high-fat diet–induced obesity model

BackgroundObesity is associated with numerous cardiac complications, including arrhythmias, cardiac fibrosis, remodeling and heart failure. Here we evaluated the therapeutic potential of mesenchymal stromal cells (MSCs) and their conditioned medium (CM) to treat cardiac complications in a mouse model of high-fat diet (HFD)–induced obesity. MethodsAfter obesity induction and HFD withdrawal, obese mice were treated with MSCs, CM or vehicle. Cardiac function was assessed using electrocardiography, echocardiography and treadmill test. Body weight and biochemical parameters were evaluated. Cardiac tissue was used for real time (RT)-polymerase chain reaction (PCR) and histopathologic analysis. Results/DiscussionCharacterization of CM by protein array showed the presence of different cytokines and growth factors, including chemokines, osteopontin, cystatin C, Serpin E1 and Gas 6. HFD-fed mice presented cardiac arrhythmias, altered cardiac gene expression and fibrosis reflected in physical exercise incapacity associated with obesity and diabetes. Administration of MSCs or CM improved arrhythmias and exercise capacity. This functional improvement correlated with normalization of GATA4 gene expression in the hearts of MSC- or CM-treated mice. The gene expression of connexin 43, troponin I, adiponectin, transforming growth factor (TGF) β, peroxisome proliferator activated receptor gamma (PPARγ), insulin-like growth factor 1 (IGF-1), matrix metalloproteinase-9 (MMP9) and tissue inhibitor of metalloproteinases 1 (TIMP1) were significantly reduced in MSCs, but not in CM-treated mice. Moreover, MSC or CM administration reduced the intensity of cardiac fibrosis. ConclusionOur results suggest that MSCs and CM have a recovery effect on cardiac disturbances due to obesity and corroborate to the paracrine action of MSCs in heart disease models.

Down-regulated GATA-1 up-regulates interferon regulatory factor 3 in lung adenocarcinoma

Interferon regulatory factor 3 (IRF-3) is widely known for its prompt response against viral infection by activating the interferon system. We previously reported that E2F1, Sp1 and Sp3 regulated transcriptional activity of IRF-3. Recently, different expression patterns of IRF-3 were found in lung cancer, leading to the alternation of the immunomodulatory function in tumorigenesis. However, the mechanism of transcriptional regulation of IRF-3 in lung cancer has not been extensively studied. Here, we investigated the characterization of IRF-3 promoter and found that GATA-1 bound to a specific domain of IRF-3 promoter in vitro and in vivo. We found elevated IRF-3 and decreased GATA-1 gene expression in lung adenocarcinoma in Oncomine database. Additionally, higher IRF-3 gene expression was observed in human lung adenocarcinoma, accompanied by aberrant GATA-1 protein expression. We further analyzed the relationship of GATA-1 and IRF-3 expression in lung adenocarcinoma cell lines and found that inhibition of GATA-1 by siRNA increased the promoter activity, mRNA and protein levels of IRF-3, while over-expression of GATA-1 down-regulated IRF-3 gene expression. Taken together, we conclude that reduced GATA-1 could be responsible for the upregulation of IRF-3 in lung adenocarcinoma cells through binding with a specific domain of IRF-3 promoter.

Arsenic Trioxide Suppresses the Expression of EVI-1 Gene and Regulates Related Hematopoietic Transcription Factors in THP-1 Cells

IntroductionAcute myeloid leukemia (AML) is a malignant clonal hematopoietic stem cell disease. Ecotropic viral integration site-1(EVI-1) has been recognized as one of the most dominant oncogenes associated with murine and human myeloid leukemia. EVI-1 harbors several hallmark functions that are normally associated with leukemogenesis. Overexpression EVI-1 upregulates the transcription of the transcription factor gene GATA-2, which plays a critical role in the maintenance of hematopoietic stem cells.EVI-1 also binds to the transcription factors GATA-1,PU.1,RUNX-1,SCL and LMO2 thereby inhibiting their activity and blocking the differentiation of hematopoietic progenitors. Arsenic trioxide (ATO), which was used as a traditional Chinese medicine, has shown excellent therapeutic efficiency for acutepromyelocyticleukemia. A previous study has demonstrated that ATO targets EVI-1 protein to induce apoptosis . However, the molecular mechanisms underlying this regulation of AML cells by ATO have not been fully elucidated. We aim to expand our understanding of the cellular regulation and effect of ATO on EVI-1 and several related transcription factors (such as GATA1, GATA2, RUNX-1, LMO2, PU-1) in human acute monocytic leukemia THP-1 cells.MethodsThe endogenous expression of EVI-1 is higher in THP-1 cells, which was confirmed through gene analysis sequencing and comparison with Genbank.THP-1 cells were treated with various concentrations of ATO( 0,1,3,5¦ÌM) for 24h, 48h and 72h. Expressions of EVI-1,and related transcription factor genes(GATA-2 ,GATA-1, RUNX-1,MPO,LMO,PU.1,SCL2) were determined by qRT-PCR.ResultsCompared to controls, THP-1 cells with high expression of EVI-1 gene were selected to confirm that overexpression of EVI-1 can promote the expression of GATA-2 , and reduce the level of GATA-1, RUNX-1, MPO, LMO, PU.1, SCL2 simultaneously in vitro(Fig.1).To determine whether ATO is able to directly regulate EVI-1 and related transcription factor genes expression in THP-1 cells, we examined their mRNA expression by qRT-PCR after treatment with different concentrations of ATO. We found that ATO has the ability to down regulate EVI-1 gene in a dose-dependent and time-dependent manner( Fig.2a).Moreover, GATA-2 gene expression decreased when THP-1 cells were treated with ATO(Fig.2b). We examined the related transcription factors expression and found that ATO suppressed the expression levels of PU.1, RUNX-1, SCL and LMO2 (Fig.2c, 2d, 2e) in THP-1 cells. Interestingly, exposure to increasing concentrations of ATO for increasing lengths of time was associated with gradual promotion in the expression of GATA-1 mRNA( Fig 2f).SummaryWe verified that the endogenous expression of EVI-1 is higher in THP-1 cells, which confirmed THP-1 cells can be a vitro model to investigate EVI-1 gene functions. Meanwhile, related transcription factor gene GATA-2, which is regarded as EVI-1 regulatory element, is upregulated in THP-1 cells. Moreover, GATA-1, which is essential for erythroid differentiation, and other related transcription factor genes (PU.1, RUNX-1, SCL and LMO2) decreased in THP-1 cells. ATO dose-and time-dependently decreased expressions of EVI-1 and GATA-2 gene and regulated those related transcription factors in EVI-1-overexpressing cells compared with control cells.Disclosure: No relevant conflicts of interest to declare. DisclosuresLang:National Natural Science Fundation of China (no.81470312),: Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding. Chen:National Natural Science Fundation of China (no.81470312): Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding. Zhu:Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding; National Natural Science Fundation of China (no.81470312): Research Funding. Zhong:National Natural Science Fundation of China (no.81470312): Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding.

GATA-related hematologic disorders.

The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and have overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice invivo have indicated that GATA2 and GATA1 are functionally nonredundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, illustrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematologic analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematologic diseases, focusing on the regulation of GATA factor gene expression.

Mutations of NKX2.5 and GATA4 genes in the development of congenital heart disease

ObjectiveTo investigate the mutations of NKX2.5 and GATA4 genes in the development of CHD in Chinese population. MethodsBetween December 2010 and December 2014, 185 cases of CHD patients and 210 cases of healthy people were enrolled. NKX2.5 and GATA4 gene mutations and gene expression were detected via DNA sequencing and real-time PCR (RT-PCR), respectively. BMSCs were transfected with pCMV-HA-NKX2.5 and pCMV-Myc-GATA4 plasmids. Cardiac troponin T (cTnT) and connexin 43 (Cx43) and β-myosin heavy chain (β-MHC) and myosin light chain-2 (MLC-2) expressions were detected. Co-immunoprecipitation assay was used to detect the interaction of NKX2.5 and GATA4 and luciferase to detect their effect on B-type natriuretic peptide (BNP) gene promoter. ResultsNKX2.5 and GATA4 gene mutations were found in the CHD group, but not in the normal control group, and NKX2.5 and GATA4 gene expressions were significantly lower in the case group compared with the control group (both P<0.05). Compared to the control and empty vector groups, cTnT and Cx43 expressions were significantly higher in the pCMV-HA-NKX2.5 and pCMV-Myc-GATA4 plasmid groups; β-MHC at 1–4weeks and MLC-2 at 2–4weeks were higher in the pCMV-HA-NKX2.5 plasmid group; and β-MHC at 2–3weeks and MLC-2 at 3–4weeks were higher in the pCMV-Myc-GATA4 plasmid group (all P<0.05). NKX2.5 and GATA4 interacted in the BMSCs and co-transfected pCMV-Myc-GATA4 and pCMV-HA-NKX2.5 significantly enhanced the fluorescence intensity of BNP. ConclusionNKX2.5 and GATA4 gene mutations might participate in the development of CHD and can promote BMSCs differentiate into cardiomyocytes.

Hydroxyurea down-regulates BCL11A, KLF-1 and MYB through miRNA-mediated actions to induce γ-globin expression: implications for new therapeutic approaches of sickle cell disease.

BackgroundThe major therapeutic benefit of hydroxyurea, the only FDA-approved pharmacologic treatment for sickle cell disease (SCD), is directly related to fetal hemoglobin (HbF) production that leads to significant reduction of morbidity and mortality. However, potential adverse effects such as infertility, susceptibility to infections, or teratogenic effect have been subject of concerns. Therefore, understanding HU molecular mechanisms of action, could lead to alternative therapeutic agents to increase HbF with less toxicity. This paper investigated whether HU-induced HbF could operate through post-transcriptional miRNAs regulation of BCL11A, KLF-1 and MYB, potent negative regulators of HbF. Both ex vivo differentiated primary erythroid cells from seven unrelated individuals, and K562 cells were treated with hydroxyurea (100 μM) and changes in BCL11A, KLF-1, GATA-1, MYB, β- and γ-globin gene expression were investigated. To explore potential mechanisms of post-transcriptional regulation, changes in expression of seven targeted miRNAs, previously associated with basal γ-globin expression were examined using miScript primer assays. In addition, K562 cells were transfected with miScript miRNA inhibitors/anti-miRNAs followed by Western Blot analysis to assess the effect on HbF protein levels. Direct interaction between miRNAs and the MYB 3′-untranslated region (UTR) was also investigated by a dual-luciferase reporter assays.ResultsDown-regulation of BCL11A and MYB was associated with a sevenfold increase in γ-globin expression in both primary and K562 cells (p < 0.003). Similarly, KLF-1 was down-regulated in both cell models, corresponding to the repressed expression of BCL11A and β-globin gene (p < 0.04). HU induced differential expression of all miRNAs in both cell models, particularly miR-15a, miR-16, miR-26b and miR-151-3p. An HU-induced miRNAs-mediated mechanism of HbF regulation was illustrated with the inhibition of miR-26b and -151-3p resulting in reduced HbF protein levels. There was direct interaction between miR-26b with the MYB 3′-untranslated region (UTR).ConclusionsThese experiments have shown the association between critical regulators of γ-globin expression (MYB, BCL11A and KLF-1) and specific miRNAs; in response to HU, and demonstrated a mechanism of HbF production through HU-induced miRNAs inhibition of MYB. The role of miRNAs-mediated post-transcriptional regulation of HbF provides potential targets for new treatments of SCD that may minimize alterations to the cellular transcriptome.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-016-0092-7) contains supplementary material, which is available to authorized users.

The Human GATA1 Gene Retains a 5' Insulator That Maintains Chromosomal Architecture and GATA1 Expression Levels in Splenic Erythroblasts.

GATA1 is a key transcription factor for erythropoiesis. GATA1 gene expression is strictly regulated at the transcriptional level. While the regulatory mechanisms governing mouse Gata1 (mGata1) gene expression have been studied extensively, how expression of the human GATA1 (hGATA1) gene is regulated remains to be elucidated. To address this issue, we generated hGATA1 bacterial artificial chromosome (BAC) transgenic mouse lines harboring a 183-kb hGATA1 locus covering the hGATA1 exons and distal flanking sequences. Transgenic hGATA1 expression coincides with endogenous mGata1 expression and fully rescues hematopoietic deficiency in mGata1 knockdown mice. The transgene exhibited copy number-dependent and integration position-independent expression of hGATA1, indicating the presence of chromatin insulator activity within the transgene. We found a novel insulator element at 29 kb 5' to the hGATA1 gene and refer to this element as the 5' CCCTC-binding factor (CTCF) site. Substitution mutation of the 5' CTCF site in the hGATA1 BAC disrupted the chromatin architecture and led to a reduction of hGATA1 expression in splenic erythroblasts under conditions of stress erythropoiesis. Our results demonstrate that expression of the hGATA1 gene is regulated through the chromatin architecture organized by 5' CTCF site-mediated intrachromosomal interactions in the hGATA1 locus.