GATA-1 Protein Research Articles

Impairment of the Hypothalamus-Pituitary-Thyroid Axis Caused by Naturally Occurring GATA2 Mutations In Vitro.

The transcription factor GATA2 regulates gene expression in several cells and tissues, including hematopoietic tissues and the central nervous system. Recent studies revealed that loss-of-function mutations in GATA2 are associated with hematological disorders. Our earlier in vitro studies showed that GATA2 plays an essential role in the hypothalamus–pituitary–thyroid axis (HPT axis) by regulating the genes encoding prepro-thyrotropin-releasing hormone (preproTRH) and thyroid-stimulating hormone β (TSHβ). However, the effect of GATA2 mutants on the transcriptional activity of their promoters remains unelucidated. In this study, we created five human GATA2 mutations (R308P, T354M, R396Q, R398W, and S447R) that were reported to be associated with hematological disorders and analyzed their functional properties, including transactivation potential and DNA-binding capacity toward the preproTRH and the TSHβ promoters. Three mutations (T354M, R396Q, and R398W) within the C-terminal zinc-finger domain reduced the basal GATA2 transcriptional activity on both the preproTRH and the TSHβ promoters with a significant loss of DNA binding affinity. Interestingly, only the R398W mutation reduced the GATA2 protein expression. Subsequent analysis demonstrated that the R398W mutation possibly facilitated the GATA2 degradation process. R308P and S447R mutants exhibited decreased transcriptional activity under protein kinase C compared to the wild-type protein. In conclusion, we demonstrated that naturally occurring GATA2 mutations impair the HPT axis through differential functional mechanisms in vitro.

Loss of GATA4 C-Terminus by p.S335X Mutation Modulates Coronary Artery Vascular Smooth Muscle Cell Phenotype.

Coronary artery disease (CAD) has been the leading cause of morbidity and mortality worldwide, and its pathogenesis is closely related with the proliferation and migration of vascular smooth muscle cell (VSMC). We previously reported a truncated GATA4 protein lacking C-terminus induced by p.S335X mutation in cardiomyocyte from ventricular septal defect (VSD) patients. However, it is still unclear whether GATA4 p.S335X mutation could influence the development of CAD. GATA4 wild-type (WT) and p.S335X mutant (MU) overexpression plasmids were constructed and transfected transiently into rat coronary artery smooth muscle cell (RCSMC) to observe the proliferative and migratory abilities by MTS and wound healing assay, respectively. PCR array was used to preliminarily detect the expression of phenotypic modulation-related genes, and QRT-PCR was then carried out to verify the screened differentially expressed genes (DEGs). The results showed that, when stimulated by fetal bovine serum (10%) for 24 h or tumor necrosis factor-α (10 or 30 ng/ml) for 10 or 24 h, deletion of GATA4 C-terminus by p.S335X mutation in GATA4 enhanced the proliferation of RCSMC, without alteration of the migration capability. Twelve DEGs, including Fas, Hbegf, Itga5, Aimp1, Cxcl1, Il15, Il2rg, Il7, Tnfsf10, Il1r1, Irak1, and Tlr3, were screened and identified as phenotypic modulation-related genes. Our data might be beneficial for further exploration regarding the mechanisms of GATA4 p.S335X mutation on the phenotypic modulation of coronary VSMC.

Abstract 131: Gata4 Ablation In Smooth Muscle Cells Reduces Aortic Root Dilation In A Murine Model Of Loeys-Dietz Syndrome

Loeys-Dietz syndrome (LDS) is a connective tissue disorder characterized by predisposition to aneurysm. Although dilation can develop in all segments of the arterial tree, the aortic root is especially prone to disease. This aortic region is primarily composed of vascular smooth muscle cells (VSMCs) derived from secondary heart field (SHF) progenitors, while VSMCs derived from the cardiac neural crest (CNC) predominate in the more distal ascending aorta. Although intrinsic differences have been found between these two types of VSMCs, it remains unclear how these may contribute to localized risk. To identify transcripts associated with the vulnerability of the aortic root in LDS, we performed single cell transcriptomic analysis on proximal aortas from 16-week-old control and Tgfbr1 M318R/+ LDS mice, which carry a kinase-inactivating mutation in TGF-β Receptor 1. We identified four major VSMC clusters regardless of genotype, with two clusters being defined by CNC-enriched transcripts and two clusters by SHF-enriched transcripts. The latter were defined by expression of Gata4 , which codes for a transcription factor required for development of SHF-derived vascular structures, as well as by higher expression of transcripts previously involved in aneurysm pathogenesis such as Thbs1 , Tgfb1, and Tgfb3. In situ RNA hybridization confirmed that Gata4 -expressing VSMCs were enriched in the aortic root and proximal ascending aorta, with almost no expression in the more distal ascending aorta, suggesting that this transcript defined the aortic region most susceptible to the effects of the Tgfbr1 M318R/+ mutation. GATA4 protein levels were also upregulated in the aortic root of LDS mice. To test if excessive GATA4 expression or activity in VSMCs contributed to aortic root dilation in LDS, we generated LDS mice in which Gata4 was specifically deleted in VSMCs by tamoxifen administration at 6 weeks of age. We found that this intervention significantly reduced aortic root growth from 8 to 20 weeks of age. Although the mechanisms remain under investigation, these data suggest that expression of Gata4 might sensitize specific subsets of aortic VSMCs to the effects of an LDS mutation, rendering the aortic root more susceptible to disease.

Expression of GATA3 and bcl-11b in peripheral T-cell lymphoma and their clinical significance

Objective: To study the expression of GATA3 and bcl-11b in peripheral T-cell lymphoma (PTCL) and their correlation with clinicopathological features. Methods: The Oncomine and GEO databases were used for analyzing the expression levels of GATA3 and bcl-11b mRNA in PTCL. Immunohistochemistry was used to detect the expression of GATA3 and bcl-11b proteins in 127 cases of PTCL diagnosed at Shanxi Provincial Cancer Hospital from January 2010 to June 2020, as well as 40 cases of lymph node with reactive hyperplasia. Results: The data in Oncomine and GEO databases showed that the expression of GATA3 and bcl-11b mRNA in PTCL was lower than that in normal tissues (P<0.05). Immunohistochemistry showed that the positive rates of GATA3 in PTCL and lymph nodes with reactive hyperplasia were 60.6% (77/127) and 85.0% (34/40, P<0.05), respectively. The expression rates of bcl-11b in PTCL and lymph nodes with reactive hyperplasia were 55.1% (70/127) and 75.0% (30/40, P<0.05), respectively. The expression of GATA3 was related to the pathological classification of the patients with PTCL, and was inversely related to the Ann Arbor stage of the patient, while the expression of bcl-11b was inversely correlated with the IPI score of the patient (P<0.05). The expression of GATA3 and bcl-11b was related to the patients' age, gender, LDH level, and B symptoms. Other clinicopathological characteristics were irrelevant. Spearman correlation analysis shows that the expression of GATA3 protein was associated with that of bcl-11b protein in PTCL. Conclusion: GATA3 and bcl-11b are closely related to the prognosis of PTCL, and may be important factors involved in the occurrence and development of PTCL.

Network pharmacology of triptolide in cancer cells: implications for transcription factor binding

SummaryBackground Triptolide is an active natural product, which inhibits cell proliferation, induces cell apoptosis, suppresses tumor metastasis and improves the effect of other therapeutic treatments in several cancer cell lines by affecting multiple molecules and signaling pathways, such as caspases, heat-shock proteins, DNA damage and NF-ĸB. Purpose We investigated the effect of triptolide towards NF-ĸB and GATA1. Methods We used cell viability assay, compare and cluster analyses of microarray-based mRNA transcriptome-wide expression data, gene promoter binding motif analysis, molecular docking, Ingenuity pathway analysis, NF-ĸB reporter cell assay, and electrophoretic mobility shift assay (EMSA) of GATA1. Results Triptolide inhibited the growth of drug-sensitive (CCRF-CEM, U87.MG) and drug-resistant cell lines (CEM/ADR5000, U87.MGΔEGFR). Hierarchical cluster analysis showed six major clusters in dendrogram. The sensitive and resistant cell lines were statistically significant (p = 0.65 × 10–2) distributed. The binding motifs of NF-κB (Rel) and of GATA1 proteins were significantly enriched in regions of 25 kb upstream promoter of all genes. IPA showed the networks, biological functions, and canonical pathways influencing the activity of triptolide towards tumor cells. Interestingly, upstream analysis for the 40 genes identified by compare analysis revealed ZFPM1 (friend of GATA protein 1) as top transcription regulator. However, we did not observe any effect of triptolide to the binding of GATA1 in vitro. We confirmed that triptolide inhibited NF-κB activity, and it strongly bound to the pharmacophores of IκB kinase β and NF-κB in silico. Conclusion Triptolide showed promising inhibitory effect toward NF-κB, making it a potential candidate for targeting NF-κB.

Abstract 1193: Digital quantification of GATA6 expression from immunohistochemistry is associated with overall survival after surgery for pancreatic cancer in the ESPAC trials

Abstract Introduction: GATA6, a transcription factor involved in pancreaticogenesis, correlates with major transcriptomic subtypes of pancreatic cancer, and early evidence suggests a potential role as a prognostic biomarker. In this study, we tested whether semi-automated digital quantification of GATA6 immunohistochemistry (IHC) was associated with overall survival (OS) after surgical resection of pancreatic cancer in randomized trials of adjuvant chemotherapy. Materials and Methods: Tissue microarrays of pancreatic adenocarcinoma specimens were stained for GATA6 expression using IHC. Specimens came from patients in the ESPAC-3 and ESPAC-4 randomized phase III trials comparing adjuvant chemotherapy regimens. Two pathologists blinded to clinical data independently assessed GATA6 protein expression in cores using a customized computer algorithm from the open-source digital pathology software QuPath. Briefly, the digital assistant provided GATA6 IHC semiquantitative scores (0-4) for individual cells, which were made available to the pathologist for review. This semi-automated digital quantification of GATA6 expression was compared to manual quantification as previously reported (Grant et al., AACR Pancreatic Cancer, 2020). Concordance between pathologists across tissue cores was calculated using a weighted kappa statistic for scores 0 through 4, and an unweighted kappa statistic for high (3-4) and low (0-2) expression levels. Discrepancies were resolved through consensus. Median OS was estimated in GATA6 high and low subgroups using Kaplan-Meier curves. OS was compared using Cox proportional hazard regressions adjusting for adjuvant chemotherapy treatment, lymph node status, and post-operative CA-19-9. Results: GATA6 expression was quantified in 230 patients using the semi-automated digital and manual approaches. Inter-observer concordance markedly improved with digital assistance compared to the manual approach (weighted kappa statistic across all scores 0.69 versus 0.41, unweighted kappa statistic comparing high and low expression 0.71 versus 0.25). GATA6 expression measured using the semi-automated digital approach was significantly associated with OS: median OS was 25.7 months in GATA6 low cancers versus 42.4 months in GATA6 high cancers, with an adjusted hazard ratio of 1.58 (95% confidence interval 1.10-2.28, P=0.014). Conclusion: GATA6 IHC is a prognostic biomarker for pancreatic cancers treated with surgical resection and adjuvant chemotherapy that can be consistently and reliably quantified using digital assistance. Citation Format: Robert C. Grant, Kai Duan, Richard Jackson, William Greenhalf, Eithne Costello-Goldring, Paula Ghaneh, Christopher Halloran, Daniel Palmer, Thilo Hackert, Markus Buchler, Shawn Hutchison, Stephanie Ramotar, Anna Dodd, Julie Wilson, Faiyaz Notta, Grainne O'Kane, Jennifer Knox, John Neoptolemos, Steven Gallinger, Sandra Fischer. Digital quantification of GATA6 expression from immunohistochemistry is associated with overall survival after surgery for pancreatic cancer in the ESPAC trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1193.

Impact of Mutant Haemopoietic Transcription Factor GATA1s on Haemopoiesis

Neonates and children with Down Syndrome (DS), with constitutional Trisomy 21 (T21), have a 150-fold increased risk of developing Myeloid Leukaemia (ML-DS), with differentiation arrest of immature megakaryocyte-erythroid cells. Virtually all ML-DS patients have in utero-acquired somatic mutations in the gene encoding the megakaryocyte-erythroid transcription factor GATA1 leading to the production of an 84 amino acid N-terminal truncated form of the GATA1 protein (GATA1s). We, and others, have previously shown that this N-terminal domain is necessary to prevent excessive megakaryocytic proliferation. However, the mechanisms by which GATA1, but not GATA1s, restrains megakaryocyte proliferation are unclear.We generated knock-in murine ES cell models expressing biotinylated forms of either full length GATA1 or GATA1s protein. We established a large scale in vitro differentiation assay to study embryonic-fetal megakaryocyte differentiation to define the normal megakaryocytic differentiation pathway in GATA1-expressing cells. ES cells were differentiated into embryoid bodies (EBs), which were disaggregated after 6 days and CD41+c-kit+ cells were cultured on OP9 feeder layers with TPO, IL6 and IL11. Detailed examination of the differentiation kinetics including FACS-sorting of specific populations followed by re-culture or Biomark qRT-PCR analysis, showed complex differentiation pathways as wild type cells differentiated into both megakaryocyte and non-megakaryocyte (both erythroid and myeloid) fates. By contrast, GATA1s-expressing cells almost exclusively differentiated into megakaryocyte lineage. Furthermore, as immature CD41+ haemopoietic cells differentiate into the megakaryocyte lineage, they lose c-kit expression and CD41 expression increases. In the GATA1s-expressing cells compared to GATA1-expressing cells, there is a significant accumulation (5 to 10-fold) of a specific immature megakaryocyte CD41++c-kit+ population, partially blocked in differentiation. Cell cycle analysis showed an increase in cells in S-phase specifically in this population in GATA1s-expressing cells (44% compared to 27% in normal cells) together with a decrease in apoptosis (5% compared to 11% in normal cells). To determine the relevance of these findings in vivo , corresponding mouse models were generated and experiments on yolk sacs isolated at different stages of development are ongoing.Taken together, these data suggest that our in vitro murine ES cell differentiation model is a relevant tool to dissect the molecular mechanisms underlying GATA1s function in abnormal megakaryocytic differentiation and they are likely to provide insight into general mechanisms of how altered function of a critical lineage-affiliated transcription factor can lead to multiple changes in progenitor cell cycle and differentiation. DisclosuresVyas:Celgene Corporation: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau.

Deubiquitinase USP7 Regulates Erythroid Development Via Deubiquinating GATA1

Erythropoiesis is the process which produces mature red blood cells from haematopoietic stem cell. Ubiquitination/deubiquititation plays important roles in many cellular processes. Previous studies have documented the roles of ubiquitination and several ubiquitinases in erythropoiesis. In contrast, the roles of deubiquitiation/deubiquitinases in erythropoiesis remain unexplored. In this study, we found that the expression of deubiquitinase USP7 was significantly upregulated during erythropoiesis. We then employed shRNA mediated knockdown approach to explore the role USP7, we found that USP7 knockdown significantly delayed terminal erythroid differentiation, inhibited the hemoglobin expression and cell proliferation, and induced apoptosis. To explore the underlying mechanisms for the observed phenotypic changes, we compared the expression of several key erythroid transcription factors including GATA1, KLF1, LSD1, SOX1 and IKAROS between control and USP7-knockdown cells by western blot analysis. We found that only the protein level of GATA1 but not other transcription factors was significantly decreased following USP7 knockdown. Moreover, GATA1 mRNA level was not affected by USP7 knockdown. The effect of USP7 on the stability of GATA1 protein was further demonstrated by the findings that inhibition of USP7 by USP7 inhibitors also resulted in decreased GATA1 protein levels and that knockdown/inhibition of USP7 shorten the GATA1 half-life and increased the ubiquitination of GATA1. Furthermore, we performed Co-IP, pull down and in vitro ubiquitization experiment which documented that USP7 directly binds to and deubiquinates GATA1. Our findings demonstrate that GATA1 is the substrate of USP7 and that USP7 stabilizes GATA1 by deubiquinating GATA1. To examine whether the impaired erythropoiesis following USP7 knockdown is due to decreased GATA1, we ectopically expressed GATA1 in USP7 knockdown cells and showed that the delayed differentiation was rescued by GATA1 overexpression. To demonstrate the role of USP7 in vivo, we established a stress erythropoiesis mouse model by phlebotomy and then treated the mice by intraperitoneal injection of USP7 inhibitor. We found USP7 inhibitor delayed the recovery of anemia. Collectively, our findings not only identified previously unknown roles of USP7 in erythropoiesis but also identified a novel regulatory mechanism for GATA1 expression.AcknowledgmentsThis work was supported in part by grants 81270576, 81470362 and 81530005 from Natural Science Foundation of China, by NIH grants DK100810 and DK26263, and by postgraduate innovation project of Central South University of China 2016zzts165.The authors declare no conflict of interest. DisclosuresNo relevant conflicts of interest to declare.

A Novel Mutation Causing GATA2 Haploinsufficiency Activates a Cryptic Splice Site in Exon 3

A 48-year old woman presented with clinical symptoms consistent with GATA2 haploinsufficiency: recurrent human papilloma virus associated cervical intraepithelial neoplasia, extensive deep vein thrombosis of the lower extremity, a history of hepatitis C virus infection and recurrent pneumonias complicated by cryptogenic organizing pneumonia. Infectious agents isolated from the lungs included mycoplasma pneumonia, serratia marcescens, mycobacterium intracellulare and aspergillus nidulans . Laboratory findings revealed monocytopenia, B, NK and CD4 T cell lymphopenia. However, analysis of the bone marrow ruled out myelodysplastic syndrome and showed a normal karyotype. Sanger sequencing from genomic DNA isolated from peripheral blood did not reveal any published missense, nonsense or complex mutation associated with GATA2 haploinsufficiency, but showed a novel, synonymous mutation in exon 3 (c.351C>G, p.T117T). In order to elucidate the pathomechanism of the mutation, we performed an in depth in silico evaluation of the mutated sequence and analysed cDNA from the patient bone marrow and fibroblasts for aberrant splicing patterns by GATA2 specific RT-PCR and Sanger sequencing.In silico studies of the mutation showed that the mutated cytosine at position 351 is conserved across most species (www.ensembl.org) and predicted aberrations of splicing, especially the activation of a crypticsplice site in exon 3 (www.fruitfly.org). GATA2 specific RT-PCR showed comparable results for the patient and controls and did not show any aberrant bands at visual inspection. Sanger sequencing of these RT-PCR amplified GATA2 specific transcripts from patient bone marrow and blood identified a clear “C” call for the wild type allele and a very weak “G” call for the mutated allele. In order to test if the mutated allele was expressed at minor frequency we cloned the transcripts into a topoisomerase I-activated pCR™4-TOPO® TA Vector. Sequencing of 60 clones showed an overrepresentation of the wild type allele (42/60 clones, 70%) in comparison to the mutated allele (8/70 clones, 13%). Further clones (10/60, 17%) showed an aberrantly spliced cDNA using the predicted cryptic splice donor site at cDNA position 351 and a cryptic splice acceptor site in exon 3 (cDNA position 487). The aberrant transcript showed a deletion of 136bp in exon 3 and introduction of a premature stop codon after 517bp. This presumably leads to a truncated GATA2 protein disrupting the DNA binding domains. The data support that GATA2 haploinsufficiency in our patient is caused by decreased expression of the mutated allele and abrogation of functional protein expression from the aberrantly spliced mRNA.In summary, we report a novel mutation type leading to GATA2 haploinsufficiency. The described, synonymous mutation activates a cryptic splice donor site in GATA2 exon 3. This mutation type and mutations affecting splicing in general have so far not been described in GATA2 haploinsufficiency. Our finding underscores the need to thoroughly investigate also rare “silent” mutations in genetic counselling and diagnosis. DisclosuresNo relevant conflicts of interest to declare.

Insilco Analysis of Non-Synonymous Single Nucleotide Polymorphisms of GATA 2 binding factor Human Gene

GATA2 or GATA-binding factor 2 is a transcription factor, which regulates the expression of genes that are critical for the embryonic development, self-renewal, maintenance, and functionality of blood-forming lymphatic system-forming, and other tissue-forming stem cells. GATA2 protein is encoded by the GATA2 gene which often suffers germ line and somatic mutations which lead to a wide range of familial and sporadic diseases. Association of data including protein and genetic interactions, pathways, co-expression, co-localization and protein domain similarity were predicted using Gene MANIA software. This gene was investigated in NCBI database and the Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs) were analyzed by computational software, (SIFT, PROVEAN, Polyphen-2, SNPs&amp;GO, PHD-SNPs, I-Mutant and MUpro). The Protein structural analysis was done by modelling of amino acid substitutions using Project Hope for all predicted pathological polymorphisms. GeneMANIA results showed that GATA 2 gene was associated with 20 other genes. It interacts mainly with ZFPM1 gene (Zinc Finger Protein), FOG family member 1and HDAC5 histone deacetylase5. A total of 246 nsSNPs were obtained from the SNPs database in NCBI consisting of 47 Deleterious predicted by SIFT software. Using PROVEAN 41 SNPs were predicted to have deleterious effect, 17 SNPs were predicted to be damaging by (Polyphen- 2), while 4 SNPs were benign, 14 SNPs were found to be disease related using SNPs&amp;GO and 30 SNPS were found to be disease related using PHD- SNP. By using I-MUTANT and MUPRO software the stability of the protein was predicted. From the results of SIFT, PROVEAN and PolyPhen-2 with respect to PHD-SNPs and SNPs&amp;GO software programs, 6 nsSNPs (rs148942346 (R344I), rs371096438 (P385N), rs387906629 (R398W), rs387906629 (R384W), rs387906633 (C359R) and rs387906633 (C373R)) were predicted to be deleterious, damaging, affecting the protein stability and related to the formation of a disease variants. These results might be beneficial for a precise diagnosis of GATA2 deficiency and related myeloid neoplasm.

In Silico Analysis of Nonsynonumus Single Nucleotide Polymorphisms(nsSNPs) in Human GAAGene

GATA2 or GATA-binding factor 2 is a transcription factor, which regulates the expression of genes that are critical for the embryonic development, self-renewal, maintenance, and functionality of blood-forming lymphatic system-forming, and other tissue-forming stem cells. GATA2 protein is encoded by the GATA2 gene which often suffers germ line and somatic mutations which lead to a wide range of familial and sporadic diseases. Association of data including protein and genetic interactions, pathways, co-expression, co-localization and protein domain similarity were predicted using Gene MANIA software. This gene was investigated in NCBI database and the Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs) were analyzed by computational software, (SIFT, PROVEAN, Polyphen-2, SNPs &amp; GO, PHD-SNPs, I-Mutant and MUpro). The Protein structural analysis was done by modelling of amino acid substitutions using Project Hope for all predicted pathological polymorphisms. GeneMANIA results showed that GATA 2 gene was associated with 20 other genes. It interacts mainly with ZFPM1 gene (Zinc Finger Protein), FOG family member 1and HDAC5 histone deacetylase5. A total of 246 nsSNPs were obtained from the SNPs database in NCBI consisting of 47 Deleterious predicted by SIFT software. Using PROVEAN 41 SNPs were predicted to have deleterious effect, 17 SNPs were predicted to be damaging by (Polyphen- 2), while 4 SNPs were benign, 14 SNPs were found to be disease related using SNPs&amp;GO and 30 SNPS were found to be disease related using PHD- SNP. By using I-MUTANT and MUPRO software the stability of the protein was predicted. From the results of SIFT, PROVEAN and PolyPhen-2 with respect to PHD-SNPs and SNPs&amp;GO software programs, 6 nsSNPs (rs148942346 (R344I), rs371096438 (P385N), rs387906629 (R398W), rs387906629 (R384W), rs387906633 (C359R) and rs387906633 (C373R)) were predicted to be deleterious, damaging, affecting the protein stability and related to the formation of a disease variants. These results might be beneficial for a precise diagnosis of GATA2 deficiency and related myeloid neoplasm.

LncRNA SNHG16 Mediates Cell Proliferation and Apoptosis in Cholangiocarcinoma by Directly Targeting miR-146a-5p/GATA6 Axis.

Cholangiocarcinoma (CCA) is a malignant tumour with high recurrence and mortality rates and poor prognosis. However, the pathogenic mechanism remains unclear. In the present study, we aimed to investigate the roles and regulatory mechanism of SNHG16 in the occurrence and development of CCA. Gene Expression Profiling Interactive Analysis (GEPIA) was used to predict the expressions of SNHG16 and GATA6 in CCA samples from TCGA database. The levels of SNHG16, miR-146a-5p and GATA6 were evaluated using qRT-PCR. CCK-8 and flow cytometry assays were conducted to evaluate cell proliferation and apoptosis, respectively. Western blotting was applied to analyse the protein levels of GATA6 and apoptosis-related proteins. SNHG16 was significantly elevated in CCA tissues from TCGA database and CCA cell lines. Moreover, downregulation of SNHG16 restricted cell proliferation and increased apoptotic rate of RBE and HuCCT1 cells. miR-146a-5p, a downstream target of SNHG16, was shown to be an intermediate mediator of GATA6 expression regulated by SNHG16. In addition, either the miR-146a-5p inhibitor or overexpression of GATA6 obviously impaired the regulatory effects of SNHG16 downregulation in RBE and HuCCT1 cells.These data demonstrated that SNHG16 promoted cell proliferation and repressed apoptosis by regulating the miR-146a-5p/GATA6 axis, which provides some helpful insights for the diagnosis and treatment of CCA.

Ambient air PM2.5 exposure induces heart injury and cardiac hypertrophy in rats through regulation of miR-208a/b, α/β-MHC, and GATA4

Ambient air fine particulate matter (PM2.5) may increase cardiovascular disease risks. In this study, we investigated the miR-208/GATA4/myosin heavy chain (MHC) regulation mechanisms on cardiac injury in rats after PM2.5 exposure via an animal inhalation device. The results showed that PM2.5 exposure for 2 months caused pathological heart injury, reduced nucleus-cytoplasm ratio, and increased the levels of CK-MB and cTnI, showing cardiac hypertrophy. Oxidative stress and inflammatory responses were also observed in rats’ hearts exposed to PM2.5. Of note, PM2.5 exposure for 2-month significantly elevated GATA4 and β-MHC mRNA and protein expression compared with the corresponding controls, along with the high-expression of miR-208b. The ratios of β-MHC/α-MHC expression induced by PM2.5 were remarkably raised in comparison to their controls. It suggested that the up-regulation of miR-208b/β-MHC and GATA4 and the conversion from α-MHC to β-MHC may be the important causes of cardiac hypertrophy in rats incurred by PM2.5.

MAPK4 promotes prostate cancer by concerted activation of androgen receptor and AKT.

Prostate cancer (PCa) is the second leading cause of cancer death in American men. Androgen receptor (AR) signaling is essential for PCa cell growth/survival and remains a key therapeutic target for lethal castration-resistant PCa (CRPC). GATA2 is a pioneer transcription factor crucial for inducing AR expression/activation. We recently reported that MAPK4, an atypical MAPK, promotes tumor progression via noncanonical activation of AKT. Here, we demonstrated that MAPK4 activated AR by enhancing GATA2 transcriptional expression and stabilizing GATA2 protein through repression of GATA2 ubiquitination/degradation. MAPK4 expression correlated with AR activation in human CRPC. Concerted activation of both GATA2/AR and AKT by MAPK4 promoted PCa cell proliferation, anchorage-independent growth, xenograft growth, and castration resistance. Conversely, knockdown of MAPK4 decreased activation of both AR and AKT and inhibited PCa cell and xenograft growth, including castration-resistant growth. Both GATA2/AR and AKT activation were necessary for MAPK4 tumor-promoting activity. Interestingly, combined overexpression of GATA2 plus a constitutively activated AKT was sufficient to drive PCa growth and castration resistance, shedding light on an alternative, MAPK4-independent tumor-promoting pathway in human PCa. We concluded that MAPK4 promotes PCa growth and castration resistance by cooperating parallel pathways of activating GATA2/AR and AKT and that MAPK4 is a novel therapeutic target in PCa, especially CRPC.

Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli.

Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development. In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers. To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E. coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography. The identity of GATA4 protein was confirmed using western blotting and mass spectrometry. Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices. Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus. Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy.

GATA4 blocks squamous epithelial cell gene expression in human esophageal squamous cells

GATA4 promotes columnar epithelial cell fate during gastric development. When ectopically expressed in the developing mouse forestomach, the tissue emerges as columnar-like rather than stratified squamous with gene expression changes that parallel those observed in the pre-malignant squamous to columnar metaplasia known as Barrett’s esophagus (BE). GATA4 mRNA up-regulation and gene amplification occur in BE and its associated cancer, esophageal adenocarcinoma (EAC), and GATA4 gene amplification correlates with poor patient outcomes. Here, we explored the effect of ectopic expression of GATA4 in mature human esophageal squamous epithelial cells. We found that GATA4 expression in esophageal squamous epithelial cells compromised squamous cell marker gene expression and up-regulated expression of the canonical columnar cell cytokeratin KRT8. We observed GATA4 occupancy in the p63, KRT5, and KRT15 promoters, suggesting that GATA4 directly represses expression of squamous epithelial cell marker genes. Finally, we verified GATA4 protein expression in BE and EAC and found that exposure of esophageal squamous epithelial cells to acid and bile, known BE risk factors, induced GATA4 mRNA expression. We conclude that GATA4 suppresses expression of genes marking the stratified squamous epithelial cell lineage and that this repressive action by GATA4 may have implications in BE and EAC.

Mutations in exon 2 of GATA1 are early events in megakaryocytic malignancies associated with trisomy 21

AbstractPatients with Down syndrome (DS) frequently develop 2 kinds of clonal megakaryocytosis: a common, congenital, spontaneously resolving, transient myeloproliferative disorder (TMD) and, less commonly, childhood acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in exon 2 of GATA1, an X-linked gene encoding a transcription factor that promotes megakaryocytic differentiation, were described in 6 DS patients with AMKL. The mutations prevent the synthesis of the full-length GATA1, but allow the synthesis of a shorter GATA1 protein (GATA1s) that lacks the transactivation domain. To test whether mutated GATA1 is involved in the initiation of clonal megakaryoblastic proliferation or in the progression to AMKL, we screened 35 DS patients with either AMKL or TMD and 7 non-DS children with AMKL for mutations in exon 2 of GATA1. Mutations were identified in 16 of 18 DS patients with AMKL, in 16 of 17 DS patients with TMD, and in 2 identical twins with AMKL and acquired trisomy 21. Analysis revealed various types of mutations in GATA1, including deletion/insertions, splice mutations, and nonsense and missense point mutations, all of which prevent the generation of full-length GATA1, but preserve the translation of GATA1s. We also show that the likely mechanism of generation of GATA1 isoforms is alternative splicing of exon 2 rather than, or in addition to, alternative translation initiation, as was proposed before. These findings suggest that acquired intrauterine inactivating mutations in GATA1 and generation of GATA1s cooperate frequently with trisomy 21 in initiating megakaryoblastic proliferation, but are insufficient for progression to AMKL.

Exercise and cardiomyocyte regeneration

Cardiovascular disease is the most common cause of death. Many researchers have evaluated the effect of exercise on heart function improvement, but studies about how exercise can affect heart regeneration are rare. Most of the previous studies only assess biomarkers that indicate heart damage. GATA-4 Transcription Factor is one of the transcription factors that form heart cells, showing the heart's ability to function properly under pressure. Cardiac-restricted zinc-finger TF GATA-4 is a survival factor that can break the vicious cycle of post-MI heart failure through increased myocardial angiogenesis, decreased apoptosis, and increased c-kit cell generation. Further research is needed because of its critical role in heart regeneration, whether GATA-4 can be used as an excellent cardiac biomarker in the future, and how the role of exercise or physical exercise on GATA-4 protein and its expression in cardiac regeneration.

Effect of MiR-451a on Erythroid Differentiation of K562 Cells under Hypoxia

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. 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. 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). 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.

G ATA2 mediates the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded T3 receptor β2 in the rat hypothalamic paraventricular nucleus.

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) β2 is known to mediate the negative regulation of the prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene suggest that there is a tethering mechanism, whereby liganded TRβ2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSHβ gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TRβ2 and GATA2 in cultured neuronal cells. Here, we confirmed the expression of the GATA2 protein in the TRH neuron of the rat PVN using immunohistochemistry with an anti-GATA2 antibody. According to an experimental study from transgenic mice, a region of the rat prepro-TRH promoter from nt. -547 to nt. +84 was able to mediate its expression in the PVN. We constructed a chloramphenicol acetyltransferase (CAT) reporter gene containing this promoter sequence (rTRH(547)-CAT) and showed that GATA2 activated the promoter in monkey kidney-derived CV1 cells. Deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. Unexpectedly, T3-dependent negative regulation was maintained after mutation of the reported negative T3-responsive element, site 4. T3 also inhibited the GATA2-dependent transcription enhanced by cAMP agonist, 8-bromo-cAMP. A rat thyroid medullary carcinoma cell line, CA77, is known to express the preproTRH mRNA. Using a chromatin immunoprecipitation assay with this cell line where GATA2 expression plasmid was transfected, we observed the recognition of the GATA-RE by GATA2. We also confirmed GATA2 binding using gel shift assay with the probe for the GATA-RE. In CA77 cells, the activity of rTRH(547)-CAT was potentiated by overexpression of GATA2, and it was inhibited in a T3-dependent manner. These results suggest that GATA2 transactivates the rat prepro-TRH gene and that liganded TRβ2 interferes with this activation via a tethering mechanism as in the case of the TSHβ gene.