HEL cells, a human erythroleukemia cell line, mainly express the fetal (r)globin gene and trace amount of the embryonic (E)globin gene, but not adult (B) globin gene. Here we show that hydroxyurea (HU) can induce HEL ...HEL cells, a human erythroleukemia cell line, mainly express the fetal (r)globin gene and trace amount of the embryonic (E)globin gene, but not adult (B) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (B) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human B- globin gene, including the proximal regulatory element (the B- promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU- induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human B-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human β- globin gene in HU-induced HEL cells.展开更多
Seed dormancy is an important agronomic trait in crops, and plants with low dormancy are prone to preharvest sprouting(PHS) under high-temperature and humid conditions. In this study,we report that the GATA transcript...Seed dormancy is an important agronomic trait in crops, and plants with low dormancy are prone to preharvest sprouting(PHS) under high-temperature and humid conditions. In this study,we report that the GATA transcription factor TaGATA1 is a positive regulator of seed dormancy by regulating TaABI5 expression in wheat.Our results demonstrate that TaGATA1 overexpression significantly enhances seed dormancy and increases resistance to PHS in wheat. Gene expression patterns, abscisic acid(ABA) response assay, and transcriptome analysis all indicate that TaGATA1 functions through the ABA signaling pathway. The transcript abundance of TaABI5, an essential regulator in the ABA signaling pathway,is significantly elevated in plants overexpressing TaGATA1. Chromatin immunoprecipitation assay(ChIP) and transient expression analysis showed that TaGATA1 binds to the GATA motifs at the promoter of TaABI5 and induces its expression.We also demonstrate that TaGATA1 physically interacts with the putative demethylase TaELF6-A1, the wheat orthologue of Arabidopsis ELF6.ChIP–qPCR analysis showed that H3K27me3 levels significantly decline at the TaABI5 promoter in the TaGATA1-overexpression wheat line and that transient expression of TaELF6-A1 reduces methylation levels at the TaABI5 promoter, increasing TaABI5 expression. These findings reveal a new transcription module, including TaGATA1–TaELF6-A1–TaABI5, which contributes to seed dormancy through the ABA signaling pathway and epigenetic reprogramming at the target site. TaGATA1 could be a candidate gene for improving PHS resistance.展开更多
To gain a better understanding of the regulation of Zn homeostasis in plants and the degree of conservation of Zn homeostasis between plants and yeast, a cDNA library from the Zn/Cd hyperaccumulating plant species, No...To gain a better understanding of the regulation of Zn homeostasis in plants and the degree of conservation of Zn homeostasis between plants and yeast, a cDNA library from the Zn/Cd hyperaccumulating plant species, Noccaea caerulescens, was screened for its ability to restore growth under Zn limiting conditions in the yeast mutant zaplA. ZAP1 is a transcription factor that activates the Zn dependent transcription of yeast genes involved in Zn uptake, including ZRT1, the yeast high affinity Zn transporter. From this screen two members of the E2F family of transcription factors were found to activate ZRT1 expression in a Zn independent manner. The activation of ZRTI by the plant E2F proteins involves E2F- mediated activation of a yeast GATA transcription factor which in turn activates ZRT1 expression. A ZRT1 promoter region necessary for activation by E2F and GATA proteins is upstream of two zinc responsive elements previously shown to bind ZAP1 in ZRT1. This activation may not involve direct binding of E2F to the ZRT1 promoter. The expression of E2F genes in yeast does not replace function of ZAP1; instead it appears to activate a novel GATA regulatory pathway involved in Zn uptake and homeostasis that is not Zn responsive.展开更多
OBJECTIVE: To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. METHODS: By transfecting EDRF1 sense and ant...OBJECTIVE: To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. METHODS: By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2. RESULTS: It was shown that when EDRF1 was overexpressed, production of alpha-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of alpha-, gamma-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression. CONCLUSION: The results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.展开更多
文摘HEL cells, a human erythroleukemia cell line, mainly express the fetal (r)globin gene and trace amount of the embryonic (E)globin gene, but not adult (B) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (B) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human B- globin gene, including the proximal regulatory element (the B- promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU- induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human B-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human β- globin gene in HU-induced HEL cells.
基金funded by grants from the National Natural Science Foundation of China (31671692 and 32072005)。
文摘Seed dormancy is an important agronomic trait in crops, and plants with low dormancy are prone to preharvest sprouting(PHS) under high-temperature and humid conditions. In this study,we report that the GATA transcription factor TaGATA1 is a positive regulator of seed dormancy by regulating TaABI5 expression in wheat.Our results demonstrate that TaGATA1 overexpression significantly enhances seed dormancy and increases resistance to PHS in wheat. Gene expression patterns, abscisic acid(ABA) response assay, and transcriptome analysis all indicate that TaGATA1 functions through the ABA signaling pathway. The transcript abundance of TaABI5, an essential regulator in the ABA signaling pathway,is significantly elevated in plants overexpressing TaGATA1. Chromatin immunoprecipitation assay(ChIP) and transient expression analysis showed that TaGATA1 binds to the GATA motifs at the promoter of TaABI5 and induces its expression.We also demonstrate that TaGATA1 physically interacts with the putative demethylase TaELF6-A1, the wheat orthologue of Arabidopsis ELF6.ChIP–qPCR analysis showed that H3K27me3 levels significantly decline at the TaABI5 promoter in the TaGATA1-overexpression wheat line and that transient expression of TaELF6-A1 reduces methylation levels at the TaABI5 promoter, increasing TaABI5 expression. These findings reveal a new transcription module, including TaGATA1–TaELF6-A1–TaABI5, which contributes to seed dormancy through the ABA signaling pathway and epigenetic reprogramming at the target site. TaGATA1 could be a candidate gene for improving PHS resistance.
文摘To gain a better understanding of the regulation of Zn homeostasis in plants and the degree of conservation of Zn homeostasis between plants and yeast, a cDNA library from the Zn/Cd hyperaccumulating plant species, Noccaea caerulescens, was screened for its ability to restore growth under Zn limiting conditions in the yeast mutant zaplA. ZAP1 is a transcription factor that activates the Zn dependent transcription of yeast genes involved in Zn uptake, including ZRT1, the yeast high affinity Zn transporter. From this screen two members of the E2F family of transcription factors were found to activate ZRT1 expression in a Zn independent manner. The activation of ZRTI by the plant E2F proteins involves E2F- mediated activation of a yeast GATA transcription factor which in turn activates ZRT1 expression. A ZRT1 promoter region necessary for activation by E2F and GATA proteins is upstream of two zinc responsive elements previously shown to bind ZAP1 in ZRT1. This activation may not involve direct binding of E2F to the ZRT1 promoter. The expression of E2F genes in yeast does not replace function of ZAP1; instead it appears to activate a novel GATA regulatory pathway involved in Zn uptake and homeostasis that is not Zn responsive.
文摘OBJECTIVE: To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. METHODS: By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2. RESULTS: It was shown that when EDRF1 was overexpressed, production of alpha-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of alpha-, gamma-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression. CONCLUSION: The results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.
