DNA-binding Activity Of GATA-1 Research Articles

HDAC1 Can Deacetylate GATA-1 and Regulates Its Activity through a FOG-1 Independent Manner

The transcription factor GATA-1 is essential for erythroid and megakaryocytic cell differentiation and maturation. It has been well documented that GATA-1 can indirectly interact with histone deacetylase 1 (HDAC1) containing NuRD corepressor complexes through an association with FOG-1. Our previous work also shows that HDAC1 acetylation modulates the activity of FOG-1 associated NuRD complexes. Earlier studies show GATA-1 can be acetylated by p300/CBP, and the acetylation modulates GATA-1 binding activity to chromatin. However, it is not clear whether the acetylation can be reversed by a deacetylase. In this study, we found that GATA-1 can directly interact with HDAC1 in a FOG-1 independent manner. The interaction results in the deacetylation of GATA-1. We have identified two arginine sites within GATA-1 that are important for its interaction with HDAC1. The arginine to alanine mutation on these sites (2RA) can largely decrease the interaction of these two proteins, but doesn’t affect its interaction with FOG-1, indicating that the direct interactions with HDAC1 and FOG-1 dependent association of NuRD complexes are separate events. The mutations also do not affect GATA-1 DNA binding activity in vitro. To further investigate the function of this interaction in erythropoiesis, we introduced wild type or the 2RA mutant of GATA-1 fused with estrogen receptor ligand binding domains into G1E cells, a GATA-1-null erythroid progenitor cell line. Upon estradiol induction, GATA-1 2RA inhibits the differentiation in G1E cells. We performed RNA sequencing to study the effect of GATA-1 2RA in gene expression in comparison with wild type GATA-1 and GATA-1-V205M (a mutation abolished binding with FOG-1) on a genome wild scale. GATA-1 2RA affects GATA-1 function in both GATA-1 activated and repressed genes. Although there is some overlap between 2RA and V205M mutations in both activated and repressed genes, more genes affected by these two mutations are different. Thus, this study unveils a novel regulation for GATA-1 by its direct interaction with HDAC1 during hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 420: Critical role of MMP-2 and GATA-1 nuclear interaction in the regulation of ionizing radiation (IR)-induced IL-10/Stat3-mediated invasion and migration in glioblastoma.

Abstract Glioblastoma multiforme (GBM) is the most lethal human brain tumor with high recurrence rates despite the existence of multimodal treatments including chemotherapy and radiation after surgical tumor resection. Augmented tumor metastasis after irradiation (IR) has been attributed to enhanced extracellular protease levels in tumors. Secreted or cleaved matrix metalloproteinase-2 (MMP-2) levels were reported to be elevated in irradiated tumors. Interestingly, previous in silico prediction analyses identified the presence of a putative nuclear localization signal (NLS) in MMP-2 and reported the cleavage of PARP by nuclear-MMP-2. However, MMP-2 nuclear localization in possible correlation with tumor progression has not been identified to date. In addition to membrane and cytoplasmic expression of MMP-2, we observed prominent MMP-2 nuclear localization, which is further elevated in IR (8 Gy)-treated human glioma xenograft cells 4910 and 5310. Interestingly, The protein-transcription factor (TF) interaction arrays using 4910 nuclear extracts revealed the potential binding of nuclear-MMP-2 with several transcription factors including CEF-2, c-Rel, FOXO4, GAS, GATA-1, MUSF-1, p300, PARP and RREB1. Co-IP experiments showed the direct binding between MMP-2/GATA-1 in glioma. Further, we also observed the presence of MMP-2 in the GATA-1 DNA-binding complex and its recruitment onto the promoter of IL-10, a GATA-1 target gene. ChIP-cloning with anti-MMP-2 antibody confirmed the specific binding of MMP-2 with GATA-1 consensus elements. We also observed that the MMP-2/GATA-1 nuclear co-localization was elevated in correlation with increasing GBM pathological grades. Downregulation of MMP-2 inhibited the GATA-1 expression and DNA-binding activity and abrogated IR-induced migration and invasion in these cells. Conversely, treatment with recombinant MMP-2 protein elevated the GATA-1 DNA-binding activity and increased IL-10 and phospho-Stat3 expression in correlation with increased invasion and migration. In vivo orthotopic experiments with MMP-2.siRNA decreased tumor growth in nude mice and inhibited nuclear MMP-2 and GATA-1 expression levels in tumors. To our knowledge, this is the first study demonstrating significant IR-induced nuclear translocation of MMP-2 and shows the functional association between MMP-2/GATA-1 in the regulation of IL-10/Stat3-mediated migration and invasion. Our results suggest the potential therapeutic efficacy of targeting MMP-2/GATA-1 liaison in glioma treatment. Citation Format: Divya Kesanakurti, Dilip Maddirela, Meena Gujrati, Jasti S. Rao. Critical role of MMP-2 and GATA-1 nuclear interaction in the regulation of ionizing radiation (IR)-induced IL-10/Stat3-mediated invasion and migration in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 420. doi:10.1158/1538-7445.AM2013-420

DNA Binding Diversity Achieved Through the Interaction of GATA1 N-Finger and GATA Motif Is Important for Embryonic Erythropoiesis

AbstractAbstract 3441Transcription factor GATA1 regulates a set of genes essential for the erythroid and megakaryocytic cell differentiation through the interaction with GATA motifs (consensus sequence: A/TGATAA/T). Two zinc fingers within GATA1 have been identified to be important in the DNA binding of GATA1, which are referred to as C-finger (CF) and N-finger (NF) domains. It has been shown that transactivation activity of GATA1 is completely abolished upon deletion of the CF domain, indicating that the CF domain is a requisite for the DNA binding of GATA1. While conventional reporter transactivation analyses hardly clarified the importance of the NF domain for the DNA binding, substitution mutations on 216th arginine (R216) located in the DNA-interacting surface of the NF domain have been identified to cause familial diseases of thrombocytopenia, thalassemia, and porphyria. As a consequence of the substitution of R216 to glutamine (Q) or tryptophan (W), DNA binding activity of GATA1 to a palindromic configuration of two GATA motifs (palindromic GATA) was largely diminished, while that to a single GATA motif was maintained.In this study we have examined the DNA binding diversity of GATA1 caused by the difference in the configuration of GATA motifs. We performed surface plasmon resonance (SPR) analyses of GATA1 to a single GATA, a palindromic GATA, and a repeating configuration of two GATA motifs (tandem GATA). We found that GATA1 binds to the palindromic GATA motif in a bivalent way, while it binds to the single GATA motif in a monovalent mode. We also found that a double quantity of GATA1 is associated with the tandem GATA motif and GATA1 lacking the NF domain binds to any configurations of GATA motif in a monovalent way.To further investigate contribution of the NF domain to the binding mode of GATA1, we have constructed two types of GATA1 mutants; one type was the substitution mutations on R216 (R216Q and R216W) that were mouse homologues of the human mutations, while the other type was the alanine substitution mutation on three lysine residues (K245, K246 and K312; referred to as 3KA mutant), whereby dimerization potential of GATA1 was reduced to trace level similar to the case for GATA1 lacking the NF domain. Impotantly, R216Q and R216W mutants bind the palindromic GATA motif in a monovalent way, while these mutants bind normally to the other configuration of GATA motifs. In contrast, we found that one molecule of 3KA mutant bound to the tandem GATA motif and this observation seems to explain well the fact that dimerization potential of GATA1 is an important requisite for the full-function of GATA1 in embryos. The binding modes of this 3KA mutant to the other configurations were not influenced.These results thus demonstrate that the both NF and CF domains recognize the multiple configurations of GATA motifs and specify the binding modes of GATA1. Importantly, GATA1-deficient mice rescued with R216Q were lethal during late gestation period due to abnormality in erythroid differentiation, indicating that the contribution of the NF domain to the recognition of the palindromic GATA motif configuration indeed functions in vivo. These results thus support our contention that the NF domain acts to regulate a proper spatio-temporal gene expression of a subset of GATA1 target genes utilizing the variations in the GATA motif configuration. Disclosures:No relevant conflicts of interest to declare.

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

Notch Signals Inhibit the Development of Erythroid/Megakaryocytic Cells by Suppressing GATA-1 Activity through the Induction of HES1

The effects of Notch signals on the erythroid/megakaryocytic differentiation of hematopoietic cells were examined. Activation of Notch signals by the intracellular Notch1 or an estradiol-inducible form of Notch1/ER suppressed the expression of the erythroid marker glycophorin A in an erythroid/megakaryocytic cell line K562. Although Mock-transfected K562 cells underwent megakaryocytic differentiation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA), estradiol-activated Notch1/ER induced apoptosis during TPA treatment in the transfectant, which was accompanied by the reduced expression of an antiapoptotic molecule Bcl-XL. Even when apoptosis was prevented by the overexpression of Bcl-XL, activated Notch signals still inhibited TPA-induced megakaryocytic differentiation. As for this mechanism, Notch1/recombination signal binding protein J-kappa-induced HES1 but not HES5 was found to inhibit the function of an erythroid/megakaryocytic lineage-specific transcription factor GATA-1. Although HES1 did not affect the DNA binding activity of GATA-1 in gel shift and chromatin immunoprecipitation assays, it directly bound to GATA-1 and dissociated a critical transcriptional cofactor, p300, from GATA-1. Furthermore, overexpressed HES1 inhibited the development of erythroid and megakaryocytic cells in colony assays. Also, the Notch ligand Jagged1 expressed on NIH3T3 cells suppressed the development of erythroid and megakaryocytic cells from cocultured Lin-Sca-1+ hematopoietic stem/progenitor cells. These results suggest that Notch1 inhibits the development of erythroid/megakaryocytic cells by suppressing GATA-1 activity through HES1.

Antisense EDRF1 gene inhibited GATA-1 transcription factor DNA-binding activity in K562 cells

Our previous studies showed that EDRF1 influenced expression of alpha-globin mRNA and synthesis of hemoglobin in K562 cells and modulated self-renewal of K562 cells. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. By using microarray and dot blot assay, 60 cytokine receptors and some oncogenes sharing important functions in cell proliferation and differentiation were investigated. The results of this study demonstrated that IL-6 receptor, GM-CSF receptor, c-Jun/c-Fos, c-Myc and c-kit genes were regulated by antisense EDRF1 expression. The regulation was confirmed by RNA blot assay. GATA-1 mRNA expression was modulated by EDRF1 gene transfection. Electrophoretic mobility shift assay suggested that the DNA-binding activity of GATA-1 was remarkably inhibited in K562 cells expressing EDRF1 antisense gene. DNA binding activity of NF-E2 was at the same level as control experiment. Therefore EDRF1 may play a role in erythroid proliferation and differentiation by affecting the interaction between GATA-1 and its cis-elements.

Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells.

We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythroleukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N -acetylcysteine (NAC). Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases. However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythroleukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAC, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells. In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data. The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphoryl-ation and DNA binding of GATA-1 are already optimalin these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor. Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding. Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.

Cell-Cycle–Dependent Regulation of Erythropoietin Receptor Gene

AbstractTo understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF ), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.

GATA-1 DNA Binding Activity Is Down-regulated in Late S Phase in Erythroid Cells

We have set out to test a model for tissue-specific gene expression that relies on the early replication of expressed genes to sequester limiting activating transcription factors. Using an erythroid cell line, we have tested the changes in the DNA binding activity of the lineage-restricted transcription factor GATA-1 through the cell cycle. We find that GATA-1 activity is low in G1, peaks in mid-S phase, and then decreases in G2/M. In contrast, the binding activities of two ubiquitous transcription factors, Oct1 and Sp1, remain high in G2/M. GATA-1 protein and mRNA vary in a similar manner through the cell cycle, suggesting that the expression of the gene or the stability of its message is regulated. Although a number of transcription factors involved in the control of the cell cycle or DNA replication have been shown to peak in S phase, this is the first example of a lineage-restricted transcription factor displaying S phase-specific DNA binding activity. One interpretation of these data leads to a model in which the peak in GATA-1 DNA binding amplifies the effect of early replication on the activation of erythroid-specific genes at the same time as preventing activation of non-erythroid genes containing GATA-responsive elements. These results may also relate to recent data implicating GATA-1 function in apoptosis and cell cycle progression.

Murine erythroleukemia cells contain two distinct GATA-binding proteins that have different patterns of expression during cellular differentiation

Abstract GATA-1 is a major transcription factor of the erythroid lineage that has been implicated in the induced expression of a variety of red cell-specific genes during terminal differentiation of murine erythroleukemia cells. Although the GATA-1 protein is present at nearly equal levels before and after differentiation of murine erythroleukemia cells, in this study it was found that in the early commitment stages of the differentiation program there is a transient decrease in the GATA-1 mRNA and DNA binding activity levels due to a temporary block in transcription of the gene. Moreover, using a whole cell extraction procedure it was discovered that murine erythroleukemia cells contain a second GATA binding activity (denoted GATA-rel) which appears to be distinct from the GATA-1 factor based on its non-reactivity to two GATA-1 antisera. This protein has a limited tissue specificity, as it could not be detected in extracts from CHO, NIH 3T3, or COS cells. Similarly to the GATA-1 DNA-binding activity, the GATA-rel activity decreased during the early stages of differentiation. However, unlike GATA-1, GATA-rel activity did not return to pre-induced levels at later times. These results suggest that changes in gene expression during erythroid terminal differentiation may involve an interplay on levels of different GATA-binding factors.

Erythropoietin triggers a burst of GATA-1 in normal human erythroid cells differentiating in tissue culture.

GATA-1 is a central transcription-activator of erythroid differentiation. In the present work we have studied the kinetics of its expression and activity during development of normal human erythroid progenitors, grown in primary cultures. In response to the addition of erythropoietin (Epo), the cells undergo proliferation and differentiation in a synchronized fashion. This recently developed experimental system allows biochemical dissection of erythroid differentiation in a physiological meaningful environment. No DNA-binding activity of GATA-1 could be detected before the addition of Epo, although a very low level of mRNA was observed. Following Epo addition there was a sharp parallel rise in both mRNA and DNA-binding activity, consistent with positive autoregulation of the GATA-1 gene. After reaching a peak on day 7-9, both mRNA and protein activity decreased. The binding activity of the ubiquitous factor SP1 showed a biphasic pattern; its second peak usually coincided with the GATA-1 peak, suggesting that SP1 also plays a specific role in erythroid maturation. The highest activity of GATA-1 per erythroid cell was found on day 6-8, immediately preceding the major rise in globin gene mRNA and in the number of hemoglobinized cells. The results imply that a high level of GATA-1 activity is necessary for globin gene expression and erythroid maturation, suggesting that a requirement for a threshold concentration of GATA-1 is part of the mechanism that determines the final steps of erythroid maturation.