GATA1 Changes DNA-Binding Fashion in a Binding-Site-Specific Manner and Alters Transcriptional Activity during Erythropoiesis

Abstract GATA1 is a transcription factor that coordinately regulates multiple target genes during the development and differentiation of erythroid and megakaryocytic lineages through binding to GATA motif (A/T)GATA(A/G). GATA1 has four functional domains, i.e., two transactivation domains reside in amino- and carboxyl- terminus, which transactivate GATA1 target genes redundantly and/or cooperatively, and two zinc-finger domains in the middle of the protein. The two zinc finger domains of GATA1 have been characterized extensively and their links to human diseases have also been identified. Carboxyl-terminal side zinc (C)-finger is essential for the DNA binding of GATA1, whereas amino-terminal side zinc (N)-finger retains insufficient binding activity to the GATA motifs by itself, but contributes to stabilize the binding of C-finger to a double GATA site arranged in a palindromic manner. Of note, while this two-finger structure is conserved in six distinct vertebrate GATA factors, there exist GATA factors with single zinc finger in non-vertebrates, indicating that only the C-finger and following basic tail region are evolutionary conserved in both vertebrate and non-vertebrate GATA factors. In our transgenic rescue analyses, GATA1 lacking the N-finger (ΔNF-GATA1) supports, if not completely, the erythropoiesis in mice, but mice without C-finger (ΔCF-GATA1) die in utero showing similar phenotype to the mice with complete loss-of-GATA1-function. Therefore, roles that the N-finger plays have been assumed to be evolutionally acquired features during molecular evolution. In this study, we have examined GATA-motif configuration-specific modulation of GATA1 function by using composite GATA elements in which two GATA motifs aligned side-by-side, either tandem or palindromic. We have defined changes in the GATA1 binding and transactivation activity in accordance with the arrangement of cis -acting GATA motifs. While GATA1 binds to Single-GATA in a monovalent way via C-finger without the influence of N-finger, the N-finger appears to contribute to specific bivalent binding of GATA1 to Pal-GATA, i.e., the N- and C-fingers in a single GATA1 molecule individually bind to two GATA motifs aligned in a palindromic orientation. Showing very good agreement with the human case analyses, the transgenic expression of G1R216Q that lacks N-finger-DNA interaction potential hardly rescues the GATA1-deficient mice due to defects in definitive erythropoiesis, indicating that roles owed by R216 residue are vital for the GATA1 activity in vivo. The N-finger also contributes to GATA1 homodimer formation, which is a prerequisite for two GATA1 binding to two GATA motifs aligned in a tandem orientation. Each GATA1 C-finger in the dimeric GATA1 protein binds to each GATA motif in Tandem-GATA. In this regard, we previously found in a transgenic complementation rescue assay that mutant GATA1 molecule G13KA, which lacks the dimerization potential but possesses most of the other N- and C-finger functions, hardly rescues the GATA1-deficient mice from embryonic lethality, indicating that the GATA1 dimerization is important to attain full GATA1 activity. We surmise based on these observations that the configuration of cis -acting GATA motifs located in the regulatory regions of the GATA1 target genes critically influences the DNA-binding of GATA1 and controls transcription of the genes. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v120.21.3441.3441 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3441-3441

Author(s):

Atsushi Hasegawa ◽

Ritsuko Shimizu ◽

Hirofumi Kurokawa ◽

Masayuki Yamamoto

Keyword(s):

Dna Binding ◽

Target Genes ◽

Conflicts Of Interest ◽

Consensus Sequence ◽

Late Gestation ◽

The Other ◽

Binding Modes ◽

Substitution Mutations ◽

Gata Motif

Abstract Abstract 3441 Transcription 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.

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A GATA family transcription factor is expressed along the embryonic dorsoventral axis in Drosophila melanogaster

Development ◽

10.1242/dev.119.4.1055 ◽

1993 ◽

Vol 119 (4) ◽

pp. 1055-1065 ◽

Cited By ~ 14

Author(s):

J. Winick ◽

T. Abel ◽

M.W. Leonard ◽

A.M. Michelson ◽

I. Chardon-Loriaux ◽

...

Keyword(s):

Dna Binding ◽

Cell Fate ◽

Zinc Finger ◽

Sequence Similarity ◽

Vertebrate Development ◽

Drosophila Development ◽

Gata Factors ◽

Factors Analysis ◽

Dorsal Portion ◽

Dorsal Cell Fate

The GATA transcription factors are a family of C4 zinc finger-motif DNA-binding proteins that play defined roles in hematopoiesis as well as presumptive roles in other tissues where they are expressed (e.g., testis, neuronal and placental trophoblast cells) during vertebrate development. To investigate the possibility that GATA proteins may also be involved in Drosophila development, we have isolated and characterized a gene (dGATAa) encoding a factor that is quite similar to mammalian GATA factors. The dGATAa protein sequence contains the two zinc finger DNA-binding domain of the GATA class but bears no additional sequence similarity to any of the vertebrate GATA factors. Analysis of dGATAa gene transcription during Drosophila development revealed that its mRNA is expressed at high levels during early embryogenesis, with transcripts first appearing in the dorsal portion of the embryo just after cellularization. As development progresses, dGATAa mRNA is present at high levels in the dorsal epidermis, suggesting that dGATAa may be involved in determining dorsal cell fate. The pattern of expression in a variety of dorsoventral polarity mutants indicates that dGATAa lies downstream of the zygotic patterning genes decapentaplegic and zerknullt.

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Human leukemia mutations corrupt but do not abrogate GATA-2 function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813015115 ◽

2018 ◽

Vol 115 (43) ◽

pp. E10109-E10118 ◽

Cited By ~ 14

Author(s):

Koichi R. Katsumura ◽

Charu Mehta ◽

Kyle J. Hewitt ◽

Alexandra A. Soukup ◽

Isabela Fraga de Andrade ◽

...

Keyword(s):

Dna Binding ◽

Progenitor Cells ◽

Zinc Finger ◽

Erythroid Differentiation ◽

Cell Types ◽

Myeloid Differentiation ◽

Human Leukemia ◽

Hematopoietic Stem ◽

Amino Terminal ◽

Disease Mutations

By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous GATA2 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA2 disease mutations commonly disrupt amino acid residues that mediate DNA binding or cis-elements within a vital GATA2 intronic enhancer, suggesting a haploinsufficiency mechanism of pathogenesis. Mutations also occur in GATA2 coding regions distinct from the DNA-binding carboxyl-terminal zinc finger (C-finger), including the amino-terminal zinc finger (N-finger), and N-finger function is not established. Whether distinct mutations differentially impact GATA-2 mechanisms is unknown. Here, we demonstrate that N-finger mutations decreased GATA-2 chromatin occupancy and attenuated target gene regulation. We developed a genetic complementation assay to quantify GATA-2 function in myeloid progenitor cells from Gata2 −77 enhancer-mutant mice. GATA-2 complementation increased erythroid and myeloid differentiation. While GATA-2 disease mutants were not competent to induce erythroid differentiation of Lin−Kit+ myeloid progenitors, unexpectedly, they promoted myeloid differentiation and proliferation. As the myelopoiesis-promoting activity of GATA-2 mutants exceeded that of GATA-2, GATA2 disease mutations are not strictly inhibitory. Thus, we propose that the haploinsufficiency paradigm does not fully explain GATA-2–linked pathogenesis, and an amalgamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex phenotypes of GATA-2–dependent pathologies.

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Determination of DNA binding specificities of mutated zinc finger domains

FEBS Letters ◽

10.1016/0014-5793(91)80545-e ◽

1991 ◽

Vol 283 (1) ◽

pp. 23-26 ◽

Cited By ~ 11

Author(s):

Hans-Jürgen Thiesen ◽

Christian Bach

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Zinc Finger Domains

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KDM2B in polycomb repressive complex 1.1 functions as a tumor suppressor in the initiation of T-cell leukemogenesis

Blood Advances ◽

10.1182/bloodadvances.2018028522 ◽

2019 ◽

Vol 3 (17) ◽

pp. 2537-2549 ◽

Cited By ~ 6

Author(s):

Yusuke Isshiki ◽

Yaeko Nakajima-Takagi ◽

Motohiko Oshima ◽

Kazumasa Aoyama ◽

Mohamed Rizk ◽

...

Keyword(s):

T Cell ◽

Tumor Suppressor ◽

Zinc Finger ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Dependent Manner ◽

Polycomb Repressive Complex ◽

Human T Cell ◽

T Cell Leukemogenesis ◽

Deficient Mice

Abstract KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.

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The structural role of the zinc ion can be dispensable in prokaryotic zinc-finger domains

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0810003106 ◽

2009 ◽

Vol 106 (17) ◽

pp. 6933-6938 ◽

Cited By ~ 34

Author(s):

Ilaria Baglivo ◽

Luigi Russo ◽

Sabrina Esposito ◽

Gaetano Malgieri ◽

Mario Renda ◽

...

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Coordination Sphere ◽

Zinc Ion ◽

Binding Domain ◽

Hydrophobic Core ◽

Zinc Finger Domain ◽

High Sequence Identity ◽

Zinc Coordination ◽

Zinc Finger Domains

The recent characterization of the prokaryotic Cys2His2 zinc-finger domain, identified in Ros protein from Agrobacterium tumefaciens, has demonstrated that, although possessing a similar zinc coordination sphere, this domain is structurally very different from its eukaryotic counterpart. A search in the databases has identified ≈300 homologues with a high sequence identity to the Ros protein, including the amino acids that form the extensive hydrophobic core in Ros. Surprisingly, the Cys2His2 zinc coordination sphere is generally poorly conserved in the Ros homologues, raising the question of whether the zinc ion is always preserved in these proteins. Here, we present a functional and structural study of a point mutant of Ros protein, Ros56–142C82D, in which the second coordinating cysteine is replaced by an aspartate, 5 previously-uncharacterized representative Ros homologues from Mesorhizobium loti, and 2 mutants of the homologues. Our results indicate that the prokaryotic zinc-finger domain, which in Ros protein tetrahedrally coordinates Zn(II) through the typical Cys2His2 coordination, in Ros homologues can either exploit a CysAspHis2 coordination sphere, previously never described in DNA binding zinc finger domains to our knowledge, or lose the metal, while still preserving the DNA-binding activity. We demonstrate that this class of prokaryotic zinc-finger domains is structurally very adaptable, and surprisingly single mutations can transform a zinc-binding domain into a nonzinc-binding domain and vice versa, without affecting the DNA-binding ability. In light of our findings an evolutionary link between the prokaryotic and eukaryotic zinc-finger domains, based on bacteria-to-eukaryota horizontal gene transfer, is discussed.

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Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method

Nucleic Acids Research ◽

10.1093/nar/gkm385 ◽

2007 ◽

Vol 35 (11) ◽

pp. e81 ◽

Cited By ~ 19

Author(s):

Xiangdong Meng ◽

Stacey Thibodeau-Beganny ◽

Tao Jiang ◽

J. Keith Joung ◽

Scot A. Wolfe

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Zinc Finger Domains

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A Novel GATA1 Mutation (Ter414Arg) in a Family with the Rare X-Linked Blood Group Lu(a-b-) Phenotype.

Blood ◽

10.1182/blood.v114.22.1979.1979 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1979-1979 ◽

Cited By ~ 7

Author(s):

Belinda K Singleton ◽

David Roxby ◽

John Stirling ◽

Frances A Spring ◽

Carolyn Wilson ◽

...

Keyword(s):

Blood Group ◽

Zinc Finger ◽

Conflicts Of Interest ◽

Large Family ◽

Rflp Analysis ◽

Blood Group Antigens ◽

Zinc Finger Domain ◽

Carboxy Terminus ◽

Amino Terminal ◽

Gata1 Mutation

Abstract Abstract 1979 Poster Board I-1001 The X-borne transcription factor GATA-1 is essential for erythroid and megakaryocyte development. In 1986, Norman et al. (Vox Sang 51:49) described a large family in which the rare Lu(a-b-) blood group phenotype is inherited as the result of an X-borne gene. Serologic and flow cytometric analyses confirmed the suppression of Lutheran blood group antigens on the red blood cells from the original male propositus. Analysis of DNA from the propositus revealed a mutation (1240T>C) in the termination codon of GATA1 converting TGA to a codon for arginine (CGA). The mutation predicts a translated GATA-1 protein containing an additional 41 amino acids at the carboxy terminus. DNA from an unaffected sister of the propositus had a wild-type GATA1 sequence. The GATA1 mutation was not present in 78 random blood donors as determined by restriction fragment length polymorphism (RFLP) analysis using BspHI. The propositus, who is now 64, has an Hb of 122 g/l, a low platelet count (86 × 109/l) with occasional macrothrombocytes (diameter 4-5 um) and a history of bruising easily. Several mutations in the amino terminal zinc finger domain of GATA-1 have been linked with thrombocytopenia and thalassemia or dsyerythropoietic anemia and the presence of macrothrombocytes. Our data provide evidence that mutations outside the zinc finger domain can affect GATA-1 functions in erythroid and megakaryocyte differentiation. In this case, the predicted extended carboxy terminus may interfere with GATA-1 interactions involving other DNA-binding proteins. Disclosures: No relevant conflicts of interest to declare.

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Mutation In Erythroid Specific Transcription Factor KLF1 Causes Hereditary Spherocytosis In the Nan (Neonatal Anemia) Hemolytic Anemia Mouse Model

Blood ◽

10.1182/blood.v116.21.3217.3217 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3217-3217

Author(s):

Robert A White ◽

Daniel P. Heruth ◽

Troy Hawkins ◽

Derek Logsdon ◽

Margaret Gibson ◽

...

Keyword(s):

Amino Acid ◽

Hemolytic Anemia ◽

Zinc Finger ◽

Target Genes ◽

Zinc Finger Protein ◽

Hereditary Spherocytosis ◽

Competitive Inhibition ◽

Conflicts Of Interest ◽

Dominant Negative ◽

Zinc Finger Domain

Abstract Abstract 3217 The zinc finger protein Erythroid Krüuppel-like factor (EKLF, KLF1) regulates definitive erythropoiesis and terminal differentiation of red blood cells. KLF1 facilitates transcription through high affinity binding to CACCC elements within its erythroid-specific target genes which include genes encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins lead to the hemolytic anemia Hereditary Spherocytosis (HS). We have identified a new HS gene by studying the hemolytic anemia mouse mutant Nan (Neonatal Anemia). Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in Nan mice. The causative nature of the E339D mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1+/− knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild type KLF1. Competitive inhibition may help explain the apparent disconnect between the finding that Nan/+ heterozygous mice are anemic, whereas Klf1+/− heterozygous mice are normal and haplo-sufficient. This is the first direct association of a KLF1mutation with a disease in adult mammals. After examining a small population of HS patients, we also discovered one HS patient with a KLF1 mutation, which resulted in a significant amino acid substitution (T251I) in the activator/repressor domain, 28 amino acid residues upstream of the first zinc finger domain. This HS subject had no known mutations in the exons or intron/exon boundaries of EMS genes (SPTA1, SPTB, ANK1, SLC4A1) which comprise 85% of HS mutations in humans. The lack of a known genetic mutation in EMS genes leaves this patient's KLF1 mutation as the leading candidate defect. The identification of the gene causing the Nan mutation is significant because the Nan mutant has allowed discovery of a new HS gene which may also cause this disease in humans. In addition, the putative dominant/negative competitive inhibition of the Nan mutation makes the Nan mouse an excellent model system to study the function of KLF1. Disclosures: No relevant conflicts of interest to declare.

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Homozygous Mutation of the ETS DNA-Binding Domain of FLI1 Causes a Bleeding Disorder with Giant Alpha Granules Similar to Paris-Trousseau Thrombocytopenia

Blood ◽

10.1182/blood.v126.23.78.78 ◽

2015 ◽

Vol 126 (23) ◽

pp. 78-78

Author(s):

William S Stevenson ◽

David John Rabbolini ◽

Timothy Brighton ◽

Joel Mackay ◽

Christopher M Ward ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Conflicts Of Interest ◽

Variable Region ◽

Bleeding Disorder ◽

Hek293 Cells ◽

Luciferase Reporter ◽

Homozygous Mutation ◽

Jacobsen Syndrome ◽

Ets Domain

Abstract Deletion of a variable region on chromosome 11q containing FLI1 causes a platelet-related bleeding disorder in Paris-Trousseau thrombocytopenia and Jacobsen syndrome. We report a kindred with the autosomal recessive inheritance of an ETS domain mutation of FLI1, c.970C>T, that causes macrothrombocytopenia with large alpha granule fusion similar to that observed in Paris-Trousseau thrombocytopenia but with no other syndromal features of Paris-Trousseau or Jacobsen syndromes. Affected individuals are moderately thrombocytopenic (mean = 71 x109/L), have absent collagen-induced platelet aggregation and a lifelong mucosal bleeding history. Platelet MYH10 levels were increased in affected members of the kindred consistent with previous reports of elevated MYH10 in Paris-Trousseau thrombocytopenia. Luciferase reporter assays in HEK293 cells demonstrate that the mutant FLI1 transcript is associated with decreased transcription at the FLI target genes GP6, GP9 and ITGA2B compared to the wild-type transcript (23 vs 31, n=9, P<0.01; 3.8 vs 6.5, P<0.01, n=12; 11 vs 14, n=9, P=0.01, respectively). This transcriptional change was consistent with reduced expression of GPVI (P<0.01), GPIbIX (P<0.01) and GPIIbIIIa (P=0.04) observed on western blotting of platelet lysates from affected family members. This mutation replaces the conserved Arg324 with a tryptophan in the DNA-binding loop between the alpha-2 and alpha-3 helices of the FLI1 ETS domain. Protein modelling suggests that Arg324 does not directly bind DNA but may instead make direct contact with an N-terminal autoinhibitory domain of FLI1 that is considered to regulate DNA-binding affinity through a transition between a folded and unfolded state. This mutation may disrupt this important conformational change. These data suggest abnormalities of FLI1 function may be responsible for the complex platelet defect observed in Paris-Trousseau thrombocytopenia and Jacobsen Syndrome and confirm the role of FLI1 as an important transcriptional regulator of normal platelet development. Disclosures No relevant conflicts of interest to declare.

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