Human GATA-3 trans-activation, DNA-binding, and nuclear localization activities are organized into distinct structural domains

GATA-3 is a zinc finger transcription factor which is expressed in a highly restricted and strongly conserved tissue distribution pattern in vertebrate organisms, specifically, in a subset of hematopoietic cells, in cells within the central and peripheral nervous systems, in the kidney, and in placental trophoblasts. Tissue-specific cellular genes regulated by GATA-3 have been identified in T lymphocytes and the placenta, while GATA-3-regulated genes in the nervous system and kidney have not yet been defined. We prepared monoclonal antibodies with which we could dissect the biochemical and functional properties of human GATA-3. The results of these experiments show some anticipated phenotypes, for example, the definition of discrete domains required for specific DNA-binding site recognition (amino acids 303 to 348) and trans activation (amino acids 30 to 74). The signaling sequence for nuclear localization of human GATA-3 is a property conferred by sequences within and surrounding the amino finger (amino acids 249 to 311) of the protein, thereby assigning a function to this domain and thus explaining the curious observation that this zinc finger is dispensable for DNA binding by the GATA family of transcription factors.

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Determinants for DNA-binding site recognition by the glucocorticoid receptor.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)73988-x ◽

1992 ◽

Vol 267 (35) ◽

pp. 24941-24947

Author(s):

J Zilliacus ◽

A.P. Wright ◽

U Norinder ◽

J.A. Gustafsson ◽

J Carlstedt-Duke

Keyword(s):

Dna Binding ◽

Glucocorticoid Receptor ◽

Binding Site ◽

Dna Binding Site ◽

Site Recognition

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The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5128 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5128-5137 ◽

Cited By ~ 10

Author(s):

M M Witte ◽

R C Dickson

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Zinc Finger ◽

Binding Specificity ◽

Binding Domain ◽

Dna Binding Domain ◽

Transcription Activator ◽

Activator Proteins ◽

Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

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Toward rules relating zinc finger protein sequences and DNA binding site preferences.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.16.7345 ◽

1992 ◽

Vol 89 (16) ◽

pp. 7345-7349 ◽

Cited By ~ 144

Author(s):

J. R. Desjarlais ◽

J. M. Berg

Keyword(s):

Dna Binding ◽

Binding Site ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Protein Sequences ◽

Site Preferences ◽

Finger Protein ◽

Dna Binding Site

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A position-dependent transcription-activating domain in TFIIIA

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7496-7506.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7496-7506

Author(s):

X Mao ◽

M K Darby

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Zinc Finger ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Rna Polymerase Iii ◽

Binding Domain ◽

Dna Binding Domain ◽

5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

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Definition of a consensus DNA binding site for SRY

Nucleic Acids Research ◽

10.1093/nar/22.8.1500 ◽

1994 ◽

Vol 22 (8) ◽

pp. 1500-1501 ◽

Cited By ~ 245

Author(s):

Vincent R. Harley ◽

Robin Lovell-Badge ◽

Peter N. Goodfellow

Keyword(s):

Dna Binding ◽

Binding Site ◽

Dna Binding Site ◽

Definition Of

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A position-dependent transcription-activating domain in TFIIIA.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7496 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7496-7506 ◽

Cited By ~ 15

Author(s):

X Mao ◽

M K Darby

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Zinc Finger ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Rna Polymerase Iii ◽

Binding Domain ◽

Dna Binding Domain ◽

5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

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Genetic definition of the Escherichia coli zwf "soxbox," the DNA binding site for SoxS-mediated induction of glucose 6-phosphate dehydrogenase in response to superoxide.

Journal of Bacteriology ◽

10.1128/jb.177.7.1742-1750.1995 ◽

1995 ◽

Vol 177 (7) ◽

pp. 1742-1750 ◽

Cited By ~ 51

Author(s):

W P Fawcett ◽

R E Wolf

Keyword(s):

Escherichia Coli ◽

Dna Binding ◽

Binding Site ◽

Dna Binding Site ◽

Definition Of ◽

Glucose 6 Phosphate Dehydrogenase

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Unliganded and Liganded Estrogen Receptors Protect against Cancer Invasion via Different Mechanisms

Molecular Endocrinology ◽

10.1210/mend.14.7.0492 ◽

2000 ◽

Vol 14 (7) ◽

pp. 999-1009 ◽

Cited By ~ 32

Author(s):

Nadine Platet ◽

Séverine Cunat ◽

Dany Chalbos ◽

Henri Rochefort ◽

Marcel Garcia

Keyword(s):

Breast Cancer ◽

Amino Acids ◽

Dna Binding ◽

Cancer Cells ◽

Zinc Finger ◽

Transcriptional Activation ◽

Breast Cancer Cells ◽

Cancer Invasion ◽

Specific Gene ◽

Matrigel Invasion

Abstract While estrogens are mitogenic in breast cancer cells, the presence of estrogen receptor α (ERα) clinically indicates a favorable prognosis in breast carcinoma. To improve our understanding of ERα action in breast cancer, we used an original in vitro method, which combines transient transfection and Matrigel invasion assays to examine its effects on cell invasiveness. ERα expression in MDA-MB-231 breast cancer cells reduced their invasiveness by 3-fold in the absence of hormone and by 7-fold in its presence. Integrity of hormone and DNA-binding domains and activating function 2 were required for estradiol-induced inhibition, suggesting that transcriptional activation of estrogen target genes was involved. In contrast, these domains were dispensable for hormone-independent inhibition. Analysis of deletion mutants of ERα indicated that amino acids 179–215, containing the N-terminal zinc finger of the DNA-binding domain, were required for ligand-independent receptor action. Among different members of the nuclear receptor family, only unliganded ERα and ERβ reduced invasion. Calreticulin, a Ca2+-binding protein that could interact with amino acids 206–211 of ERα, reversed hormone-independent ERα inhibition of invasion. However, since calreticulin alone also inhibited invasion, we propose that this protein probably prevents ERα interaction with another unidentified invasion-regulating factor. The inhibitor role of the unliganded ER was also suggested in three ERα-positive cell lines, where ERα content was inversely correlated with cell migration. We conclude that ERα protects against cancer invasion in its unliganded form, probably by protein-protein interactions with the N-terminal zinc finger region, and after hormone binding by activation of specific gene transcription.

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Multimerization via Its Myosin Domain Facilitates Nuclear Localization and Inhibition of Core Binding Factor (CBF) Activities by the CBFβ-Smooth Muscle Myosin Heavy Chain Myeloid Leukemia Oncoprotein

Molecular and Cellular Biology ◽

10.1128/mcb.22.23.8278-8291.2002 ◽

2002 ◽

Vol 22 (23) ◽

pp. 8278-8291 ◽

Cited By ~ 21

Author(s):

Tanawan Kummalue ◽

Jianrong Lou ◽

Alan D. Friedman

Keyword(s):

Amino Acids ◽

Smooth Muscle ◽

Dna Binding ◽

Nuclear Localization ◽

Heavy Chain ◽

Myeloid Leukemia ◽

Dna Binding Domain ◽

Core Binding Factor ◽

Binding Factor ◽

Muscle Myosin

ABSTRACT In CBFβ-SMMHC, core binding factor beta (CBFβ) is fused to the α-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFβ-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFβ-SMMHC(ΔACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the ΔACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G1 to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFβ domain to 149 or 83 C-terminal CBFβ-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFβ-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFβ-SMMHC(83C) did not. The majority of CBFβ-SMMHC and CBFβ-SMMHC(149C) was detected in the nucleus, whereas the ΔACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFβ-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFβ-SMMHC(ΔACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFβ-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G1 to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFβ-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFβ-SMMHC expression.

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