Dominant Negative Form of PAX5 Is Generated by Multimerization of Its DNA Binding Domain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 25-25
Author(s):  
Norihiko Kawamata ◽  
Mario Pennella ◽  
Jennifer Woo ◽  
Arnold Berk ◽  
H. Phillip Koeffler
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Binding Affinity ◽  
Alpha Helix ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Negative Effect ◽  
Dna Binding Affinity

Abstract Abstract 25 We have previously cloned a number of fusion genes involving PAX5 in acute lymphoblastic leukemia (ALL) (Kawamata N. et al. PNAS, 2008). All of these fusion products exerted a dominant negative effect over the wild-type PAX5. One of these fusion PAX5 proteins, PAX5-C20orf112, was generated by the fusion between the DNA binding domain of PAX5 (PAX5DB) and the C-terminal end of C20orf112. To find the mechanism of the dominant negative effect of the PAX5-C20 fusion, we performed Fluorescence Recovery After Photobleaching (FRAP) assay using PAX5-C20 and PAX5wt constructs connected with Yellow Fluorescence Proteins (YFP). Results showed extremely strong DNA binding affinity of PAX5-C20 compared to PAX5wt. FRAP experiments using deletion mutants of PAX5-C20 showed that both the DNA binding domain and C-terminal alpha-helix region of C20 were indispensable for this strong binding to DNA. Fluorescence Resonance Energy Transfer (FRET) assay, Bi-molecule Fluorescence Complementation (BiFC) assay, and co-immunoprecipitation assay showed that C-terminal end of C20 containing an alpha-helix region encodes a homo-multimerization domain. To confirm that homo-multimerization of PAX5DB increases DNA binding affinity, PAX5DB was fused to the inducible dimerization motif of FKBP (PAX5DB-FK). PAX5DB-FK increased its DNA binding affinity with addition of FKBP ligand inducing homo-dimerization. We also fused PAX5DB to homo-dimerization of MAX (bHLH domain), or tetramerization domain of TP53. FRAP assays showed that homo-dimerization increased its DNA binding activity, and homo-tetramerization further increased its DNA binding and its dominant negative effect over PAX5wt. PAX5-ETV6, also a common fusion protein in ALL, exerts a dominant negative effect over PAX5wt. The ETV6 region of this fusion protein has a multimerization (SAM) domain and the PAX5DB-ETV6SAM mutant protein also showed a dominant negative effect and strong binding to DNA. Importantly, in further studies, co-expression of PAX5-C20 and the YFP-C20-alpha-helix-region diminished the strong DNA binding and the dominant negative activity of the fusion protein. Our data show that multimerization of the DNA binding domain of PAX5 induces strong DNA binding activity, leading to its dominant negative effect over the wild type transcription factor. We believe this represents a new paradigm explaining how a number of fusion genes containing a DB motif from one protein and a multimerization motif from the other partner, can behave in a dominant negative fashion. These observations suggest that peptides/ small molecules inhibiting the multimerization of these oncogenic fusion transcription factors can be promising reagents for treating cancers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals p53 Isoforms and Their Implications in Cancer

Cancers ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 288 ◽  
Author(s):  
Maximilian Vieler ◽  
Suparna Sanyal
Keyword(s):  
Dna Binding ◽  
Tp53 Gene ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Evolutionary Significance ◽  
Dna Binding Domain ◽  
Negative Effect ◽  
P53 Isoforms ◽  
P53 Inactivation

In this review we focus on the major isoforms of the tumor-suppressor protein p53, dysfunction of which often leads to cancer. Mutations of the TP53 gene, particularly in the DNA binding domain, have been regarded as the main cause for p53 inactivation. However, recent reports demonstrating abundance of p53 isoforms, especially the N-terminally truncated ones, in the cancerous tissues suggest their involvement in carcinogenesis. These isoforms are ∆40p53, ∆133p53, and ∆160p53 (the names indicate their respective N-terminal truncation). Due to the lack of structural and functional characterizations the modes of action of the p53 isoforms are still unclear. Owing to the deletions in the functional domains, these isoforms can either be defective in DNA binding or more susceptive to altered ‘responsive elements’ than p53. Furthermore, they may exert a ‘dominant negative effect’ or induce more aggressive cancer by the ‘gain of function’. One possible mechanism of p53 inactivation can be through tetramerization with the ∆133p53 and ∆160p53 isoforms—both lacking part of the DNA binding domain. A recent report and unpublished data from our laboratory also suggest that these isoforms may inactivate p53 by fast aggregation—possibly due to ectopic overexpression. We further discuss the evolutionary significance of the p53 isoforms.

scholarly journals The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential.

1996 ◽  
Vol 16 (3) ◽  
pp. 1203-1211 ◽  
Author(s):  
K Takebayashi ◽  
K Chida ◽  
I Tsukamoto ◽  
E Morii ◽  
H Munakata ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
T Antigen ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Negative Effect

In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T-antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF.

scholarly journals Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors from Escherichia coli and Bacillus stearothermophilus

2002 ◽  
Vol 184 (23) ◽  
pp. 6602-6614 ◽  
Author(s):  
Anahit Ghochikyan ◽  
Iovka Miltcheva Karaivanova ◽  
Michèle Lecocq ◽  
Patricia Vusio ◽  
Marie-Claire Arnaud ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Affinity ◽  
Bacillus Stearothermophilus ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
E Coli ◽  
Linker Peptide ◽  
Dna Binding Affinity ◽  
Oligomerization Domain

ABSTRACT Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the α4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the α4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

scholarly journals Role of GCR2 in transcriptional activation of yeast glycolytic genes.

1992 ◽  
Vol 12 (9) ◽  
pp. 3834-3842 ◽  
Author(s):  
H Uemura ◽  
Y Jigami
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Gene Products ◽  
Lacz Expression ◽  
Genetic Method ◽  
Potential Interactions

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome

Nature ◽  
2007 ◽  
Vol 448 (7157) ◽  
pp. 1058-1062 ◽  
Author(s):  
Yoshiyuki Minegishi ◽  
Masako Saito ◽  
Shigeru Tsuchiya ◽  
Ikuya Tsuge ◽  
Hidetoshi Takada ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dna Binding Domain ◽  
Hyper Ige Syndrome ◽  
Dominant Negative Mutations

Characterization of a dominant negativeC. elegansTwist mutant protein with implications for human Saethre-Chotzen syndrome

Development ◽  
2002 ◽  
Vol 129 (11) ◽  
pp. 2761-2772
Author(s):  
Ann K. Corsi ◽  
Thomas M. Brodigan ◽  
Erik M. Jorgensen ◽  
Michael Krause
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Dna Binding Domain ◽  
Mesodermal Cell ◽  
C Elegans ◽  
Dna Binding Activity ◽  
A Charge

Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The Caenorhabditis elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or heterodimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus.

Sign in / Sign up

Export Citation Format

Share Document