DNA-binding domain mutations in SMAD genes yield dominant-negative proteins or a neomorphic protein that can activate WG target genes in Drosophila

ABSTRACT Human tumor suppressor p53 is a sequence-specific master regulatory transcription factor that targets response elements (REs) in many genes. p53 missense mutations in the DNA-binding domain are often cancer associated. As shown with systems based on the yeast Saccharomyces cerevisiae, p53 mutants can alter the spectra and intensities of transactivation from individual REs. We address directly in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA-binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV or ionizing radiation, or spontaneously. These patterns reflected changes in the spectra and activities of target genes, as demonstrated for P21, MDM2, BAX, and MSH2. Thus, as originally proposed for “master genes of diversity,” p53 mutations in human cells can differentially influence target gene transactivation, resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy.

Download Full-text

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

Nature ◽

10.1038/nature06096 ◽

2007 ◽

Vol 448 (7157) ◽

pp. 1058-1062 ◽

Cited By ~ 683

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

Download Full-text

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

Development ◽

10.1242/dev.129.11.2761 ◽

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.

Download Full-text

An IFN regulatory factor-2 DNA-binding domain dominant negative mutant exhibits altered cell growth and gene expression

Oncogene ◽

10.1038/sj.onc.1203435 ◽

2000 ◽

Vol 19 (11) ◽

pp. 1411-1418 ◽

Cited By ~ 3

Author(s):

Y R Rubinstein ◽

P H Driggers ◽

V V Ogryzko ◽

A M Thornton ◽

K Ozato ◽

...

Keyword(s):

Gene Expression ◽

Cell Growth ◽

Dna Binding ◽

Binding Domain ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dna Binding Domain ◽

Regulatory Factor ◽

Altered Cell ◽

Negative Mutant

Download Full-text

trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3154 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3154-3163 ◽

Cited By ~ 72

Author(s):

J H Küpper ◽

M Müller ◽

M K Jacobson ◽

J Tatsumi-Miyajima ◽

D L Coyle ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Gamma Irradiation ◽

Cellular Proliferation ◽

Molecular Genetic ◽

Genetic System ◽

Binding Domain ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dna Binding Domain

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.

Download Full-text

Selective transcription of p53 target genes by zinc finger–p53 DNA binding domain chimeras

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/j.bbaexp.2004.09.011 ◽

2004 ◽

Vol 1681 (1) ◽

pp. 15-27 ◽

Cited By ~ 5

Author(s):

D. Falke ◽

M.H. Fisher ◽

R.L. Juliano

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Target Genes ◽

Binding Domain ◽

Dna Binding Domain ◽

P53 Target Genes

Download Full-text

Faculty Opinions recommendation of Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1091900.596181 ◽

2008 ◽

Author(s):

Francisco A Bonilla

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Dominant Negative ◽

Dna Binding Domain ◽

Hyper Ige Syndrome ◽

Dominant Negative Mutations

Download Full-text

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

Blood ◽

10.1182/blood.v116.21.25.25 ◽

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.

Download Full-text

Structure of the DNA-binding domain of the response regulator SaeR fromStaphylococcus aureus

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715010287 ◽

2015 ◽

Vol 71 (8) ◽

pp. 1768-1776 ◽

Cited By ~ 8

Author(s):

Xiaojiao Fan ◽

Xu Zhang ◽

Yuwei Zhu ◽

Liwen Niu ◽

Maikun Teng ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Response Regulator ◽

Regulatory System ◽

Binding Domain ◽

Dna Binding Domain ◽

Promoter Regions ◽

Winged Helix

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors inStaphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeRDBD) was solved at 2.5 Å resolution. The structure reveals that SaeRDBDexists as a monomer and has the canonical winged helix–turn–helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix–turn–helix module were verified to be important for binding to the P1 promoterin vitroand for the physiological function of SaeRin vivo.

Download Full-text