Multiple environmental signaling pathways control the differentiation of RORγt-expressing regulatory T cells

AbstractRORγt-expressing Tregs form a specialized subset of intestinal CD4+ Foxp3+ cells which is essential to maintain gut homeostasis and tolerance to commensal microbiota. Recently, c-Maf emerged as a critical factor in the regulation of RORγt expression in Tregs. However, aside from c-Maf signaling, the signaling pathways involved in the differentiation of RORγt+ Tregs and their possible interplay with c-Maf in this process are largely unknown. We show that RORγt+ Treg development is controlled by positive as well as negative signals. Along with c-Maf signaling, signals derived from a complex microbiota, as well as IL-6/STAT3- and TGF-β-derived signals act in favor of RORγt+ Treg development. Ectopic expression of c-Maf did not rescue RORγt expression in STAT3-deficient Tregs, indicating the presence of additional effectors downstream of STAT3. Moreover, we show that an inflammatory IFN-γ/STAT1 signaling pathway acts as a negative regulator of RORγt+ Treg differentiation in a c-Maf independent fashion.These data thus argue for a complex integrative signaling network that finely tunes RORγt expression in Tregs. The finding that type 1 inflammation impedes RORγt+ Treg development even in the presence of an active IL-6/STAT3 pathway further suggests a dominant negative effect of STAT1 over STAT3 in this process.

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Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired

Development ◽

10.1242/dev.122.9.2709 ◽

1996 ◽

Vol 122 (9) ◽

pp. 2709-2718 ◽

Cited By ~ 4

Author(s):

P. Miskiewicz ◽

D. Morrissey ◽

Y. Lan ◽

L. Raj ◽

S. Kessler ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Ectopic Expression ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Paired Domain ◽

Mutant Proteins ◽

Segment Polarity ◽

Negative Effect

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.

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Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3

Development ◽

10.1242/dev.121.3.755 ◽

1995 ◽

Vol 121 (3) ◽

pp. 755-765 ◽

Cited By ~ 19

Author(s):

S.B. Pierce ◽

D. Kimelman

Keyword(s):

Ectopic Expression ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Axis Formation ◽

Serine Threonine Kinase ◽

Spemann Organizer ◽

Isolation And Characterization ◽

Secondary Axis ◽

Dorsal Axis ◽

Negative Effect

Dorsal axis formation in the Xenopus embryo can be induced by the ectopic expression of several Wnt family members. In Drosophila, the protein encoded by the Wnt family gene, wingless, signals through a pathway that antagonizes the effects of the serine/threonine kinase zeste-white 3/shaggy. We describe the isolation and characterization of a Xenopus homolog of zeste-white 3/shaggy, Xgsk-3. A kinase-dead mutant of Xgsk-3, Xgsk-3K-->R, has a dominant negative effect and mimics the ability of Wnt to induce a secondary axis by induction of an ectopic Spemann organizer. Xgsk-3K-->R, like Wnt, induces dorsal axis formation when expressed in the deep vegetal cells, which do not contribute to the axis. These results indicate that the dorsal fate is actively repressed by Xgsk-3, which must be inactivated for dorsal axis formation to occur. Furthermore, our work suggests that the effects of Xgsk-3K-->R are mediated by an additional intercellular signal.

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Truncation of TRIM5 in the Feliformia Explains the Absence of Retroviral Restriction in Cells of the Domestic Cat

Journal of Virology ◽

10.1128/jvi.00670-09 ◽

2009 ◽

Vol 83 (16) ◽

pp. 8270-8275 ◽

Cited By ~ 42

Author(s):

William A. McEwan ◽

Torsten Schaller ◽

Laura M. Ylinen ◽

Margaret J. Hosie ◽

Greg J. Towers ◽

...

Keyword(s):

Mammalian Species ◽

Ectopic Expression ◽

Binding Domain ◽

Dominant Negative ◽

Domestic Cat ◽

Dominant Negative Effect ◽

Negative Effect ◽

In Cells

ABSTRACT TRIM5α mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5α. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

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A Dominant Negative Effect of eth-1r, a Mutant Allele of the Neurospora crassa S-Adenosylmethionine Synthetase-Encoding Gene Conferring Resistance to the Methionine Toxic Analogue Ethionine

Genetics ◽

10.1093/genetics/144.4.1455 ◽

1996 ◽

Vol 144 (4) ◽

pp. 1455-1462

Author(s):

José L Barra ◽

Mario R Mautino ◽

Alberto L Rosa

Keyword(s):

Neurospora Crassa ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Resistant Phenotype ◽

Negative Effect ◽

Encoding Gene ◽

Adenosylmethionine Synthetase ◽

Adomet Synthetase ◽

Identical Gene

eth-1r a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48 → N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1 +/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1 +/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1 + and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1 +/eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1 +/eth-1r AdoMet synthetase molecules.

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Evidence for a dominant‐negative effect of a missense mutation in the SERPING1 gene responsible for hereditary angioedema type I

The Journal of Dermatology ◽

10.1111/1346-8138.15930 ◽

2021 ◽

Author(s):

Shuichiro Yasuno ◽

Osamu Ansai ◽

Ryota Hayashi ◽

Sawako Nakamura ◽

Yutaka Shimomura

Keyword(s):

Missense Mutation ◽

Hereditary Angioedema ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Type I ◽

Negative Effect ◽

Serping1 Gene

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Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽

10.1093/genetics/162.2.633 ◽

2002 ◽

Vol 162 (2) ◽

pp. 633-645 ◽

Cited By ~ 2

Author(s):

Guido Cuperus ◽

David Shore

Keyword(s):

Protein A ◽

Dominant Negative ◽

Class I ◽

Dominant Negative Effect ◽

Rna Pol Ii ◽

Interacting Protein ◽

Nucleosome Remodeling ◽

Negative Effect ◽

Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

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

Blood ◽

10.1182/blood.v93.12.4154 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4154-4166 ◽

Cited By ~ 80

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.

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Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies

Antibiotics ◽

10.3390/antibiotics10070830 ◽

2021 ◽

Vol 10 (7) ◽

pp. 830

Author(s):

Prasangi Rajapaksha ◽

Isoiza Ojo ◽

Ling Yang ◽

Ankit Pandeya ◽

Thilini Abeywansha ◽

...

Keyword(s):

Efflux Pump ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Assembly Process ◽

Multidrug Efflux ◽

E Coli ◽

Multidrug Efflux Pumps ◽

Negative Effect ◽

Dynamic Assembly ◽

Pump Assembly

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

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The Diagnostic Journey of a Patient with Prader–Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature

Genes ◽

10.3390/genes12060875 ◽

2021 ◽

Vol 12 (6) ◽

pp. 875

Author(s):

Karlijn Pellikaan ◽

Geeske M. van Woerden ◽

Lotte Kleinendorst ◽

Anna G. W. Rosenberg ◽

Bernhard Horsthemke ◽

...

Keyword(s):

Intellectual Disability ◽

Single Nucleotide Polymorphism Array ◽

Genetic Defect ◽

Missense Variant ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Pituitary Hormone ◽

Negative Effect

Prader–Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader–Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the ‘Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations’ (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype–phenotype relationship in PWS.

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