Drosophila transcription factor AP-2 in proboscis, leg and brain central complex development

We report loss- and gain-of-function analyses that identify essential roles in development for Drosophila transcription factor AP-2. A mutagenesis screen yielded 16 lethal point mutant alleles of dAP-2. Null mutants die as adults or late pupae with a reduced proboscis, severely shortened legs (~30% of normal length) lacking tarsal joints, and disruptions in the protocerebral central complex, a brain region critical for locomotion. Seven hypomorphic alleles constitute a phenotypic series yielding hemizygous adults with legs ranging from 40–95% of normal length. Hypomorphic alleles show additive effects with respect to leg length and viability; and several heteroallelic lines were established. Heteroallelic adults have moderately penetrant defects that include necrotic leg joints and ectopic growths (sometimes supernumerary antennae) invading medial eye territory. Several dAP-2 alleles with DNA binding domain missense mutations are null in hemizygotes but have dominant negative effects when paired with hypomorphic alleles. In wild-type leg primordia, dAP-2 is restricted to presumptive joints. Ectopic dAP-2 in leg discs can inhibit but not enhance leg elongation indicating that functions of dAP-2 in leg outgrowth are region restricted. In wing discs, ectopic dAP-2 cell autonomously transforms presumptive wing vein epithelium to ectopic sensory bristles, consistent with an instructive role in sensory organ development. These findings reveal multiple functions for dAP-2 during morphogenesis of feeding and locomotor appendages and their neural circuitry, and provide a new paradigm for understanding AP-2 family transcription factors.

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OP0314 DOCK8 MUTATIONS IN COVID-19 AND MIS-C CYTOKINE STORM SYNDROME

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.1865 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 192.1-192

Author(s):

R. Cron ◽

M. Zhang ◽

D. Absher ◽

J. Bridges ◽

A. Schnell ◽

...

Keyword(s):

Nk Cell ◽

Foamy Virus ◽

Dominant Negative ◽

Target Cells ◽

Missense Mutations ◽

Cytokine Storm ◽

Negative Effects ◽

Research Support ◽

Rare Mutations ◽

Lytic Function

Background:We recently identified DOCK8 as a novel gene associated with cytokine storm syndrome (CSS)1. Heterozygous missense mutations in DOCK8 diminish NK cell lytic function and contribute to increased pro-inflammatory cytokine production (CSS). CSS is a potential complication of COVID-19 with severe consequences2. Children are at risk of a SARS-CoV-2 post-infectious CSS, multisystem inflammatory syndrome in children (MIS-C)3. Host genetic factors associated with COVID-19 CSS and MIS-C CSS are unknown.Objectives:The goals are to identify and functionally study rare mutations in DOCK8 in patients with SARS-CoV-2 COVID-19 and MIS-C.Methods:To date, 16 adult patients enrolled in a COVID-19 CSS clinical trial at UAB had whole genome sequencing. Four (25%) had rare heterozygous DOCK8 mutations (3 missense, 1 intronic). A COVID-19 CSS adult patient in Seattle also had a DOCK8 missense mutation. In addition, DOCK8 missense mutations were identified in five children (UAB & Northwell) hospitalized with MIS-C. DOCK8 mutations, or wild-type (WT) sequence controls, were introduced into human NK-92 cells by FOAMY virus transduction. WT and mutant DOCK8-expressing NK-92 cells were incubated with K562 target cells and compared for cytolysis and degranulation (CD107a).Results:One COVID-19 patient DOCK8 mutation (Gly523Arg) reduced NK cell degranulation by 30% and cytolysis by 23% (n=3) (Figure 1). Similar studies of 3 MIS-C patients with DOCK8 missense mutations (Arg899Trp, Ala2Thr, Pro687Leu) revealed up to 31% reduced NK cell degranulation and 48% reduction in cytolysis by 3 distinct mutations (n=3). Two-way ANOVA analysis revealed statistically significant (p<0.05) differences in NK cell degranulation and lysis for four unique DOCK8 mutations.Conclusion:Heterozygous DOCK8 missense mutations may contribute to severe COVID-19 and MIS-C CSS by partial dominant-negative effects yielding decreased NK cell cytolysis.References:[1]Schulert GS, Cron RQ. The genetics of macrophage activation syndrome. Genes Immun 2020:21:169-181.[2] Cron RQ, Chatham WW. The rheumatologist’s role in COVID-19. J Rheumatol 2020:47:639-642.[3]Reiff D, Mannion ML, Samuy N, Scalici P, Cron RQ. Distinguishing active pediatric COVID-19 from MIS-C. Pediatr Rheumatol Online J, in press.Disclosure of Interests:Randy Cron Consultant of: SOBI, Novartis, Pfizer, Sironax, Grant/research support from: SOBI, Mingce Zhang: None declared, Remy Cron: None declared, Devin Absher: None declared, John Bridges: None declared, Amanda Schnell: None declared, Pavan Bhatraju: None declared, Anshul Vagrecha: None declared, Shannon Lozinsky: None declared, Suchitra Acharya: None declared, Carolyn Levy: None declared, Winn Chatham Grant/research support from: SOBI.

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Limited Availability of General Co-Repressors Uncovered in an Overexpression Context during Wing Venation in Drosophila melanogaster

Genes ◽

10.3390/genes11101141 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1141

Author(s):

Anja C. Nagel ◽

Dieter Maier ◽

Janika Scharpf ◽

Manuela Ketelhut ◽

Anette Preiss

Keyword(s):

Cell Fate ◽

Notch Pathway ◽

Careful Consideration ◽

Dominant Negative ◽

Limiting Factors ◽

Wing Venation ◽

Negative Effects ◽

Wing Vein ◽

Limited Availability ◽

H Protein

Cell fate is determined by the coordinated activity of different pathways, including the conserved Notch pathway. Activation of Notch results in the transcription of Notch targets that are otherwise silenced by repressor complexes. In Drosophila, the repressor complex comprises the transcription factor Suppressor of Hairless (Su(H)) bound to the Notch antagonist Hairless (H) and the general co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). The latter two are shared by different repressors from numerous pathways, raising the possibility that they are rate-limiting. We noted that the overexpression during wing development of H mutants HdNT and HLD compromised in Su(H)-binding induced ectopic veins. On the basis of the role of H as Notch antagonist, overexpression of Su(H)-binding defective H isoforms should be without consequence, implying different mechanisms but repression of Notch signaling activity. Perhaps excess H protein curbs general co-repressor availability. Supporting this model, nearly normal wings developed upon overexpression of H mutant isoforms that bound neither Su(H) nor co-repressor Gro and CtBP. Excessive H protein appeared to sequester general co-repressors, resulting in specific vein defects, indicating their limited availability during wing vein development. In conclusion, interpretation of overexpression phenotypes requires careful consideration of possible dominant negative effects from interception of limiting factors.

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Mechanisms of I Ks suppression in LQT1 mutants

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.6.h3003 ◽

2000 ◽

Vol 279 (6) ◽

pp. H3003-H3011 ◽

Cited By ~ 44

Author(s):

Laura Bianchi ◽

Silvia G. Priori ◽

Carlo Napolitano ◽

Krystyna A. Surewicz ◽

Adrienne T. Dennis ◽

...

Keyword(s):

Cell Surface ◽

Dominant Negative ◽

Delayed Rectifier ◽

Poor Correlation ◽

Type I ◽

Missense Mutations ◽

Wild Type ◽

Negative Effects ◽

Clinical Phenotypes ◽

First Time

Mutations in the cardiac potassium ion channel gene KCNQ1 (voltage-gated K+ channel subtype KvLQT1) cause LQT1, the most common type of hereditary long Q-T syndrome. KvLQT1 mutations prolong Q-T by reducing the repolarizing cardiac current [slow delayed rectifier K+ current ( I Ks )], but, for reasons that are not well understood, the clinical phenotypes may vary considerably even for carriers of the same mutation, perhaps explaining the mode of inheritance. At present, only currents expressed by LQT1 mutants have been studied, and it is unknown whether abnormal subunits are transported to the cell surface. Here, we have examined for the first time trafficking of KvLQT1 mutations and correlated the results with the I Ks currents that were expressed. Two missense mutations, S225L and A300T, produced abnormal currents, and two others, Y281C and Y315C, produced no currents. However, all four KvLQT1 mutations were detected at the cell surface. S225L, Y281C, and Y315C produced dominant negative effects on wild-type I Ks current, whereas the mutant with the mildest dysfunction, A300T, did not. We examined trafficking of a severe insertion deletion mutant Δ544 and detected this protein at the cell surface as well. We compared the cellular and clinical phenotypes and found a poor correlation for the severely dysfunctional mutations.

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Generation of Novel Knockin Mouse Lines Harboring Leukemia-Associated Point Mutations at AML1/Runx1 Gene Locus.

Blood ◽

10.1182/blood.v104.11.465.465 ◽

2004 ◽

Vol 104 (11) ◽

pp. 465-465 ◽

Cited By ~ 1

Author(s):

Yoko Fukushime-Nakase ◽

Mitsushige Nakao ◽

Tohru Sugimoto ◽

Tsukasa Okuda

Keyword(s):

Transcription Factor ◽

Gene Locus ◽

Point Mutations ◽

Dominant Negative ◽

Myelogenous Leukemia ◽

Wild Type ◽

Negative Effects ◽

Runt Domain ◽

Runx1 Gene ◽

Acute Myelogenous

Abstract Acute Myeloid Leukemia 1 (AML1; also called as Runt-related transcription factor 1: Runx1) encodes the DNA binding subunit of Core-Binding Factor (CBF) transcription factor complex which plays pivotal roles in several phases of hematopoietic regulation, including initial development of definitive hematopoiesis. AML1 is known as a frequent target of leukemia-associated chromosomal translocations, where chimeric AML1 genes with strong dominant-negative effects against normal CBF function are produced. Recently, point-mutations of the AML1 gene locus have also been reported to associate with sporadic cases of acute myelogenous leukemia or myelodysplastic syndromes, and pedigrees of Familial Platelet Disorder with Predisposition to Acute Myelogenous Leukemia (FPD/AML). Hot spots of the mutations are confined to within the Runt domain, which is the signature-motif of the AML1 molecules localized near the N-terminus and is responsible for both sequence-specific DNA binding and hetero-dimerization with CBFβ subunit. Biochemical examinations have so far demonstrated that these mutants lose their function as a transcription factor and that most of them also have dominant-negative effects at some extent. Indeed, three-dimensional analyses of the Runt domain structure revealed that frequent sites of the mutation, such as, R80, R139, R174, or R177, were mapped at residues important for the DNA-contact of the molecule while mutations of these residues left AML1’s ability to associate with CBFβ being un-affected, thus providing a molecular basis of their biochemical characteristics. In contrast, however, biological properties of the mutations are mostly left to be elucidated. In order to define the biologic consequences of the point-mutations at Runx1 gene locus within the context of entire mouse, we introduced each of the point-mutations, including R139Q, R174Q, or R177Q, into mouse germline by means of a gene-knockin approach. Heterozygous mice for each of the mutations were born healthy and grew up fertile as far as they were kept under specific pathogen-free conditions similarly to their wild-type siblings or to control mouse lines harboring a knockin allele with wild-type cDNA of Runx1. These observations indicate that the dominant-negative effects of the mutations, if any present, are not that strong as is the case for the AML1-MTG8 (ETO) chimeric gene which leads to mid-gestational death when transmitted to one allele of mouse germline. In addition, in contrast to the knockin of the wild-type cDNA which results in minimal phenotype in the homozygous progeny, homozygous mice for the R174Q allele were embryonic-lethal just like the simple disruption of this gene, clearly indicating that this leukemia-associated one amino acid substitution, arginine-to-glutamine at the 174th residue, abolished its biologic function completely. We are generating homozygous progeny by breeding for R139Q and R177Q alleles to further define their biologic consequences. In addition, examinations are currently undertaken to analyze if the heterozygous mice manifest any phenotype resulted from haplo-insufficiency along with possible dominant-negative effects by these mutations. These knockin mice should contribute to better understanding of the molecular mechanisms of AML1/Runx1 activity in normal and leukemic hematopoiesis.

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Pitt–Hopkins syndrome-associated mutations in TCF4 lead to variable impairment of the transcription factor function ranging from hypomorphic to dominant-negative effects

Human Molecular Genetics ◽

10.1093/hmg/dds112 ◽

2012 ◽

Vol 21 (13) ◽

pp. 2873-2888 ◽

Cited By ~ 53

Author(s):

Mari Sepp ◽

Priit Pruunsild ◽

Tõnis Timmusk

Keyword(s):

Transcription Factor ◽

Dominant Negative ◽

Negative Effects ◽

Factor Function

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Defining relative mutational difficulty to understand cancer formation and prevention

10.1101/789313 ◽

2019 ◽

Author(s):

Lin Shan ◽

Jiao Yu ◽

Zhengjin He ◽

Shishuang Chen ◽

Mingxian Liu ◽

...

Keyword(s):

Human Cancer ◽

Low Frequency ◽

Dominant Negative ◽

Missense Mutations ◽

Nucleotide Change ◽

P53 Mutations ◽

Loss Of Function ◽

Functional Importance ◽

Negative Effects ◽

Different Types

SummaryMost mutations in human cancer are low-frequency missense mutations, whose functional status remains hard to predict. Here we show that depending on the type of nucleotide change and the surrounding sequences, the tendency to generate each type of nucleotide mutations varies greatly, even by several hundred folds. Therefore, a cancer-promoting mutation may appear only in a small number of cancer cases, if the underlying nucleotide change is too difficult to generate. We propose a method that integrates both the original mutation counts and their relative mutational difficulty. Using this method, we can accurately predict the functionality of hundreds of low-frequency missense mutations in p53, PTEN and INK4A. Many loss-of-function p53 mutations with dominant negative effects were identified, and the functional importance of several regions in p53 structure were highlighted by this analysis. Furthermore, mutational difficulty analysis also points to potential means of cancer prevention. Our study not only established relative mutational difficulties for different types of mutations in human cancer, but also showed that by incorporating such parameter, we can bring new angles to understanding cancer formation and prevention.

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Oral Glucosamine Ameliorates Aggravated Neurological Phenotype in Mucopolysaccharidosis III Type C Mouse Model Expressing Misfolded HGSNAT Variant

10.1101/2021.08.26.457793 ◽

2021 ◽

Author(s):

Xuefang Pan ◽

Mahsa Taherzadeh ◽

Poulomee Bose ◽

Rachel Heon-Roberts ◽

Annie L.A. Nguyen ◽

...

Keyword(s):

Heparan Sulfate ◽

Missense Variant ◽

Dominant Negative ◽

Synaptic Proteins ◽

Missense Mutations ◽

Negative Effects ◽

Object Recognition Test ◽

Y Maze ◽

Neurological Phenotype

Objective: Over 55% of mucopolysaccharidosis IIIC (MPS IIIC) patients have at least one allelic missense variant responsible for misfolding of heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT). These variants are potentially treatable with pharmacological chaperones, such as a competitive HGSNAT inhibitor, glucosamine. Since the constitutive HGSNAT knockout mice, we generated previously cannot be used to test such strategy in vivo, we generated a novel model, the HgsnatP304L strain, expressing misfolded mutant HGSNAT with human missense mutation Pro311Leu (Pro304Leu in the mouse enzyme). Results: HgsnatP304L mice present deficits in short-term (novel object recognition test) and working/spatial (Y-maze test) memory at 4 months of age, 2-4 months earlier than previously described gene-targeted Hgsnat-Geo mice, which lack HGSNAT protein. HgsnatP304L mice also show increased severity of synaptic deficits in CA1 neurons, and accelerated course of CNS pathology including neuronal storage of heparan sulfate, accumulation of misfolded proteins, increase of simple gangliosides, and neuroinflammation as compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat-Geo neurons aggravated reduction of synaptic proteins. Memory deficits and majority of pathological changes in the brain were rescued in mice receiving daily doses of oral glucosamine. Interpretation: Altogether, our data for the first time demonstrate dominant-negative effects of the misfolded HGSNAT Pro304Leu variant and show that these effects are treatable by oral administration of glucosamine, suggesting that patients, affected with missense mutations preventing normal folding of the enzyme, could benefit from chaperone therapy.

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Robinow syndrome skeletal phenotypes caused by the WNT5AC83S variant are due to dominant interference with chondrogenesis

Human Molecular Genetics ◽

10.1093/hmg/ddz071 ◽

2019 ◽

Vol 28 (14) ◽

pp. 2395-2414

Author(s):

Sarah J Gignac ◽

Sara Hosseini-Farahabadi ◽

Takashi Akazawa ◽

Nathan J Schuck ◽

Katherine Fu ◽

...

Keyword(s):

Planar Cell Polarity ◽

Dominant Negative ◽

Missense Mutations ◽

Negative Effects ◽

Biochemical Assays ◽

Cartilage Differentiation ◽

Robinow Syndrome ◽

Variant Protein ◽

Bone Dimensions ◽

Human Wild Type

Abstract Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.

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The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3853 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3853-3865 ◽

Cited By ~ 173

Author(s):

L Galarneau ◽

J F Paré ◽

D Allard ◽

D Hamel ◽

L Levesque ◽

...

Keyword(s):

Transcription Factor ◽

Nuclear Receptor ◽

Single Copy ◽

Dominant Negative ◽

Orphan Nuclear Receptor ◽

Negative Effects ◽

Gene Encoding ◽

Amino Terminal ◽

Sequence Homologies ◽

Afp Promoter

The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

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