The homeoprotein Xiro1 is required for midbrain-hindbrain boundary formation

Development ◽  
2002 ◽  
Vol 129 (7) ◽  
pp. 1609-1621 ◽  
Author(s):  
Alvaro Glavic ◽  
José Luis Gómez-Skarmeta ◽  
Roberto Mayor
Keyword(s):  
Fusion Proteins ◽  
Dominant Negative ◽  
Transcriptional Repressors ◽  
Wild Type ◽  
Expression Domain ◽  
Vitro Assay ◽  
Novel Interactions ◽  
Repressor Element ◽  
Molecular Nature

The isthmic organizer, which patterns the anterior hindbrain and midbrain, is one of the most studied secondary organizers. In recent years, new insights have been reported on the molecular nature of its morphogenetic activity. Studies in chick, mouse and zebrafish have converged to show that mutually repressive interactions between the homeoproteins encoded by Otx and Gbx genes position this organizer in the neural primordia. We present evidence that equivalent, in addition to novel, interactions between these and other genes operate in Xenopus embryos to position the isthmic organizer. We made use of fusion proteins in which we combined Otx2 or Gbx2 homeodomains with the E1A activation domain or the EnR repressor element which were then injected into embryos. Our results show that Otx2 and Gbx2 are likely to be transcriptional repressors, and that these two proteins repress each other transcription. Our experiments show that the interaction between these two proteins is required for the positioning of the isthmic organizer genes Fgf8, Pax2 and En2. In this study we also developed a novel in vitro assay for the study of the formation of this organizer. We show that conjugating animal caps previously injected with Otx2 and Gbx2 mRNAs recreate the interactions required for the induction of the isthmic organizer. We have used this assay to determine which cells produce and which cells receive the Fgf signal. Finally, we have added a novel genetic element to this process, Xiro1, which encode another homeoprotein. We show that the Xiro1 expression domain overlaps with territories expressing Otx2, Gbx2 and Fgf8. By expressing wild-type or dominant negative forms of Xiro1, we show that this gene activates the expression of Gbx2 in the hindbrain. In addition, Xiro1 is required in the Otx2 territory to allow cells within this region to respond to the signals produced by adjacent Gbx2 cells. Moreover, Xiro1 is absolutely required for Fgf8 expression at the isthmic organizer. We discuss a model where Xiro1 plays different roles in regulating the genetic cascade of interactions between Otx2 and Gbx2 that are necessary for the specification of the isthmic organizer.

Novel REST Truncation Mutations Causing Hereditary Gingival Fibromatosis

2021 ◽  
pp. 002203452199662
Author(s):  
J.T. Chen ◽  
C.H. Lin ◽  
H.W. Huang ◽  
Y.P. Wang ◽  
P.C. Kao ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Genetic Disorder ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Negative Effect ◽  
Localization Signal ◽  
Gingival Fibromatosis

Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

scholarly journals Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

scholarly journals Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding.

1992 ◽  
Vol 12 (12) ◽  
pp. 5581-5592 ◽  
Author(s):  
E Shaulian ◽  
A Zauberman ◽  
D Ginsberg ◽  
M Oren
Keyword(s):  
Dna Binding ◽  
Point Mutation ◽  
P53 Gene ◽  
Dominant Negative ◽  
Mutant P53 ◽  
Wild Type ◽  
Transforming Activity ◽  
Wild Type P53 ◽  
Negative Dominance

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

A Conserved Mechanism of Transcription Factor Competition Controls Hematopoietic Progenitor Self-Renewal.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 91-91
Author(s):  
Shane R. Horman ◽  
Chinamenveni S. Velu ◽  
Tristan Bourdeau ◽  
Avinash Baktula ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Conditional Deletion

Abstract An intrinsic mechanism of self-renewal is critical for the maintenance of hematopoietic stem cells (HSC), but this HSC function is extinguished during differentiation of progenitors. Here we show that the self-renewal capacity of hematopoietic progenitor cells is regulated through physical competition for occupancy of select DNA binding sites. Initially, we found that conditional deletion of the Growth factor independent-1 (Gfi1) gene results in the accumulation of abnormally persistent myeloid progenitors in vivo. Specifically, while germline Gfi1 deletion induces defective HSC self renewal and a block to granulopoiesis, we find that conditional deletion of Gfi1 induces a severe but transient block to neutrophil development with repopulation of the bone marrow by the remaining wild type HSC within 8 weeks post deletion. However, even though normal levels of granulocyte colony forming units (G-CFU) returned by 8 weeks post deletion, an abnormal Gfi1−/− myeloid progenitor remained in the bone marrow in vivo. Subsequently, we find in vitro that both wild-type bone marrow cells expressing Gfi1-dominant-negative mutants, and Gfi1−/− Lin- bone marrow contain cells that replate indefinitely. We hypothesized that Gfi1 is critical to extinguish self renewal in hematopoietic progenitors. In seemingly unrelated work, we discovered antagonism between the drosophila orthologs of Gfi1 and the Hoxa9/Pbx1/Meis1 transcription factor complex during drosophila embryo segmentation. We extended our drosophila findings to discover that a subset of mammalian DNA regulatory sequences encode DNA binding sites for both Gfi1 and Hoxa9/Pbx1/Meis1. These DNA sequences are able to bind either factor, and function as a molecular switch. Interestingly, composite Gfi1/ Hoxa9/Pbx1/Meis1 binding sites are present in the regulatory regions of the gene encoding Hoxa9. We note that Gfi1 expression is normally induced, while Hoxa9 expression is down-regulated, during the transition from common myeloid progenitor (CMP) to the granulocyte-monocyte progenitor (GMP). CMP have greater self renewal potential than GMP. Conditional deletion of Gfi1 in sorted CMP or GMP both increases Hoxa9 expression and generates progenitors capable of replating indefinitely in vitro. Thus, Gfi1 is critical to limit self renewal in these progenitors. Deregulated Hoxa9 expression or activity appears pivotal to this new Gfi1-null phenotype, because Gfi1 dominant-negative mutants immortalize wild-type (or Hoxa7−/−) but not Hoxa9−/− bone marrow cells in vitro. An abnormal gain of self-renewal can unleash the leukemic potential of progenitor cells. We find that both limiting Gfi1 gene dosage and expression of Gfi1 dominant-negative mutants significantly increases Nup98-Hoxa9-mediated colony formation. In contrast, forced expression of Gfi1 prevents Nup98-Hoxa9 immortalization. Notably, the expression of Hoxa9 (independent of cases with Nup98-Hoxa9 fusions) has been reported to be of significant prognostic value in human acute myeloid leukemia. In conclusion, Gfi1 and the Hoxa9/Pbx1/Meis1 complex compete to control the expression of genes (such as Hoxa9) which are critical to extinguish self renewal and limit the leukemogenic potential of hematopoietic progenitors. The antagonism between these transcription factor complexes is conserved from drosophila segment formation to mammalian hematopoietic progenitor biology.

scholarly journals Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

2004 ◽  
Vol 11 (1) ◽  
pp. 21-28 ◽  
Author(s):  
James J. McSharry ◽  
Ann C. McDonough ◽  
Betty A. Olson ◽  
George L. Drusano
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Virus Yield ◽  
Drug Resistant ◽  
Wild Type ◽  
Vitro Assay ◽  
Drug Concentrations ◽  
Clinical Resistance ◽  
Na Gene

ABSTRACT A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.

JAK kinases promote invasiveness in VHL-mediated renal cell carcinoma by a suppressor of cytokine signaling-regulated, HIF-independent mechanism

2007 ◽  
Vol 293 (6) ◽  
pp. F1836-F1846 ◽  
Author(s):  
Karen L. Wu ◽  
Hui Miao ◽  
Shenaz Khan
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Dominant Negative ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Cancer Syndrome ◽  
Independent Mechanism ◽  
Vhl Disease

von Hippel-Lindau (VHL) disease is a cancer syndrome, which includes renal cell carcinoma (RCC), and is caused by VHL mutations. Most, but not all VHL phenotypes are due to failure of mutant VHL to regulate constitutive proteolysis of hypoxia-inducible factors (HIFs). Janus kinases (JAK1, 2, 3, and TYK2) promote cell survival and proliferation, processes tightly controlled by SOCS proteins, which have sequence and structural homology to VHL. We hypothesized that in VHL disease, RCC pathogenesis results from enhanced SOCS1 degradation, leading to upregulated JAK activity. We find that baseline JAK2, JAK3, and TYK2 activities are increased in RCC cell lines, even after serum deprivation or coincubation with cytokine inhibitors. Furthermore, JAK activity is sustained in RCC stably expressing HIF2α shRNA. Invasion through Matrigel and migration in wound-healing assays, in vitro correlates of metastasis, are significantly greater in VHL mutant RCC compared with wild-type cells, and blocked by dominant-negative JAK expression or JAK inhibitors. Finally, we observe enhanced SOCS2/SOCS1 coprecipitation and reduced SOCS1 expression due to proteasomal degradation in VHL-null RCC compared with wild-type cells. The data support a new HIF-independent mechanism of RCC metastasis, whereby SOCS2 recruits SOCS1 for ubiquitination and proteasome degradation, which lead to unrestricted JAK-dependent RCC invasion. In addition to commonly proposed RCC treatment strategies that target HIFs, our data suggest that JAK inhibition represents an alternative therapeutic approach.

scholarly journals Biochemical and Mutational Analyses of AcuA, the Acetyltransferase Enzyme That Controls the Activity of the Acetyl Coenzyme A Synthetase (AcsA) in Bacillus subtilis

2008 ◽  
Vol 190 (14) ◽  
pp. 5132-5136 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Effective Means ◽  
Wild Type ◽  
Modification System ◽  
Vitro Assay ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.

scholarly journals Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

2013 ◽  
Vol 79 (15) ◽  
pp. 4727-4733 ◽  
Author(s):  
Kathrin Emmi Scholz ◽  
Benita Kopka ◽  
Astrid Wirtz ◽  
Martina Pohl ◽  
Karl-Erich Jaeger ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fusion Proteins ◽  
Enzyme Stability ◽  
Enzyme Analysis ◽  
Phase System ◽  
Wild Type ◽  
Hydroxynitrile Lyase ◽  
Fluorescent Reporter

ABSTRACTHydroxynitrile lyase fromArabidopsis thaliana(AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescencein vivoandin vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almost 2 orders of magnitude-increased half-lives in the weakly acidic pH range compared to findings for the wild-type enzyme. Analysis of the quaternary structure of the respective FbFP-AtHNL fusion proteins suggested that this increased stability is apparently caused by oligomerization mediated via the FbFP tag. Moreover, the increased stability of the fusion proteins enabled the efficient synthesis of (R)-mandelonitrile in an aqueous-organic two-phase system at a pH of <5. Remarkably, (R)-mandelonitrile synthesis is not possible using wild-typeAtHNL under the same conditions due to the inherent instability of this enzyme below pH 5. The fusion strategy presented here reveals a surprising means for the stabilization of enzymes and stresses the importance of a thoroughin vitrocharacterization ofin vivo-employed fluorescent fusion proteins.

scholarly journals The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

1998 ◽  
Vol 143 (2) ◽  
pp. 523-532 ◽  
Author(s):  
Janne Balsamo ◽  
Carlos Arregui ◽  
TinChung Leung ◽  
Jack Lilien
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Wild Type ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
Cytoplasmic Proteins ◽  
Inactive Form

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.

scholarly journals Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7

1999 ◽  
Vol 67 (5) ◽  
pp. 2225-2232 ◽  
Author(s):  
Gregory Govoni ◽  
François Canonne-Hergaux ◽  
Cheryl G. Pfeifer ◽  
Sandra L. Marcus ◽  
Scott D. Mills ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Functional Expression ◽  
Integral Membrane Protein ◽  
Biochemical Properties ◽  
Raw264.7 Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Vitro Assay

ABSTRACT Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes.Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-typeNramp1G169 cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1D169 allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane ofSalmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defectiveS. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.

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