scholarly journals Adaptive Significance of ERα Splice Variants in Killifish (Fundulus heteroclitus) Resident in an Estrogenic Environment

Endocrinology ◽  
2016 ◽  
Vol 157 (6) ◽  
pp. 2294-2308 ◽  
Author(s):  
Kellie A. Cotter ◽  
Diane Nacci ◽  
Denise Champlin ◽  
Alan T. Yeo ◽  
Thomas D. Gilmore ◽  
...  
Keyword(s):  
Estrogen Receptor Alpha ◽  
Fundulus Heteroclitus ◽  
Splice Variants ◽  
Dominant Negative ◽  
Polluted Environment ◽  
Gene Encoding ◽  
Three Generations ◽  
Clean Environment ◽  
Heritable Change

The possibility that chronic, multigenerational exposure to environmental estrogens selects for adaptive hormone-response phenotypes is a critical unanswered question. Embryos/larvae of killifish from an estrogenic-polluted environment (New Bedford Harbor, MA [NBH]) compared with those from a reference site overexpress estrogen receptor alpha (ERα) mRNA but are hyporesponsive to estradiol. Analysis of ERα mRNAs in the two populations revealed differences in splicing of the gene encoding ERα (esr1). Here we tested the transactivation functions of four differentially expressed ERα mRNAs and tracked their association with the hyporesponsive phenotype for three generations after transfer of NBH parents to a clean environment. Deletion variants ERαΔ6 and ERαΔ6–8 were specific to NBH killifish, had dominant negative functions in an in vitro reporter assay, and were heritable. Morpholino-mediated induction of ERαΔ6 mRNA in zebrafish embryos verified its role as a dominant negative ER on natural estrogen-responsive promoters. Alternate long (ERαL) and short (ERαS) 5′-variants were similar transcriptionally but differed in estrogen responsiveness (ERαS ≫ ERαL). ERαS accounted for high total ERα expression in first generation (F1) NBH embryos/larvae but this trait was abolished by transfer to clean water. By contrast, the hyporesponsive phenotype of F1 NBH embryos/larvae persisted after long-term laboratory holding but reverted to a normal or hyper-responsive phenotype after two or three generations, suggesting the acquisition of physiological or biochemical traits that compensate for ongoing expression of negative-acting ERαΔ6 and ERαΔ6–8 isoforms. We conclude that a heritable change in the pattern of alternative splicing of ERα pre-mRNA is part of a genetic adaptive response to estrogens in a polluted environment.

Integration of FGF and TWIST in calvarial bone and suture development

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1845-1855 ◽  
Author(s):  
D.P. Rice ◽  
T. Aberg ◽  
Y. Chan ◽  
Z. Tang ◽  
P.J. Kettunen ◽  
...  
Keyword(s):  
Expression Analysis ◽  
Splice Variants ◽  
Bone Development ◽  
Dominant Negative ◽  
Calvarial Bone ◽  
Fgf Receptor ◽  
Helix Loop Helix ◽  
Suture Closure ◽  
Potential Ligand

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241–1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(−) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.

In Vitro Knock-Down of Myh9 Leads to a Myh9-Related Disorder Phenotype in Human Megakaryocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4596-4596
Author(s):  
Yolande Chen ◽  
Siham Boukour ◽  
Monia Romdhane ◽  
Ababacar Seye ◽  
Olivier Bluteau ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myosin Ii ◽  
In Vitro Study ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Gene Encoding ◽  
Related Disorder ◽  
Normal Platelet ◽  
Negative Effect

Abstract Abstract 4596 Normal platelet production is dependent on the formation of branched long cytoplasmic extensions, called proplatelets (PPT). Mutations of the Myh9 gene (encoding for the nonmuscle myosin heavy chain IIA) result in autosomal dominant disorders, where patients develop various degrees of macrothrombocytopenia, with sometimes glomerular impairment, hearing loss and cataracts. There has been questioning as to whether the mechanism for the macrothrombocytopenia is haploinsufficiency, or a dominant negative effect of the mutated gene. We performed an in vitro study to investigate PPF from patient megakaryocytes (MK). By this approach, a decrease in PPF from patient CD34 derived MKs was observed in comparison to normal cultured MK. Surprisingly this defect of PPF observed in patients was rescued by blebbistatin, an inhibitor of class II myosin. Immunofluorescence studies performed showed that besides clusterization of GPIb in patient's platelets, no major repartition abnormalities were seen in cultured MKs derived from patient's CD34 for myosin, actin, tubulin, vWF, and Rac (except in one patient where actin and Rac formed aggregates to some extend, in a small number of MKs). In order to better understand the role of myosin during normal and abnormal PPF, we used a shRNA strategy to disrupt the Myh9 expression during normal MK differentiation and compared shRNA-treated MKs with MKs derived from patient CD34. Megakaryocytes treated with a shRNA that knocks down the protein of about 50%, did not alter MK ploidization, but decreased in vitro PPF, as previously observed for cells issued from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as patient MKs. Addition of Blebbistatin to shRNA treated MKs led to an increase of PPF, suggesting that the remaining myosin II might be hyperactivated and inhibit PPF. Altogether this study strongly suggests that the thrombocytopenia of the Myh9 syndrome is essentially related to haploinsufficiency in myosin II. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Naturally occurring C-terminal splice variants of nuclear receptors

2009 ◽  
Vol 7 (1) ◽  
pp. nrs.07007 ◽  
Author(s):  
Michiel van der Vaart ◽  
Marcel J.M. Schaaf
Keyword(s):  
Nuclear Receptors ◽  
Splice Variants ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Aberrant Expression ◽  
C Terminus ◽  
Alternative Mrna Splicing ◽  
Negative Effect

Alternative mRNA splicing in the region encoding the C-terminus of nuclear receptors results in receptor variants lacking the entire ligand-binding domain (LBD), or a part of it, and instead contain a sequence of splice variant-specific C-terminal amino acids. A total of thirteen such splice variants have been shown to occur in vertebrates, and at least nine occur in humans. None of these receptor variants appear to be able to bind endogenous ligands and to induce transcription on promoters containing the response element for the respective canonical receptor variant. Interestingly, ten of these C-terminal splice variants have been shown to display dominant-negative activity on the transactivational properties of their canonical equivalent. Research on most of these splice variants has been limited, and the dominant-negative effect of these receptor variants has only been demonstrated in reporter assays in vitro, using transiently transfected receptors and reporter constructs. Therefore, the in vivo function and relevance of most C-terminal splice variants remains unclear. By reviewing the literature on the human glucocorticoid receptor β-isoform (hGRβ), we show that the dominant-negative effect of hGRβ is well established using more physiologically relevant readouts. The hGR β-isoform may alter gene transcription independent from the canonical receptor and increased hGRβ levels correlate with glucocorticoid resistance and the occurrence of several immune-related diseases. Thus, available data suggests that C-terminal splice variants of nuclear receptors act as dominant-negative inhibitors of receptor-mediated signaling in vivo, and that aberrant expression of these isoforms may be involved in the pathogenesis of a variety of diseases.

Flow Cytometry Analysis of Type IIB Procollagen as Quality Control of Chondrogenic Commitment of MSCs

2021 ◽  
Author(s):  
Emeline Perrier-Groult ◽  
Elisabeth Aubert-Foucher ◽  
Marielle Pasdeloup ◽  
Jérôme Lafont ◽  
Hugo Fabre ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Splice Variants ◽  
Type Ii ◽  
Flow Cytometry Analysis ◽  
Gene Encoding ◽  
Marrow Mesenchymal Stem Cells ◽  
Reliable Marker

Type II collagen is the major collagen protein in cartilage, synthesized as precursor forms (procollagens). Several splice variants of the gene encoding type II procollagen have been identified such as IIA and IIB isoforms. Interestingly, a shift from IIA to IIB transcripts has been reported to occur during cartilage development and during chondrogenic differentiation of mesenchymal stem cells in vitro. Thus, type IIB procollagen represents a reliable marker of chondrocyte differentiation. We characterized previously the first antibody (referred as anti-pNIIB52) able to selectively detect the IIB form of human type II procollagen in Western-blot or immunohistochemistry analysis. More recently, we used anti-pNIIB52 in flow cytometry to quantify chondrogenic induction of bone marrow-mesenchymal stem cells cultivated in agarose hydrogel, after release of the cells from the gel. Here, we use imaging flow cytometry and anti-pNIIB52 to visualize directly intracellular accumulation of type IIB procollagen in cells undergoing chondrogenesis. Our data together show that flow cytometry analysis using anti-pNIIB52 represents an efficient and rapid diagnostic tool of good chondrogenic conversion, at the cellular level.

scholarly journals Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1102
Author(s):  
Galina N. Raldugina ◽  
Sergey V. Evsukov ◽  
Liliya R. Bogoutdinova ◽  
Alexander A. Gulevich ◽  
Ekaterina N. Baranova
Keyword(s):  
Choline Oxidase ◽  
Culture Methods ◽  
Gene Encoding ◽  
Oxidase Gene ◽  
Bacterial Enzyme ◽  
Transgenic Lines ◽  
High Regeneration ◽  
Whole Plants ◽  
Implicit Response

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

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

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 633-645 ◽  
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.

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 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 ◽  
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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