scholarly journals JAK2 is required for induction of the murine DUB-1 gene.

1997 ◽  
Vol 17 (6) ◽  
pp. 3364-3372 ◽  
Author(s):  
R Jaster ◽  
Y Zhu ◽  
M Pless ◽  
S Bhattacharya ◽  
B Mathey-Prevot ◽  
...  
Keyword(s):  
Amino Acid ◽  
Target Genes ◽  
Dominant Negative ◽  
Early Gene ◽  
Deubiquitinating Enzyme ◽  
Enhancer Element ◽  
Dominant Negative Form ◽  
C Subunit ◽  
Dose Dependent ◽  
Negative Form

Cytokine receptors activate multiple signal transduction pathways, resulting in the induction of specific target genes. We have recently identified a hematopoietic cell-specific immediate-early gene, DUB-1, that encodes a growth-regulatory deubiquitinating enzyme. The DUB-1 gene contains a 112-bp enhancer element that is specifically induced by the beta c subunit of the interleukin-3 (IL-3) receptor. To investigate the mechanism of DUB-1 induction, we examined the effects of dominant-negative forms of JAK kinases, STAT transcription factors, and Raf-1 in transient transfection assays. In Ba/F3 cells, IL-3 induced a dose-dependent activation of DUB-1-luciferase (luc) and GAS-luc reporter constructs. A dominant-negative form of JAK2 (truncated at amino acid 829) inhibited the induction of DUB-1-luc and GAS-luc by IL-3. A dominant-negative form of STAT5 (truncated at amino acid 650) inhibited the induction of GAS-luc but not DUB-1-luc. A dominant-negative form of Raf-1 inhibited the induction of DUB-1-luc but had no effect on the induction of GAS-luc by IL-3. The requirement for JAK2 in the stimulation of the DUB-1 enhancer was further supported by the suppression of DUB-1 induction in Ba/F3 cells stably expressing the dominant-negative JAK2 polypeptide. We hypothesize that IL-3 activates a JAK2/Raf-1 signaling pathway that is required for DUB-1 induction and is independent of STAT5.

ChIP-on-Chip Analysis Using Dominant Negative Form of PAX5 Fusion Gene Revealed Genes Associated with Tumorigenesis Were Directly Regulated by PAX5 in a Human B Cell Line, NALM6

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3585-3585
Author(s):  
Norihiko Kawamata ◽  
Fabienne Isken ◽  
Stefanie Goellner ◽  
C. Müller-Tidow ◽  
H. Phillip Koeffler
Keyword(s):  
B Cell ◽  
Specific Antibody ◽  
Target Genes ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Wild Type ◽  
Dominant Negative Form ◽  
On Chip ◽  
Chip Analysis ◽  
Negative Form

Abstract PAX5 is a transcriptional factor playing an important role in B-cell development. Overexpression of PAX5 induced by translocation to the enhancer region of immunoglobulin heavy chain gene occurs in non-Hodgkin lymphomas (NHL), suggesting that PAX5 can be also associated with development of NHL. To identify genes associated with tumorigenesis in malignancies overexpressing PAX5, we performed ChIP-on-chip analysis using PAX5 specific antibody. Non-specifically immunoprecipitated DNA by antibodies can cause false positive results using ChIP-on-chip analysis (background). To reduce the background in ChIP-on chip analysis, we used a dominant negative form of PAX5 and a wild-type PAX5 specific antibody for our ChIP-on-chip analysis. We have previously found a PAX5 chimeric protein expressed in acute lymphoblastic leukemia in which the C-terminal end of PAX5 was replaced by C20ORF112 protein (Kawamata N et al, Proc Natl Acad Sci U S A. Aug. 12, 2008). We have also found that this chimeric protein behaved in a dominant negative fashion over the wild-type PAX5 and suppressed expression of target genes of wild-type PAX5. PAX5 chimeric protein can compete with wild-type PAX5 for binding on the promoter region of direct down-stream target genes. To identify the genes directly regulated by PAX5 in human B-cells, we transfected the dominant-negative form of PAX5 chmeric protein, PAX5-C20ORF112 (PAX5-C20S) into NALM6 human B-cell leukemia cells which constitutively express abundant PAX5. Transfected cells were collected and chromatin immunoprecipitation (ChIP) assay was performed using PAX5 C-terminal specific antibody which can recognize only wild-type PAX5, but not the chimeric PAX5 protein, PAX5C20S. As a control, we also performed ChIP assay using NALM6 cells transfected with an empty vector. Immunoprecipitated DNA was recovered and amplified using the whole genome amplification technique. The DNAs were hybridized with oligonucleotide probes containing the promoter regions of the human genome. The levels of hybridized DNA were quantified and genes directly bound by PAX5 were identified. Comparison between NALM6 cells transfected with the empty vector and PAX5C20S significantly reduced the background and allowed identification of genes directly regulated by PAX5 in NALM6, including BUB1B, SSSCA1, CEP68, and BAG1. BUB1B, CEP68 and SSSCA1 are proteins involved in mitosis; BAG1 is a protein associated with apoptosis. Dysregulation of these genes by overexpressed PAX5 may be associated with development of B-cell malignancies.

scholarly journals Pax6 Regulates the Proglucagon Processing Enzyme PC2 and Its Chaperone 7B2

2009 ◽  
Vol 29 (8) ◽  
pp. 2322-2334 ◽  
Author(s):  
Liora S. Katz ◽  
Yvan Gosmain ◽  
Eric Marthinet ◽  
Jacques Philippe
Keyword(s):  
Target Genes ◽  
Dominant Negative ◽  
Prohormone Convertase ◽  
Endocrine Differentiation ◽  
Processing Enzyme ◽  
Dominant Negative Form ◽  
Prohormone Convertase 2 ◽  
Interfering Rna ◽  
Negative Form

ABSTRACT Pax6 is important in the development of the pancreas and was previously shown to regulate pancreatic endocrine differentiation, as well as the insulin, glucagon, and somatostatin genes. Prohormone convertase 2 (PC2) is the main processing enzyme in pancreatic α cells, where it processes proglucagon to produce glucagon under the spatial and temporal control of 7B2, which functions as a molecular chaperone. To investigate the role of Pax6 in glucagon biosynthesis, we studied potential target genes in InR1G9 α cells transfected with Pax6 small interfering RNA and in InR1G9 clones expressing a dominant-negative form of Pax6. We now report that Pax6 controls the expression of the PC2 and 7B2 genes. By binding and transactivation studies, we found that Pax6 indirectly regulates PC2 gene transcription through cMaf and Beta2/NeuroD1 while it activates the 7B2 gene both directly and indirectly through the same transcription factors, cMaf and Beta2/NeuroD1. We conclude that Pax6 is critical for glucagon biosynthesis and processing by directly and indirectly activating the glucagon gene through cMaf and Beta2/NeuroD1, as well as the PC2 and 7B2 genes.

scholarly journals Repression of c-myc Is Necessary but Not Sufficient for Terminal Differentiation of B Lymphocytes In Vitro

2000 ◽  
Vol 20 (23) ◽  
pp. 8684-8695 ◽  
Author(s):  
Kuo-I Lin ◽  
Yi Lin ◽  
Kathryn Calame
Keyword(s):  
Cell Differentiation ◽  
Target Genes ◽  
Ectopic Expression ◽  
Constitutive Expression ◽  
Dominant Negative ◽  
B Cell Differentiation ◽  
Dominant Negative Form ◽  
Proliferation And Differentiation ◽  
Negative Form

ABSTRACT The importance of c-myc as a target of the Blimp-1 repressor has been studied in BCL-1 cells, in which Blimp-1 is sufficient to trigger terminal B-cell differentiation. Our data show that Blimp-1-dependent repression of c-myc is required for BCL-1 differentiation, since constitutive expression of c-Myc blocked differentiation. Furthermore, ectopic expression of cyclin E mimicked the effects of c-Myc on both proliferation and differentiation, indicating that the ability of c-Myc to drive proliferation is responsible for blocking BCL-1 differentiation. However, inhibition of c-Myc by a dominant negative form was not sufficient to drive BCL-1 differentiation. Thus, during Blimp-1-dependent plasma cell differentiation, repression of c-myc is necessary but not sufficient, demonstrating the existence of additional Blimp-1 target genes.

Multiple Signaling Pathways Control Tbx6 Expression during Xenopus Myogenesis

2004 ◽  
Vol 36 (6) ◽  
pp. 390-396 ◽  
Author(s):  
Pan-Feng Fang ◽  
Rui-Ying Hu ◽  
Xing-Yue He ◽  
Xiao-Yan Ding
Keyword(s):  
Signaling Pathways ◽  
Molecular Mechanism ◽  
Bmp Signaling ◽  
Dominant Negative ◽  
Paraxial Mesoderm ◽  
Dominant Negative Form ◽  
Multiple Signaling ◽  
Dose Dependent ◽  
Negative Form

Abstract Tbx6 is critical for somite specification and myogenesis initiation. It has been shown that Activin/Nodal, VegT/Nodal, FGF, and BMP signaling pathways are involved early in specifying mesoderm or later in patterning mesoderm, and Xnot plays roles in setting up the boundary between notochord and paraxial mesoderm. In this study, we introduce the dominant negative form of above genes into embryos to evaluate if they are responsible for regulating Tbx6 expression. The results show that: (1) Activin/Nodal and VegT/Nodal signals are necessary for both initiation and maintenance of Tbx6 expression, and Nodal is sufficient to induce ectopic Tbx6 expression; (2) FGF signal is necessary for the initiation and maintenance of Tbx6, but it is not sufficient to induce Tbx6 expression; (3) BMP is also necessary for the expression of Tbx6, and the induction of Tbx6 expression by BMP is dose dependent; (4) Xnot has no effect on the expression of Tbx6. Our results suggest that several signaling pathways are involved in regulating Tbx6 expression, and pave the route to reveal the molecular mechanism of initiating myogenesis.

scholarly journals Modulation of flight and feeding behaviours requires presynaptic IP3Rs in dopaminergic neurons

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Anamika Sharma ◽  
Gaiti Hasan
Keyword(s):  
Dopaminergic Neurons ◽  
Calcium Release ◽  
Neural Circuit ◽  
Dominant Negative ◽  
Neuronal Function ◽  
Axonal Projections ◽  
Dominant Negative Form ◽  
Internal States ◽  
Innate Behaviour ◽  
Negative Form

Innate behaviours, although robust and hard wired, rely on modulation of neuronal circuits, for eliciting an appropriate response according to internal states and external cues. Drosophila flight is one such innate behaviour that is modulated by intracellular calcium release through inositol 1,4,5-trisphosphate receptors (IP3Rs). Cellular mechanism(s) by which IP3Rs modulate neuronal function for specific behaviours remain speculative, in vertebrates and invertebrates. To address this, we generated an inducible dominant negative form of the IP3R (IP3RDN). Flies with neuronal expression of IP3RDN exhibit flight deficits. Expression of IP3RDN helped identify key flight-modulating dopaminergic neurons with axonal projections in the mushroom body. Flies with attenuated IP3Rs in these presynaptic dopaminergic neurons exhibit shortened flight bouts and a disinterest in seeking food, accompanied by reduced excitability and dopamine release upon cholinergic stimulation. Our findings suggest that the same neural circuit modulates the drive for food search and for undertaking longer flight bouts.

scholarly journals Suppression of thymic development by the dominant-negative form of Gads

2001 ◽  
Vol 13 (6) ◽  
pp. 777-783 ◽  
Author(s):  
Kazu Kikuchi ◽  
Yoshitada Kawasaki ◽  
Naoto Ishii ◽  
Yoshiteru Sasaki ◽  
Hironobu Asao ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Thymic Development ◽  
Dominant Negative Form ◽  
Negative Form
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