The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate

Development ◽  
1997 ◽  
Vol 124 (8) ◽  
pp. 1485-1495 ◽  
Author(s):  
C.A. Micchelli ◽  
E.J. Rulifson ◽  
S.S. Blair
Keyword(s):  
Gene Expression ◽  
Ventral Side ◽  
Dominant Negative ◽  
Specific Marker ◽  
Wing Margin ◽  
Dynamic Feedback ◽  
Compartment Boundary ◽  
Wingless Signaling ◽  
Negative Role

We have investigated the role of the Notch and Wingless signaling pathways in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified about the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells are flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. We show first that cut acts to maintain margin wingless expression, providing a potential explanation of the cut mutant phenotype. Next, we examined the regulation of cut expression. Our results indicate that Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; our results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Finally, we show that Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. We propose that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression.

scholarly journals Expression of the runt homology domain of RUNX1 disrupts homeostasis of hematopoietic stem cells and induces progression to myelodysplastic syndrome

Blood ◽  
2012 ◽  
Vol 120 (19) ◽  
pp. 4028-4037 ◽  
Author(s):  
Shinobu Matsuura ◽  
Yukiko Komeno ◽  
Kristen E. Stevenson ◽  
Joseph R. Biggs ◽  
Kentson Lam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Gene Signature ◽  
Dominant Negative ◽  
Hematopoietic Stem ◽  
Expression Of Genes ◽  
Negative Role ◽  
Mechanisms Of Development

Abstract Mutations of RUNX1 are detected in patients with myelodysplastic syndrome (MDS). In particular, C-terminal truncation mutations lack a transcription regulatory domain and have increased DNA binding through the runt homology domain. The expression of the runt homology domain, RUNX1(41-214), in mouse hematopoietic cells induced progression to MDS and acute myeloid leukemia. Analysis of premyelodysplastic animals found expansion of c-Kit+Sca-1+Lin− cells and skewed differentiation to myeloid at the expense of the lymphoid lineage. These abnormalities correlate with the phenotype of Runx1-deficient animals, as expected given the reported dominant-negative role of C-terminal mutations over the full-length RUNX1. However, MDS is not observed in Runx1-deficient animals. Gene expression profiling found that RUNX1(41-214) c-Kit+Sca-1+Lin− cells have an overlapping yet distinct gene expression profile from Runx1-deficient animals. Moreover, an unexpected parallel was observed between the hematopoietic phenotype of RUNX1(41-214) and aged animals. Genes deregulated in RUNX1(41-214), but not in Runx1-deficient animals, were inversely correlated with the aging gene signature of HSCs, suggesting that disruption of the expression of genes related to normal aging by RUNX1 mutations contributes to development of MDS. The data presented here provide insights into the mechanisms of development of MDS in HSCs by C-terminal mutations of RUNX1.

scholarly journals Characterization of the Role of AMP-Activated Protein Kinase in the Regulation of Glucose-Activated Gene Expression Using Constitutively Active and Dominant Negative Forms of the Kinase

2000 ◽  
Vol 20 (18) ◽  
pp. 6704-6711 ◽  
Author(s):  
Angela Woods ◽  
Dalila Azzout-Marniche ◽  
Marc Foretz ◽  
Silvie C. Stein ◽  
Patricia Lemarchand ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Fatty Acid Synthase ◽  
Physiological Role ◽  
Rat Hepatocytes ◽  
Dominant Negative ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Constitutively Active

ABSTRACT In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism, e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP-activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (α1312) acts as a constitutively active kinase, while the other (α1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of α1312 increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of α1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

scholarly journals Nrf Transcription Factors in Keratinocytes Are Essential for Skin Tumor Prevention but Not for Wound Healing

2006 ◽  
Vol 26 (10) ◽  
pp. 3773-3784 ◽  
Author(s):  
Ulrich auf dem Keller ◽  
Marcel Huber ◽  
Tobias A. Beyer ◽  
Angelika Kümin ◽  
Christina Siemes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Wound Repair ◽  
Target Genes ◽  
Dominant Negative ◽  
Skin Tumor ◽  
Cellular Stress Response ◽  
Skin Development

ABSTRACT The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ.

The Role of Dominant-Negative IKAROS Mutations In the Pathogenesis and Treatment Responsiveness of BCR-ABL1 positive Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 540-540 ◽  
Author(s):  
J. Racquel Collins-Underwood ◽  
Nidal Boulos ◽  
Debbie Payne-Turner ◽  
Shann-Ching Chen ◽  
Richard Williams ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Marrow Transplant ◽  
Dominant Negative ◽  
Empty Vector ◽  
Transplant Model ◽  
Treatment Responsiveness

Abstract Abstract 540 Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified common deletions of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. The IKAROS alterations commonly involve coding exons 3–6, resulting in expression of a dominant negative IKAROS isoform, IK6. Moreover, the presence of IKZF1 alterations is associated with poor outcome in BCR-ABL1 ALL. We have previously shown in a retroviral bone marrow transplant model of BCR-ABL1 ALL that Ikzf1 loss results in increased penetrance of leukemia, but the role of IK6 in the pathogenesis of ALL has not been studied. Here, we have examined the effect of the expression of Ik6 in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from C57BL/6 Arf null mice was transduced with MSCV retrovirus coexpressing p185 BCR-ABL1 and luciferase, plated for 8 days to derive pre-B cells, then transduced with MSCV retrovirus expressing either wildtype Ikaros (Ik1-RFP), Ik6-RFP, or empty vector. Expression of Ik1 was not tolerated and resulted in cell death and apoptosis. IK6 expression led to increased proliferation of p185+Arf null cells with reduced sensitivity to the BCR-ABL1 kinase inhibitor dasatinib compared to cells transduced with empty vector. Intracellular phosphosignaling analysis of Crkl phosphorylation demonstrated that this reduced sensitivity to dasatinib was independent of ABL1 inhibition. Gene expression profiling of p185+Arf null-Ik6 cells revealed a gene expression signature similar to that of human BCR-ABL1+ ALL with enrichment of hematopoietic stem cells genes as well as genes involved in B-cell receptor, Notch, and Jak-Stat signaling pathways. To test the role of Ik6 in leukemogenesis and treatment responsiveness in vivo, p185 BCR-ABL1-luciferase Arf null cells were transduced with MSCV retrovirus expressing GFP alone, Ik1-GFP, or Ik6-GFP then transplanted into lethally irradiated C57BL/6 recipients. Expression of Ik6 in vivo led to accelerated tumorigenesis and decreased survival with tumors uniformly of pre-B immunophenotype. Moreover, mice transplanted with Ik6-expressing marrow were less sensitive to dasatinib therapy (10mg/kg QD initiated 7 days post-BMT) compared to control mice (19d vs. 31.5d, p<0.001), suggesting that expression of the dominant-negative Ikaros isoform Ik6 may play a key role in resistance to therapy and poor outcome in human BCR-ABL1 positive ALL. These results indicate that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 positive ALL, and that expression of dominant negative IKZF1 isoforms influences tumor responsiveness. Disclosures: No relevant conflicts of interest to declare.

Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-ε

1999 ◽  
Vol 277 (5) ◽  
pp. G1041-G1047 ◽  
Author(s):  
D.-H. Hong ◽  
G. Petrovics ◽  
W. B. Anderson ◽  
J. Forstner ◽  
G. Forstner
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Wild Type ◽  
Mucin Gene ◽  
Time Period ◽  
Mucin Expression ◽  
Mucin Genes

Treatment of HT-29 cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces MUC2 expression. To investigate the role of PKC in regulating mucin genes in intestinal cells, we examined the regulation of MUC1, MUC2, MUC5AC, MUC5B, and MUC6 expression in two human mucin-producing colonic cell lines, T84 and HT29/A1. T84 and HT29/A1 cells (at 80–90% confluency) were exposed to 100 nM PMA for 0, 3, and 6 h. Twofold or greater increases in mRNA levels for MUC2 and MUC5AC were observed in both cell lines during this time period, whereas the levels of MUC1, MUC5B, and MUC6 mRNAs were only marginally affected. These results indicated that PKC differentially regulates mucin gene expression and that it may be responsible for altered mucin expression. Our previous results suggested that the Ca2+-independent PKC-ε isoform appeared to mediate PMA-regulated mucin exocytosis in these cell lines. To determine if PKC-ε was also involved in MUC2/MUC5AC gene induction, HT29/A1 cells were stably transfected with either a wild-type PKC-ε or a dominant-negative ATP-binding mutant of PKC-ε (PKC-ε K437R). Overexpression of the dominant-negative PKC-ε K437R blocked induction of both mucin genes, whereas PMA-induced mucin gene expression was not prevented by overexpression of wild-type PKC-ε. PMA-dependent MUC2 mucin secretion was also blocked in cells overexpressing the dominant-negative PKC-ε K437R. On the basis of these observations, PKC-ε appears to mediate the expression of two major gastrointestinal mucins in response to PMA as well as PMA-regulated mucin exocytosis.

Pref-1 in brown adipose tissue: specific involvement in brown adipocyte differentiation and regulatory role of C/EBPδ

2012 ◽  
Vol 443 (3) ◽  
pp. 799-810 ◽  
Author(s):  
Jordi Armengol ◽  
Josep A. Villena ◽  
Elayne Hondares ◽  
María C. Carmona ◽  
Hei Sook Sul ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Adipocyte Differentiation ◽  
Proximal Promoter ◽  
Brown Adipocyte ◽  
Specific Marker ◽  
Brown Adipose ◽  
Factor C

Pref-1 (pre-adipocyte factor-1) is known to play a central role in regulating white adipocyte differentiation, but the role of Pref-1 in BAT (brown adipose tissue) has not been analysed. In the present study we found that Pref-1 expression is high in fetal BAT and declines progressively after birth. However, Pref-1-null mice showed unaltered fetal development of BAT, but exhibited signs of over-activation of BAT thermogenesis in the post-natal period. In C/EBP (CCAAT/enhancer-binding protein) α-null mice, a rodent model of impaired fetal BAT differentiation, Pref-1 was dramatically overexpressed, in association with reduced expression of the Ucp1 (uncoupling protein 1) gene, a BAT-specific marker of thermogenic differentiation. In brown adipocyte cell culture models, Pref-1 was mostly expressed in pre-adipocytes and declined with brown adipocyte differentiation. The transcription factor C/EBPδ activated the Pref-1 gene transcription in brown adipocytes, through binding to the proximal promoter region. Accordingly, siRNA (small interfering RNA)-induced C/EBPδ knockdown led to reduced Pref-1 gene expression. This effect is consistent with the observed overexpression of C/EBPδ in C/EBPα-null BAT and high expression of C/EBPδ in brown pre-adipocytes. Dexamethasone treatment of brown pre-adipocytes suppressed Pref-1 down-regulation occurring throughout the brown adipocyte differentiation process, increased the expression of C/EBPδ and strongly impaired expression of the thermogenic markers UCP1 and PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-α]. However, it did not alter normal fat accumulation or expression of non-BAT-specific genes. Collectively, these results specifically implicate Pref-1 in controlling the thermogenic gene expression program in BAT, and identify C/EBPδ as a novel transcriptional regulator of Pref-1 gene expression that may be related to the specific role of glucocorticoids in BAT differentiation.

scholarly journals FOXA1 plays a role in regulating secretoglobin 1a1 expression in the absence of CCAAT/enhancer binding protein activities in lung in vivo

2011 ◽  
Vol 300 (3) ◽  
pp. L441-L452 ◽  
Author(s):  
Taketomo Kido ◽  
Takeshi Tomita ◽  
Minoru Okamoto ◽  
Yan Cai ◽  
Yoshimi Matsumoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Regulation Of Gene Expression ◽  
Dominant Negative ◽  
Clara Cell ◽  
Small Molecular Weight ◽  
Dominant Negative Form

Secretoglobin (SCGB) 1A1, also called Clara cell secretor protein (CCSP) or Clara cell-specific 10-kDa protein (CC10), is a small molecular weight secreted protein mainly expressed in lung, with anti-inflammatory/immunomodulatory properties. Previous in vitro studies demonstrated that CCAAT/enhancer-binding proteins (C/EBPs) are the major transcription factors for the regulation of Scbg1a1 gene expression, whereas FOXA1 had a minimum effect on the transcription. To determine the in vivo role of C/EBPs in the regulation of SCGB1A1 expression, experiments were performed in which A-C/EBP, a dominant-negative form of C/EBP that interferes with DNA binding activities of all C/EBPs, was specifically expressed in lung. Surprisingly, despite the in vitro findings, expression of SCGB1A1 mRNA was not decreased in vivo in the absence of C/EBPs. This may be due to a compensatory role assumed by FOXA1 in the regulation of Scgb1a1 gene expression in lung in the absence of active C/EBPs. This disconnect between in vitro and in vivo results underscores the importance of studies using animal models to determine the role of specific transcription factors in the regulation of gene expression in intact multicellular complex organs such as lung.

Inhibitor-κB kinase-β regulates LPS-induced TNF-α production in cardiac myocytes through modulation of NF-κB p65 subunit phosphorylation

2005 ◽  
Vol 289 (5) ◽  
pp. H2103-H2111 ◽  
Author(s):  
Gentzon Hall ◽  
Ishwar S. Singh ◽  
Lisa Hester ◽  
Jeffery D. Hasday ◽  
Terry B. Rogers
Keyword(s):  
Gene Expression ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Myocardial Dysfunction ◽  
Gene Promoter ◽  
Fold Increase ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Tnf Α

TNF-α is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-κB family of transcription factors are essential regulators of myocardial TNF-α gene expression, recent developments in our understanding of the modulation of NF-κB activity through posttranslational modification of NF-κB subunits suggest that the present view of NF-κB-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-α production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-α production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser536, a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-β reduced LPS-induced TNF-α production 38-fold, TNF-α mRNA levels 6-fold, and IκB-α phosphorylation 5-fold and degraded IκB-α 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-α production 3.5-fold, whereas overexpression of dominant-negative IKK-β reduced LPS-induced TNF-α production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-α had no effect on p65 phosphorylation or TNF-α production, revealing that IKK-β, not IKK-α, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-α production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-α gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-κB signaling in TNF-α gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction.

Differential requirement for NF-κB-inducing kinase in the induction of NF-κB by IL-1β, TNF-α, and Fas

2002 ◽  
Vol 283 (1) ◽  
pp. C347-C357 ◽  
Author(s):  
Maria P. Russo ◽  
Brydon L. Bennett ◽  
Anthony M. Manning ◽  
David A. Brenner ◽  
Christian Jobin
Keyword(s):  
Gene Expression ◽  
Hela Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Cytokine Signaling ◽  
Iκb Kinase ◽  
Tnf Α ◽  
Iκbα Phosphorylation ◽  
Ht 29

In this study, we examined the role of the nuclear factor-κB (NF-κB)-inducing kinase (NIK) in distinct signaling pathways leading to NF-κB activation. We show that a dominant-negative form of NIK (dnNIK) delivered by adenoviral (Ad5dnNIK) vector inhibits Fas-induced IκBα phosphorylation and NF-κB-dependent gene expression in HT-29 and HeLa cells. Interleukin (IL)-1β- and tumor necrosis factor-α (TNF-α)-induced NF-κB activation and κB-dependent gene expression are inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells. Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF-α-induced apoptosis at an early time point. However, cytokine- and Fas-induced signals to NF-κB are finally integrated by the IκB kinase (IKK) complex, since IκBα phosphorylation, NF-κB DNA binding activity, and IL-8 gene expression were strongly inhibited in HT-29 and HeLa cells overexpressing dominant-negative IKKβ (Ad5dnIKKβ). Our findings support the concept that cytokine signaling to NF-κB is redundant at the level of NIK. In addition, this study demonstrates for the first time the critical role of NIK and IKKβ in Fas-induced NF-κB signaling cascade.

Sign in / Sign up

Export Citation Format

Share Document