scholarly journals Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos

2003 ◽  
Vol 23 (22) ◽  
pp. 8295-8305 ◽  
Author(s):  
Keizo Nishikawa ◽  
Makoto Kobayashi ◽  
Atsuko Masumi ◽  
Susan E. Lyons ◽  
Brant M. Weinstein ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Germ Line ◽  
Zebra Fish ◽  
Wild Type ◽  
Lysine Residues ◽  
Self Association ◽  
Gata Motif

ABSTRACT Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3′-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.

scholarly journals Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-κB family member RelB

2008 ◽  
Vol 416 (1) ◽  
pp. 117-127 ◽  
Author(s):  
Julia Leidner ◽  
Lysann Palkowitsch ◽  
Uta Marienfeld ◽  
Dietmar Fischer ◽  
Ralf Marienfeld
Keyword(s):  
Dna Methyltransferase ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Nuclear Factor Κb ◽  
Death Domain ◽  
293 Cells ◽  
Lysine Residues ◽  
Negative Effect ◽  
The Stability

RelB is the key component of the alternative NF-κB (nuclear factor κB) signalling pathway. However, RelB exerts also a negative effect via the recruitment of a DNMT1 (DNA methyltransferase 1)–Daxx (death domain-associated protein) complex to NF-κB target genes. Importantly, the molecular mechanisms which determine the functions of RelB are still largely unknown. In the present study, we aimed to analyse whether ubiquitination of RelB might be involved in the regulation of RelB. Indeed, RelB is constitutively polyubiquitinated in the B-cell lines Namalwa and 70Z/3. Although a PMA+ionomycin-induced augmentation of RelB polyubiquitination was linked to its proteasomal degradation in B-cells, the constitutive RelB polyubiquitination seems to affect non-proteasomal functions. Consistently, a significant RelB polyubiquitination in HEK (human embryonic kidney)-293 cells correlated with an augmentation of the transcriptional activity of RelB. Yet, neither nuclear localization nor DNA binding was enhanced by RelB polyubiquitination. Interestingly, basal RelB polyubiquitination depends neither on Lys48 nor on Lys63 conjugates, but might involve unconventional ubiquitin conjugates. Mapping of the ubiquitination target sites in RelB revealed the existence of various lysine residues, which serve as ubiquitination acceptors. However, only the substitution of Lys273/274 and Lys305/308 significantly decreased the basal RelB activity and the ubiquitin-induced augmentation of the RelB activity. Collectively, these results imply a dual role of RelB polyubiquitination for the stability and activity of this transcription factor.

scholarly journals Prox1 Is a Novel Coregulator of Ff1b and Is Involved in the Embryonic Development of the Zebra Fish Interrenal Primordium

2003 ◽  
Vol 23 (20) ◽  
pp. 7243-7255 ◽  
Author(s):  
Yi-Wen Liu ◽  
Wei Gao ◽  
Hui-Ling Teh ◽  
Jee-Hian Tan ◽  
Woon-Khiong Chan
Keyword(s):  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Physical Interaction ◽  
Homeodomain Protein ◽  
Zebra Fish ◽  
Steroidogenic Factor 1 ◽  
Study Results ◽  
Biochemical Analyses

ABSTRACT Steroidogenic factor 1 (SF-1) plays an essential role in adrenal development, although the exact molecular mechanisms are unclear. Our previous work established that Ff1b is the zebra fish homologue of SF-1 and that its disruption by antisense morpholinos leads to a complete ablation of the interrenal organ. In this study, results of biochemical analyses suggest that Ff1b and other Ff1 members interact with Prox1, a homeodomain protein. Fine mapping using site-directed mutants showed that this interaction requires an intact Ff1b heptad 9 and AF2, as well as Prox1 NR Box I. In vivo, this physical interaction led to the inhibition of Ff1-mediated transactivation of pLuc3XFRE, indicating that Prox1 acts to repress the transcriptional activity of Ff1b. In situ hybridization demonstrates that prox1 colocalizes with ff1a and ff1b in the liver and interrenal primordia, respectively. Embryos microinjected with prox1 morpholino displayed a consistent partial reduction of 3β-Hsd activity in the interrenal organ, while ff1b morpholino led to a disappearance of prox1. Based on these results, we propose that during the course of interrenal organogenesis, Prox1 functions as a tissue-specific coregulator of Ff1b and that the subsequent inhibition of Ff1b activity, after its initial roles in the specification of interrenal primordium, is critical for the maturation of the interrenal organ.

scholarly journals Lysine acetyltransferase Tip60 acetylates the APP adaptor Fe65 to increase its transcriptional activity

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Sabine Probst ◽  
Florian Riese ◽  
Larissa Kägi ◽  
Maik Krüger ◽  
Natalie Russi ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Adaptor Protein ◽  
Proteolytic Processing ◽  
Class Iii ◽  
Lysine Deacetylase ◽  
Lysine Residues ◽  
Lysine Acetyltransferase ◽  
Complex Forms ◽  
Spot Formation

AbstractProteolytic processing of the amyloid precursor protein (APP) releases the APP intracellular domain (AICD) from the membrane. Bound to the APP adaptor protein Fe65 and the lysine acetyltransferase (KAT) Tip60, AICD translocates to the nucleus. Here, the complex forms spherical condensates at sites of endogenous target genes, termed AFT spots (AICD-Fe65-Tip60). We show that loss of Tip60 KAT activity prevents autoacetylation, reduces binding of Fe65 and abolishes Fe65-mediated stabilization of Tip60. Autoacetylation is a prerequisite for AFT spot formation, with KAT-deficient Tip60 retained together with Fe65 in speckles. We identify lysine residues 204 and 701 of Fe65 as acetylation targets of Tip60. We do not detect acetylation of AICD. Mutation of Fe65 K204 and K701 to glutamine, mimicking acetylation-induced charge neutralization, increases the transcriptional activity of Fe65 whereas Tip60 inhibition reduces it. The lysine deacetylase (KDAC) class III Sirt1 deacetylates Fe65 and pharmacological modulation of Sirt1 activity regulates Fe65 transcriptional activity. A second acetylation/deacetylation cycle, conducted by CBP and class I/II KDACs at different lysine residues, regulates stability of Fe65. This is the first report describing a role for acetylation in the regulation of Fe65 transcriptional activity, with Tip60 being the only KAT tested that supports AFT spot formation.

scholarly journals Shh Plays an Inhibitory Role in Cusp Patterning by Regulation of Sostdc1

2018 ◽  
Vol 98 (1) ◽  
pp. 98-106 ◽  
Author(s):  
J. Kim ◽  
Y. Ahn ◽  
D. Adasooriya ◽  
E.J. Woo ◽  
H.J. Kim ◽  
...  
Keyword(s):  
Pattern Formation ◽  
Wnt Signaling ◽  
Molecular Mechanisms ◽  
Wild Type ◽  
Shh Signaling ◽  
Shh Pathway ◽  
Inhibitory Role ◽  
Crown Shape ◽  
Cusp Formation

Crown shapes in mammalian teeth vary considerably from species to species, and morphological characters in crown shape have been used to identify species. Cusp pattern is one of the characters in crown shape. In the processes governing the formation of cusp pattern, the Shh pathway has been implicated as an important player. Suppression of Shh signaling activity in vitro in explant assays appears to induce supernumerary cusp formation in wild-type tooth germs. However, the in vivo role of Shh signaling in cusp pattern formation and the molecular mechanisms by which Shh regulates cusp patterning are not clear. Here, through in vivo phenotypic analyses of mice in which Shh activity was suppressed and compared with wild-type mice, we characterized differences in the location, number, incidence, and shape of supernumerary cusps in molars at embryonic day 15.5. We found that the distances between cusps were reduced in molars of Shh activity–suppressed mice in vivo. These findings confirm and extend the previous idea that Shh acts as an inhibitor in the reaction-diffusion model for cusp pattern formation by negatively regulating the intercuspal distance. We uncovered a significant reduction of expression level of Sostdc1, which encodes a secreted modulator of Wnt signaling, after suppression of Shh activity. The supernumerary cusp formation in Sostdc1−/− mice and compound Sostdc1 and Lrp mutant mice indicates a strong association between Wnt and Shh signaling pathways in cusp patterning. In further support of this idea, there is a high degree of similarity in the supernumerary cusp patterns of mice lacking Sostdc1 or Shh at embryonic day 15.5. These results suggest that Shh plays an inhibitory role in cusp pattern formation by modulating Wnt signaling through the positive regulation of Sostdc1.

scholarly journals In vivo and in vitro oncogenic effects of HIF2A mutations in pheochromocytomas and paragangliomas

2013 ◽  
Vol 20 (3) ◽  
pp. 349-359 ◽  
Author(s):  
Rodrigo A Toledo ◽  
Yuejuan Qin ◽  
Subramanya Srikantan ◽  
Nicole Paes Morales ◽  
Qun Li ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Target Genes ◽  
Germline Mutations ◽  
Vascular Tumors ◽  
Wild Type ◽  
Pheochromocytoma Pc12 ◽  
Hereditary Tumors ◽  
Tumor Dna

Pheochromocytomas and paragangliomas are highly vascular tumors of the autonomic nervous system. Germline mutations, including those in hypoxia-related genes, occur in one third of the cases, but somatic mutations are infrequent in these tumors. Using exome sequencing of six paired constitutive and tumor DNA from sporadic pheochromocytomas and paragangliomas, we identified a somatic mutation in the HIF2A (EPAS1) gene. Screening of an additional 239 pheochromocytomas/paragangliomas uncovered three other HIF2A variants in sporadic (4/167, 2.3%) but not in hereditary tumors or controls. Three of the mutations involved proline 531, one of the two residues that controls HIF2α stability by hydroxylation. The fourth mutation, on Ser71, was adjacent to the DNA binding domain. No mutations were detected in the homologous regions of the HIF1A gene in 132 tumors. Mutant HIF2A tumors had increased expression of HIF2α target genes, suggesting an activating effect of the mutations. Ectopically expressed HIF2α mutants in HEK293, renal cell carcinoma 786-0, or rat pheochromocytoma PC12 cell lines showed increased stability, resistance to VHL-mediated degradation, target induction, and reduced chromaffin cell differentiation. Furthermore, mice injected with cells expressing mutant HIF2A developed tumors, and those with Pro531Thr and Pro531Ser mutations had shorter latency than tumors from mice with wild-type HIF2A. Our results support a direct oncogenic role for HIF2A in human neoplasia and strengthen the link between hypoxic pathways and pheochromocytomas and paragangliomas.

Genetic evidence for the transcriptional-activating function of Homothorax during adult fly development

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3405-3413 ◽  
Author(s):  
Adi Inbal ◽  
Naomi Halachmi ◽  
Charna Dibner ◽  
Dale Frank ◽  
Adi Salzberg
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Genetic Evidence ◽  
In Vivo Studies ◽  
Loss Of Function ◽  
Native Form ◽  
Downstream Target ◽  
Activating Function

Homothorax (HTH) is a homeobox-containing protein, which plays multiple roles in the development of the embryo and the adult fly. HTH binds to the homeotic cofactor Extradenticle (EXD) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that HTH can bind DNA as a part of ternary HTH/EXD/HOX complexes, but little is known about the transcription regulating function of HTH-containing complexes in the context of the developing fly. Here we present genetic evidence, from in vivo studies, for the transcriptional-activating function of HTH. The HTH protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (EN) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate EXD to the nucleus. Expression of the repressing form of HTH in otherwise wild-type imaginal discs phenocopied hth loss of function. Thus, the repressing form was working as an antimorph, suggesting that normally HTH is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of HTH caused typical hth gain-of-function phenotypes and could rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homologue of HTH, extending the known functional similarity between the two proteins. Competition experiments demonstrated that the repressing forms of HTH or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of HTH. We also describe the phenotypic consequences of HTH antimorph activity in derivatives of the wing, labial and genital discs. Some of the described phenotypes, for example, a proboscis-to-leg transformation, were not previously associated with alterations in HTH activity. Observing the ability of HTH antimorphs to interfere with different developmental pathways may direct us to new targets of HTH. The HTH antimorph described in this work presents a new means by which the transcriptional activity of the endogenous HTH protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

scholarly journals Interdependent Recruitment of SAGA and Srb Mediator by Transcriptional Activator Gcn4p

2005 ◽  
Vol 25 (9) ◽  
pp. 3461-3474 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Fan Zhang ◽  
Gwo Jiunn Hwang ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Wild Type ◽  
General Transcription Factors ◽  
Tata Element ◽  
High Level ◽  
Target Promoters ◽  
Activation Sequence

ABSTRACT Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

Identification of an octamer element required for in vivo expression of the TIE1 gene in endothelial cells

2001 ◽  
Vol 360 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Stephane C. BOUTET ◽  
Thomas QUERTERMOUS ◽  
Bahaa M. FADEL
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Molecular Mechanisms ◽  
Vascular Development ◽  
Common Mechanism ◽  
Tyrosine Kinase Receptor ◽  
Wild Type ◽  
Binding Studies ◽  
In Vivo Expression

TIE1, an endothelial-cell-specific tyrosine kinase receptor, is required for the survival and growth of microvascular endothelial cells during the capillary sprouting phase of vascular development. To investigate the molecular mechanisms that regulate the expression of TIE1 in the endothelium, we analysed transgenic mouse embryos carrying wild-type or mutant TIE1 promoter/LacZ constructs. Our data indicate that an upstream DNA octamer element (5′-ATGCAAAT-3′) is required for the in vivo expression of TIE1 in embryonic endothelial cells. Transgenic embryos carrying the wild-type TIE1 promoter (−466 to +78bp) fused to LacZ and spanning the octamer element demonstrate endothelial-cell-specific expression of the reporter transgene. Point mutations introduced within the octamer element result in a significant decrease of endothelial LacZ expression, suggesting that the octamer site functions as a positive regulator for TIE1 gene expression in endothelial cells. DNA–protein binding studies show that the octamer element exhibits an endothelial-cell-specific pattern of binding via interaction with endothelial-cell-restricted factor(s). Our findings suggest an important role for the octamer element in regulating the expression of the TIE1 receptor in the embryonic endothelium and suggest a common mechanism for the regulation of the angiogenic and cell-specific TIE1 and TIE2 genes during vascular development.

scholarly journals Modulating PKCα Activity to Target Wnt/β-Catenin Signaling in Colon Cancer

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 693 ◽  
Author(s):  
Sébastien Dupasquier ◽  
Philippe Blache ◽  
Laurence Picque Lasorsa ◽  
Han Zhao ◽  
Jean-Daniel Abraham ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Tumor Development ◽  
Orphan Receptor ◽  
Nuclear Accumulation ◽  
Kinase C ◽  
Extracellular Signal

Inactivating mutations of the tumor suppressor Adenomatosis Polyposis Coli (APC), which are found in familial adenomatosis polyposis and in 80% of sporadic colorectal cancers (CRC), result in constitutive activation of the Wnt/β-catenin pathway and tumor development in the intestine. These mutations disconnect the Wnt/β-catenin pathway from its Wnt extracellular signal by inactivating the APC/GSK3-β/axin destruction complex of β-catenin. This results in sustained nuclear accumulation of β-catenin, followed by β-catenin-dependent co-transcriptional activation of Wnt/β-catenin target genes. Thus, mechanisms acting downstream of APC, such as those controlling β-catenin stability and/or co-transcriptional activity, are attractive targets for CRC treatment. Protein Kinase C-α (PKCα) phosphorylates the orphan receptor RORα that then inhibits β-catenin co-transcriptional activity. PKCα also phosphorylates β-catenin, leading to its degradation by the proteasome. Here, using both in vitro (DLD-1 cells) and in vivo (C57BL/6J mice) PKCα knock-in models, we investigated whether enhancing PKCα function could be beneficial in CRC treatment. We found that PKCα is infrequently mutated in CRC samples, and that inducing PKCα function is not deleterious for the normal intestinal epithelium. Conversely, di-terpene ester-induced PKCα activity triggers CRC cell death. Together, these data indicate that PKCα is a relevant drug target for CRC treatment.

Sign in / Sign up

Export Citation Format

Share Document