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.

Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3173-3183 ◽  
Author(s):  
K.L. Kroll ◽  
E. Amaya
Keyword(s):  
Large Scale ◽  
Neural Induction ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mesoderm Induction ◽  
Fgf Signaling ◽  
Fgf Receptor ◽  
Transgenic Expression

We have developed a simple approach for large-scale transgenesis in Xenopus laevis embryos and have used this method to identify in vivo requirements for FGF signaling during gastrulation. Plasmids are introduced into decondensed sperm nuclei in vitro using restriction enzyme-mediated integration (REMI). Transplantation of these nuclei into unfertilized eggs yields hundreds of normal, diploid embryos per day which develop to advanced stages and express integrated plasmids nonmosaically. Transgenic expression of a dominant negative mutant of the FGF receptor (XFD) after the mid-blastula stage uncouples mesoderm induction, which is normal, from maintenance of mesodermal markers, which is lost during gastrulation. By contrast, embryos expressing XFD contain well-patterned nervous systems despite a putative role for FGF in neural induction.

scholarly journals CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi47-vi47
Author(s):  
Marilin Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Justyna Przystal ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Profile ◽  
Transcriptional Networks ◽  
Molecular Alterations ◽  
Helix Loop Helix

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

Identification of Key Cellular Regulators That Interact with Id1 To Control Hematopoietic Stem Cell Behavior.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1319-1319
Author(s):  
Vladimir Jankovic ◽  
Alessia Ciarrocchi ◽  
Tony DeBlasio ◽  
Robert Benezra ◽  
Stephen D. Nimer
Keyword(s):  
Transcription Factors ◽  
Transcriptional Repression ◽  
Genetic Interaction ◽  
Dominant Negative ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Helix Loop Helix

Abstract The ability of hematopoietic stem cells to tightly regulate the transition from relative quiescence and self-renewal to the transiently amplifying, differentiating progenitor fate is critical for HSC homeostasis as well as their regenerative capacity. We have recently described the diminished frequency and rapid exhaustion of HSC self-renewal capacity in the absence of the dominant negative helix-loop-helix molecule Id1. Furthermore, Id1 null HSCs have an increased rate of cycling, coupled with accelerated myeloid commitment both in vivo and in vitro. This is reflected in the elevated expression of myelo-erythroid transcription factors (c/EBPalpha and GATA1) within the Lin−c-kit+Sca-1+ population - “myeloid priming”. The major targets of Id1 mediated transcriptional repression are the ubiquitous E protein E2A as well as Ets transcription factors (Ets1 and Ets2). We hypothesized that the unrestrained activity of these and/or other targets of Id1 transcriptional repression leads to premature HSC commitment in Id1 null animals. Indeed, we show that HSC differentiation in culture can be delayed by transduction of E2A directed shRNA specifically in Id1 null, but not in wild-type Id1 expressing cells. This indicates an abnormal E2A activity in Id1 null HSCs that could be responsible for their increased differentiation status. To further define the transcriptional deregulation in Id1 null HSCs, we have used the Affymetrix microarray technology. We observed ~3 fold increased expression of the CDK inhibitor p21 in freshly isolated Id1 null HSCs and have confirmed this result by multiple independent qPCR measurements. The transcriptional induction of p21 by E2A as well as its repression by Id1 have been well established. Therefore, the observed p21 induction could be explained by the elevated level of E2A activity in HSCs in the absence of Id1 expression. To explore the functional significance of Id1 mediated p21 regulation in HSCs, we have generated p21/Id1 double knockout animals. Surprisingly, despite its reported function in restricting the cell cycle entry of normal HSCs, we show that in the context of Id1 loss, p21 expression is required for the accelerated HSC cycling, and unlike Id1 single null HSCs, p21/Id1 double knockout HSCs do not show accelerated myeloid differentiation in culture. Therefore, we propose that Id1 actively represses E2A activity in HSCs, as well as the induction of p21, which could be an important component of the HSC commitment program. Further studies will be presented defining the in vivo relevance of the Id1/p21 genetic interaction for HSC growth and differentiation.

scholarly journals The pro-apoptotic protein death-associated protein 3 (DAP3) interacts with the glucocorticoid receptor and affects the receptor function

2000 ◽  
Vol 349 (3) ◽  
pp. 885-893 ◽  
Author(s):  
Sanna M. HULKKO ◽  
Hideki WAKUI ◽  
Johanna ZILLIACUS
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Heat Shock Protein 90 ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Interaction Domain ◽  
Helix Loop Helix ◽  
Aryl Hydrocarbon ◽  
Apoptotic Protein

The yeast two-hybrid system was used to isolate cDNAs encoding proteins that interact with the glucocorticoid receptor (GR) ligand-binding domain in a ligand-dependent manner. One isolated cDNA encoded a fragment of death-associated protein 3 (DAP3), which has been implicated as a positive mediator of apoptosis. In vitro experiments showed that the full-length DAP3 also interacted with GR. The main interaction domain was mapped to the N-terminal region of DAP3 that had previously been shown to function in a dominant-negative fashion, protecting cells from apoptosis. Co-transfection experiments in COS-7 cells showed that DAP3 had a stimulatory effect on the ligand-induced transcriptional activation by GR and also increased the steroid-sensitivity. Furthermore, DAP3 formed a complex with several other nuclear receptors and some basic helix–loop–helix/Per–Arnt–Sim proteins, as well as with heat-shock protein 90 (hsp90) (Arnt is the aryl-hydrocarbon-receptor nuclear translocator, and Per and Sim are the Drosophila proteins Period and Single-minded). The results suggest that DAP3 could have an important role in GR action, possibly by modulating the cytoplasmic GR–hsp90 complex. Since glucocorticoids can induce apoptosis, the pro-apoptotic DAP3 protein may be involved in this function of GR.

scholarly journals Variant Max protein, derived by alternative splicing, associates with c-Myc in vivo and inhibits transactivation.

1995 ◽  
Vol 15 (12) ◽  
pp. 6702-6709 ◽  
Author(s):  
M Arsura ◽  
A Deshpande ◽  
S R Hann ◽  
G E Sonenshein
Keyword(s):  
Alternative Splicing ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Variant Form ◽  
Factor X ◽  
Helix Loop Helix ◽  
Alternatively Spliced ◽  
Basic Region

Max (Myc-associated factor X) is a basic helix-loop-helix/leucine zipper protein that has been shown to play a central role in the functional activity of c-Myc as a transcriptional activator. Max potentiates the binding of Myc-Max heterodimers through its basic region to its specific E-box Myc site (EMS), enabling c-Myc to transactivate effectively. In addition to the alternatively spliced exon a, several naturally occurring forms of alternatively spliced max mRNAs have been reported, but variant protein products from these transcripts have not been detected. Using Western blot (immunoblot) and immunoprecipitation analysis, we have identified a variant form of Max protein (16 to 17 kDa), termed dMax, in detergent nuclear extracts of murine B-lymphoma cells, normal B lymphocytes, and NIH 3T3 fibroblasts. Cloning and sequencing revealed that dMax contains a deletion spanning the basic region and helix 1 and the loop of the helix-loop-helix region, presumably as a result of alternative splicing of max RNA. S1 nuclease analysis confirmed the presence of the mRNA for dMax in cells. The dMax protein, prepared via in vitro transcription and translation, associated with bacterially synthesized Myc-glutathione S-transferase. Coimmunoprecipitation of dMax and c-Myc indicated their intracellular association. In vitro-synthesized dMax failed to bind EMS DNA, presumably because of the absence of the basic region. Coexpression of dMax inhibited EMS-mediated transactivation by c-Myc. Thus dMax, which can interact with c-Myc, appears to function as a dominant negative regulator, providing an additional level of regulation to the transactivation potential of c-Myc.

scholarly journals Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Marilin Sophia Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Srinath Rajaraman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Subgroup ◽  
Replicative Stress ◽  
Helix Loop Helix ◽  
Molecular Events ◽  
Dominant Negative Variant

Abstract Background The overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies. Methods Glioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro. Results The induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone. Conclusions Gliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.

scholarly journals The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.

1993 ◽  
Vol 13 (12) ◽  
pp. 7874-7880 ◽  
Author(s):  
S Pesce ◽  
R Benezra
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions ◽  
Helix Loop Helix ◽  
Loop Region ◽  
Dominant Negative Activity

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.

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.

FGF-, BMP- and Shh-mediated signalling pathways in the regulation of cranial suture morphogenesis and calvarial bone development

Development ◽  
1998 ◽  
Vol 125 (7) ◽  
pp. 1241-1251 ◽  
Author(s):  
H.J. Kim ◽  
D.P. Rice ◽  
P.J. Kettunen ◽  
I. Thesleff
Keyword(s):  
Dura Mater ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Homeobox Gene ◽  
Signalling Pathways ◽  
Sagittal Suture ◽  
Calvarial Bone ◽  
Suture Closure

The development of calvarial bones is tightly co-ordinated with the growth of the brain and needs harmonious interactions between different tissues within the calvarial sutures. Premature fusion of cranial sutures, known as craniosynostosis, presumably involves disturbance of these interactions. Mutations in the homeobox gene Msx2 as well as the FGF receptors cause human craniosynostosis syndromes. Our histological analysis of mouse calvarial development demonstrated morphological differences in the sagittal suture between embryonic and postnatal stages. In vitro culture of mouse calvaria showed that embryonic, but not postnatal, dura mater regulated suture patency. We next analysed by in situ hybridisation the expression of several genes, which are known to act in conserved signalling pathways, in the sagittal suture during embryonic (E15-E18) and postnatal stages (P1-P6). Msx1 and Msx2 were expressed in the sutural mesenchyme and the dura mater. FGFR2(BEK), as well as Bmp2 and Bmp4, were intensely expressed in the osteogenic fronts and Bmp4 also in the mesenchyme of the sagittal suture and in the dura mater. Fgf9 was expressed throughout the calvarial mesenchyme, the dura mater, the developing bones and the overlying skin, but Fgf4 was not detected in these tissues. Interestingly, Shh and Ptc started to be expressed in patched pattern along the osteogenic fronts at the end of embryonic development and, at this time, the expression of Bmp4 and sequentially those of Msx2 and Bmp2 were reduced, and they also acquired patched expression patterns. The expression of Msx2 in the dura mater disappeared after birth. <P> FGF and BMP signalling pathways were further examined in vitro, in E15 mouse calvarial explants. Interestingly, beads soaked in FGF4 accelerated sutural closure when placed on the osteogenic fronts, but had no such effect when placed on the mid-sutural mesenchyme. BMP4 beads caused an increase in tissue volume both when placed on the osteogenic fronts and on the mid-sutural area, but did not effect suture closure. BMP4 induced the expression of both Msx1 and Msx2 genes in sutural tissue, while FGF4 induced only Msx1. We suggest that the local application of FGF on the osteogenic fronts accelerating suture closure in vitro, mimics the pathogenesis of human craniosynostosis syndromes in which mutations in the FGF receptor genes apparently cause constitutive activation of the receptors. Taken together, our data suggest that conserved signalling pathways regulate tissue interactions during suture morphogenesis and intramembranous bone formation of the calvaria and that morphogenesis of mouse sagittal suture is controlled by different molecular mechanisms during the embryonic and postnatal stages. Signals from the dura mater may regulate the maintenance of sutural patency prenatally, whereas signals in the osteogenic fronts dominate after birth.

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