The eve stripe 2 enhancer employs multiple modes of transcriptional synergy

Development ◽  
1996 ◽  
Vol 122 (1) ◽  
pp. 205-214 ◽  
Author(s):  
D.N. Arnosti ◽  
S. Barolo ◽  
M. Levine ◽  
S. Small
Keyword(s):  
Fusion Protein ◽  
Drosophila Embryo ◽  
Cooperative Binding ◽  
Synergistic Activity ◽  
Detailed Model ◽  
Multiple Modes ◽  
Anterior Half ◽  
Segmentation Process ◽  
Transgenic Embryos ◽  
Cis And Trans

Previous studies have provided a detailed model for the regulation of even-skipped (eve) stripe 2 expression in the Drosophila embryo. The bicoid (bcd) regulatory gradient triggers the expression of hunchback (hb); these work synergistically to activate the stripe in the anterior half of the embryo, bcd also coordinates the expression of two repressors, giant (gt) and Kruppel (Kr), which define the anterior and posterior borders of the stripe, respectively. Here, we report the findings of extensive cis- and trans- complementation analyses using a series of defective stripe 2 enhancers in transgenic embryos. This study reaches two primary conclusions. First, the strip 2 enhancer is inherently ‘sensitized’ for repression by gt. We propose that gt specifies the sharp anterior stripe border by blocking two tiers of transcriptional synergy, cooperative binding to DNA and cooperative contact of bound activators with the transcription complex. Second, we find that the synergistic activity of hb and bcd is ‘promiscuous’. For example, a maternally expressed Gal4-Sp1 fusion protein can functionally replace hb in the stripe 2 enhancer. This finding challenges previous proposals for dedicated hb and bcd interactions in the segmentation process.

Dpp signaling thresholds in the dorsal ectoderm of the Drosophila embryo

Development ◽  
2000 ◽  
Vol 127 (15) ◽  
pp. 3305-3312 ◽  
Author(s):  
H.L. Ashe ◽  
M. Mannervik ◽  
M. Levine
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Histone Acetyltransferase ◽  
Cell Types ◽  
Drosophila Embryo ◽  
Present Evidence ◽  
Drosophila Homolog ◽  
Dorsal Ectoderm ◽  
Transgenic Embryos ◽  
Different Cell Types

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGF(β) signaling molecules, Decapentaplegic (Dpp) and Screw (Scw). Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation (Sog) and a newly identified transcriptional repressor, Brinker (Brk), which are expressed in neurogenic regions that abut the dorsal ectoderm. Here we examine the expression of a number of Dpp target genes in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. We also present evidence that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase.

scholarly journals The Combinatorial Activity of Eftozanermin (ABBV-621), a Novel and Potent TRAIL Receptor Agonist Fusion Protein, in Pre-Clinical Models of Hematologic Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-41
Author(s):  
Morey L Smith ◽  
Sha Jin ◽  
Dong Chen ◽  
Haichao Zhang ◽  
Jason Huska ◽  
...  
Keyword(s):  
Cell Death ◽  
Fusion Protein ◽  
Cell Line ◽  
Receptor Agonist ◽  
B Cell Lymphoma ◽  
Synergistic Activity ◽  
Trail Receptor ◽  
Phase I Clinical Trials ◽  
Single Chain ◽  
Publicly Traded

Cell death can be initiated through activation of the extrinsic and intrinsic apoptotic signaling pathways. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, preferentially triggers the extrinsic apoptotic pathway by binding as a trimer to two closely related cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Receptor trimerization leads to the formation of the death-inducing signaling complex (DISC) to recruit and activate downstream caspases that ultimately leads to apoptotic cell death. Because TRAIL signaling induces apoptosis, several TRAIL receptor agonists have been developed for the treatment of cancer. ABBV-621 is a novel, second generation TRAIL receptor agonist that is an engineered fusion protein consisting of an IgG1-Fc linked to a single chain trimer of TRAIL subunits resulting in a total of six death receptor binding sites per molecule to maximize receptor clustering that is currently being tested in Phase I clinical trials (NCT03082209). To expand upon the potential therapeutic utility of ABBV-621, we tested the combinatorial activity of ABBV-621 with numerous standard-of-care (SoC) therapeutics and targeted agents in diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Thein vitroresults led to selection of agents to combine with ABBV-621 forin vivostudies. In DLBCL cell line-derived xenograft (CDX) preclinical models, we observed combination activity of ABBV-621 with pevonedistat (PEV) a selective NEDD8 inhibitor. Additionally, synergistic activity was observed with ABBV-621 with either bendamustine (BED) or rituximab (RTX) alone, or BED/RTX together. In AML, we observed compelling combination activity of ABBV-621 with PEV in cell line-derived xenograft (CDX) models. In MM, combination of ABBV-621 plus bortezomib (BTZ) resulted in deeper anti-tumorigenic activity than either agent alone in several CDX models. The pre-clinical data presented here support expanding the indications and settings where ABBV-621 may have utility. A clinical trial assessing the activity of ABBV-621 in combination with bortezomib and dexamethasone in R/R MM patients is planned. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Disclosures Smith: AbbVie:Current Employment, Current equity holder in publicly-traded company.Jin:AbbVie:Current Employment, Current equity holder in publicly-traded company.Chen:AbbVie:Current Employment, Current equity holder in publicly-traded company.Zhang:AbbVie:Current Employment, Current equity holder in publicly-traded company.Huska:AbbVie:Current Employment, Current equity holder in publicly-traded company.Widomski:AbbVie:Current Employment, Current equity holder in publicly-traded company.Bontcheva:AbbVie:Current Employment, Current equity holder in publicly-traded company.Buchanan:AbbVie:Current Employment, Current equity holder in publicly-traded company.Morgan-Lappe:AbbVie:Current Employment, Current equity holder in publicly-traded company.Phillips:AbbVie:Current Employment, Current equity holder in publicly-traded company.Tahir:AbbVie:Current Employment, Current equity holder in publicly-traded company.

scholarly journals Kinesin-5–dependent Poleward Flux and Spindle Length Control in Drosophila Embryo Mitosis

2009 ◽  
Vol 20 (6) ◽  
pp. 1749-1762 ◽  
Author(s):  
Ingrid Brust-Mascher ◽  
Patrizia Sommi ◽  
Dhanya K. Cheerambathur ◽  
Jonathan M. Scholey
Keyword(s):  
Gene Dosage ◽  
Time Lapse ◽  
Drosophila Embryo ◽  
Spatial Gradient ◽  
Genetic Manipulations ◽  
Chromosome Congression ◽  
Spindle Elongation ◽  
Transgenic Embryos ◽  
Force Generator ◽  
Poleward Flux

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system.

scholarly journals Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.

1992 ◽  
Vol 3 (1) ◽  
pp. 1-11 ◽  
Author(s):  
D R Kellogg ◽  
B M Alberts
Keyword(s):  
Fusion Protein ◽  
Polyclonal Antibodies ◽  
Single Step ◽  
Drosophila Embryo ◽  
Multiprotein Complex ◽  
Immunoaffinity Column ◽  
Centrosomal Protein ◽  
Partial Cdna ◽  
Partial Cdna Clone ◽  
Centrosomal Proteins

A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.

The Snail repressor positions Notch signaling in the Drosophila embryo

Development ◽  
2002 ◽  
Vol 129 (7) ◽  
pp. 1785-1793 ◽  
Author(s):  
John Cowden ◽  
Michael Levine
Keyword(s):  
Notch Signaling ◽  
Target Genes ◽  
Regulatory Gene ◽  
Ectopic Expression ◽  
Cell Types ◽  
Notch Receptor ◽  
Drosophila Embryo ◽  
Multiple Cell ◽  
Transgenic Embryos ◽  
Neurogenic Ectoderm

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. We have investigated the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2 enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in ‘naïve’ embryos that contain low uniform levels of Dorsal. We suggest that these dual activities of Snail, repression of Notch target genes and stimulation of Notch signaling, help define precise lines of sim expression within the neurogenic ectoderm.

Differential interactions between Brother proteins and Runt domain proteins in the Drosophila embryo and eye

Development ◽  
1999 ◽  
Vol 126 (15) ◽  
pp. 3313-3322
Author(s):  
L.H. Li ◽  
J.P. Gergen
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Biological Activities ◽  
Binding Activity ◽  
Dominant Negative ◽  
Drosophila Embryo ◽  
Runt Domain ◽  
Big Brother ◽  
Dna Binding Activity

Brother and Big brother were isolated as Runt-interacting proteins and are homologous to CBF(beta), which interacts with the mammalian CBF(alpha) Runt-domain proteins. In vitro experiments indicate that Brother family proteins regulate the DNA binding activity of Runt-domain proteins without contacting DNA. In both mouse and human there is genetic evidence that the CBF(alpha) and CBF(beta) proteins function together in hematopoiesis and leukemogenesis. Here we demonstrate functional interactions between Brother proteins and Runt domain proteins in Drosophila. First, we show that a specific point mutation in Runt that disrupts interaction with Brother proteins but does not affect DNA binding activity is dysfunctional in several in vivo assays. Interestingly, this mutant protein acts dominantly to interfere with the Runt-dependent activation of Sxl-lethal transcription. To investigate further the requirements for Brother proteins in Drosophila development, we examine the effects of expression of a Brother fusion protein homologous to the dominant negative CBF(beta)::SMMHC fusion protein that is associated with leukemia in humans. This Bro::SMMHC fusion protein interferes with the activity of Runt and a second Runt domain protein, Lozenge. Moreover, we find that the effects of lozenge mutations on eye development are suppressed by expression of wild-type Brother proteins, suggesting that Brother/Big brother dosage is limiting in this developmental context. Results obtained when Runt is expressed in developing eye discs further support this hypothesis. Our results firmly establish the importance of the Brother and Big brother proteins for the biological activities of Runt and Lozenge, and further suggest that Brother protein function is not restricted to enhancing DNA-binding.

scholarly journals Activation and repression by the C-terminal domain of Dorsal

Development ◽  
2001 ◽  
Vol 128 (10) ◽  
pp. 1869-1879 ◽  
Author(s):  
R.D. Flores-Saaib ◽  
S. Jia ◽  
A.J. Courey
Keyword(s):  
Fusion Protein ◽  
Concentration Gradient ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Fusion Proteins ◽  
Drosophila Embryo ◽  
Terminal Domain ◽  
Localization Signal ◽  
Pattern Forming ◽  
Rel Homology Domain

In the Drosophila embryo, Dorsal, a maternally expressed Rel family transcription factor, regulates dorsoventral pattern formation by activating and repressing zygotically active fate-determining genes. Dorsal is distributed in a ventral-to-dorsal nuclear concentration gradient in the embryo, the formation of which depends upon the spatially regulated inhibition of Dorsal nuclear uptake by Cactus. Using maternally expressed Gal4/Dorsal fusion proteins, we have explored the mechanism of activation and repression by Dorsal. We find that a fusion protein containing the Gal4 DNA-binding domain fused to full-length Dorsal is distributed in a nuclear concentration gradient that is similar to that of endogenous Dorsal, despite the presence of a constitutively active nuclear localization signal in the Gal4 domain. Whether this fusion protein activates or represses reporter genes depends upon the context of the Gal4-binding sites in the reporter. A Gal4/Dorsal fusion protein lacking the conserved Rel homology domain of Dorsal, but containing the non-conserved C-terminal domain also mediates both activation and repression, depending upon Gal4-binding site context. A region close to the C-terminal end of the C-terminal domain has homology to a repression motif in Engrailed - the eh1 motif. Deletion analysis indicates that this region mediates transcriptional repression and binding to Groucho, a co-repressor known to be required for Dorsal-mediated repression. As has previously been shown for repression by Dorsal, we find that activation by Dorsal, in particular by the C-terminal domain, is modulated by the maternal terminal pattern-forming system.

Arrest of intravitelline mitoses in Drosophila embryos by u.v. irradiation of the egg surface

Development ◽  
1984 ◽  
Vol 80 (1) ◽  
pp. 43-61
Author(s):  
Shin Togashi ◽  
Masukichi Okada
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Control Mechanism ◽  
Drosophila Embryo ◽  
Posterior Half ◽  
Simple Diffusion ◽  
Cytoplasmic Region ◽  
Anterior Half ◽  
Cytoskeletal Elements ◽  
Drosophila Embryos

The intravitelline mitosis in Drosophila was arrested at the anaphase within the span of a single cell cycle after irradiation with 300 nm u.v. Embryos at and before the 8-nucleus stage were influenced by the u.v. only when irradiated anteriorly, while at and after the 16-nucleus stage, embryos are sensitive to either anterior or posterior irradiation. In embryos anteriorly irradiated at or before the 8-nucleus stage all nuclei in the embryo were prevented from performing mitosis. When irradiated at or after the 16-nucleus stage, inhibition of the intravitelline mitosis is limited to the nuclei in approximately anterior-half region of embryos in anterior irradiation, and to those inapproximately posterior-half region in posterior irradiation, resulting in incomplete blastoderm formation. Sites sensitive to 300 nm u.v. are postulated to be present in the peripheral cytoplasmic region of the embryo and not in the nucleus, because the half-attenuation thickness of 300 nm u.v. light for the Drosophila egg cytoplasm is 3 µm and nuclei are at least 50 µm away from the periphery at the stage of irradiation. In addition lateral irradiation of a portion of an egg where there is no nucleus underneath was also effective in arresting division of nuclei in the same egg. It is suggested that the effects of 300nm u.v. may not be conveyed to the nuclei from the periphery by simple diffusion of a substance, and a hypothesis is proposed for the involvement of cytoskeletal elements associated with the u.v. sensitive sites on the surface to the control mechanism of the intravitelline mitosis of the Drosophila embryo.

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