Sea urchin goosecoid function links fate specification along the animal-vegetal and oral-aboral embryonic axes

Development ◽  
2001 ◽  
Vol 128 (22) ◽  
pp. 4393-4404 ◽  
Author(s):  
Lynne M. Angerer ◽  
David W. Oleksyn ◽  
Amy M. Levine ◽  
Xiaotao Li ◽  
William H. Klein ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Target Genes ◽  
Pigment Cell ◽  
Intercellular Signaling ◽  
Loss Of Function ◽  
Transcription Activator ◽  
Embryonic Axes ◽  
Fate Specification ◽  
Oral Ectoderm

We have identified a single homolog of goosecoid, SpGsc, that regulates cell fates along both the animal-vegetal and oral-aboral axes of sea urchin embryos. SpGsc mRNA is expressed briefly in presumptive mesenchyme cells of the ∼200-cell blastula and, beginning at about the same time, accumulates in the presumptive oral ectoderm through pluteus stage. Loss-of-function assays with morpholine-substituted antisense oligonucleotides show that SpGsc is required for endoderm and pigment cell differentiation and for gastrulation. These experiments and gain-of-function tests by mRNA injection show that SpGsc is a repressor that antagonizes aboral ectoderm fate specification and promotes oral ectoderm differentiation. We show that SpGsc competes for binding to specific cis elements with SpOtx, a ubiquitous transcription activator that promotes aboral ectoderm differentiation. Moreover, SpGsc represses transcription in vivo from an artificial promoter driven by SpOtx. As SpOtx appears long before SpGsc transcription is activated, we propose that SpGsc diverts ectoderm towards oral fate by repressing SpOtx target genes. Based on the SpGsc-SpOtx example and other available data, we propose that ectoderm is first specified as aboral by broadly expressed activators, including SpOtx, and that the oral region is subsequently respecified by the action of negative regulators, including SpGsc. Accumulation of SpGsc in oral ectoderm depends on cell-cell interactions initiated by nuclear β-catenin function, which is known to be required for specification of vegetal tissues, because transcripts are undetectable in dissociated or in cadherin mRNA-injected embryos. This is the first identified molecular mechanism underlying the known dependence of oral-aboral ectoderm polarity on intercellular signaling.

scholarly journals Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

2020 ◽  
Vol 295 (39) ◽  
pp. 13617-13629
Author(s):  
Clément Immarigeon ◽  
Sandra Bernat-Fabre ◽  
Emmanuelle Guillou ◽  
Alexis Verger ◽  
Elodie Prince ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Direct Interaction ◽  
Mediator Complex ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Rna Pol Ii ◽  
Evolutionarily Conserved

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

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 Maximal Induction of a Subset of Interferon Target Genes Requires the Chromatin-Remodeling Activity of the BAF Complex

2002 ◽  
Vol 22 (18) ◽  
pp. 6471-6479 ◽  
Author(s):  
Hong Liu ◽  
Hyeog Kang ◽  
Rui Liu ◽  
Xin Chen ◽  
Keji Zhao
Keyword(s):  
Chromatin Remodeling ◽  
Target Genes ◽  
Transcription Activator ◽  
Baf Complex ◽  
Antiproliferative Effects ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Maximal Induction ◽  
Antiproliferative Activities

ABSTRACT The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha IFN (IFN-α). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling of the promoter upon IFN-α induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3 promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling BAF complex.

scholarly journals A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Gang Ning ◽  
Qihui Zhu ◽  
Wonyoung Kang ◽  
Hamin Lee ◽  
Leigh Maher ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Lines ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Pharmacological Study ◽  
Mapk Signaling ◽  
Her2 Overexpression ◽  
Loss Of Function

Abstract Background Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 13–23% of all GC cases and patients with HER2 overexpression exhibit a poor prognosis. Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor, is an effective agent to treat HER2-amplified breast cancer but it failed in gastric cancer (GC) clinical trials. However, the molecular mechanism of lapatinib resistance in HER2-amplified GC is not well studied. Methods We employed an unbiased, genome-scale screening with pooled CRISPR library on HER2-amplified GC cell lines to identify genes that are associated with resistance to lapatinib. To validate the candidate genes, we applied in vitro and in vivo pharmacological tests to confirm the function of the target genes. Results We found that loss of function of CSK or PTEN conferred lapatinib resistance in HER2-amplified GC cell lines NCI-N87 and OE19, respectively. Moreover, PI3K and MAPK signaling was significantly increased in CSK or PTEN null cells. Furthermore, in vitro and in vivo pharmacological study has shown that lapatinib resistance by the loss of function of CSK or PTEN, could be overcome by lapatinib combined with the PI3K inhibitor copanlisib and MEK inhibitor trametinib. Conclusions Our study suggests that loss-of-function mutations of CSK and PTEN cause lapatinib resistance by re-activating MAPK and PI3K pathways, and further proved these two pathways are druggable targets. Inhibiting the two pathways synergistically are effective to overcome lapatinib resistance in HER2-amplified GC. This study provides insights for understanding the resistant mechanism of HER2 targeted therapy and novel strategies that may ultimately overcome resistance or limited efficacy of lapatinib treatment for subset of HER2 amplified GC.

95 PRODUCTION OF GENE-EDITED PIGS, CATTLE, AND LAMBS BY EMBRYO INJECTION OF TALENS OR ZFNs

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
S. C. Fahrenkrug ◽  
S. G. Lillico ◽  
C. Proudfoot ◽  
T. J. King ◽  
J. H. Pryor ◽  
...  
Keyword(s):  
Direct Injection ◽  
Blastocyst Stage ◽  
Full Term ◽  
Meat Production ◽  
Loss Of Function ◽  
Transcription Activator ◽  
Leave No Trace ◽  
Large Animals

Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) DNA editing technology enables site-directed engineering of the genome. To date, all gene-edited large animals have been produced by treatment of somatic cells and cloning to produce gene-edited offspring. Although effective, it does not take advantage of the ‘leave-no-trace' aspect of site-specific nucleases, and the derivation of food animals by cloning is negatively perceived by the public. Thus, we have investigated production of gene-edited pigs, cattle and sheep by direct injection of TALEN or ZFN mRNAs to develop loss-of-function alleles for disease resistance (RELA) or enhanced meat production (GDF8). In vitro studies demonstrated activity of TALENs by cytoplasmic injection of mRNAs from dosages of 2 to 20 ng mL–1 with an apparent increase in both editing frequency and toxicity at high dosage. Our first pregnancies were produced by transfer of pig embryos (in vivo produced) injected with 2 ng mL–1 RELA TALEN mRNA. Pregnancy was confirmed in 5 of 7 recipients 4 of which went full term giving rise to 39 piglets, 8 of which carried editing events (21%). In parallel, we injected ZFN mRNA (2 ng mL–1) targeted to a similar site of the RELA gene and 2 of 2 recipients became pregnant, resulting in the birth of 9 piglets, one of which was edited (11%). For cattle injections, we derived zygotes by ovum pickup from selected Nelore dams followed by in vitro maturation and fertilization with Nelore semen. Low (2 ng mL–1) and medium (5 ng mL–1) dosages of GDF8-targeted TALENs resulted in Day 7 development to morula/blastocyst stage in 40% (n = 18) and 10% (n = 66) of cultured embryos, respectively. A total of 20 morula/blastocysts were chosen for transfer to 11 recipients, resulting in two full-term pregnancies. One pregnancy produced two calves, both of which carried edited GDF8 alleles. Complications with parturition of the second pregnancy resulted in 2 stillborn calves, the genotypes of which are under investigation. Finally, 2 ng mL–1 of TALEN mRNA targeted to ovine GDF8 was injected into in vivo-produced sheep zygotes and transferred into nine recipients, 3 blastocysts each. The pregnancy rate, number of live-born animals, and gene editing frequency is under investigation and will be reported.

Abstract 206: Stem Cell-derived Exosomes Induce Cardiac Repair via mRNA Modification

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Prabhu Mathiyalagan ◽  
Yaxuan Liang ◽  
Adriano S Martins ◽  
Douglas W Losordo ◽  
Roger J Hajjar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Myocardial Ischemia ◽  
Target Genes ◽  
Critical Role ◽  
Cell Communication ◽  
Target Cells ◽  
Mouse Heart ◽  
Loss Of Function

Exosomes are cell-derived nanovesicles that carry and shuttle microRNAs (miRNAs) to mediate cell-cell communication. Vast majority of cell types including cardiac myocytes and progenitors actively secrete exosomes, whose miRNA contents are altered after physiological or pathological changes such as myocardial ischemia (MI). In this new study, we have discovered that chemical modification to mRNAs is a novel regulator of ischemia-induced gene expression changes in the heart. We hypothesized that the benefits of human CD34 + stem cell-derived exosomes (CD34exo) are mediated by mRNA modifications in the target cells via miRNA delivery. MiRNA profiling and bioinformatic analysis identified that CD34exo is selectively enriched with a number of miRNAs that directly target genes implicated in regulation of mRNA modifications. Interestingly, under myocardial ischemia, there was a significant increase in mRNA modifications in the mouse heart, which was decreased by about 70% with CD34exo-treatment. In line with the in vivo MI data, in vitro hypoxic stimulation in neonatal / adult rodent myocytes and non-myocytes increased mRNA modifications and controls known regulators of those mRNA modifications. Loss-of-function studies for regulators of mRNA modifications attenuated hypoxia-induced changes to epitranscriptome indicating important roles for these molecules under stress conditions. Finally, using gain-of-function and loss-of-function studies, we demonstrate that miR-126, one of the most enriched miRNAs in CD34exo, plays a critical role in regulating the mRNA modifications. We conclude that miRNAs enriched in CD34exo mediate their cardioprotective effect at least in part, by regulating the mRNA epitranscriptome of the target cell. Our new data suggests hypoxia as a novel regulator of the mRNA epitranscriptome and provides novel insights to post-transcriptional gene regulation in the heart.

Abstract 293: MicroRNA-125b-5p Protect the Mouse Heart from Ischemic Injury by Repressing Pro-apoptotic Bak1 And Klf13 in Cardiomyocytes

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Zuzana Broskova ◽  
Kyoung-mi Park ◽  
Yongchao Wang ◽  
Il-man Kim
Keyword(s):  
Target Genes ◽  
Cardiac Injury ◽  
Ischemic Injury ◽  
Mouse Heart ◽  
Loss Of Function ◽  
Increased Sensitivity ◽  
Induced Apoptosis ◽  
End Stage ◽  
And Function

Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs), small non-coding RNAs that post-transcriptionally regulate target genes. For example, miR-125a is up-regulated in patients with heart failure (HF), while miR-125b is down-regulated in patients with end-stage dilated cardiomyopathy (DCM) and ischemic DCM. Circulating levels of these two miRs have been recently proposed as potential biomarkers of HF. We previously showed that β1-adrenergic receptor-mediated cardioprotective signaling through β-arrestin1 stimulates the processing of miR-125a and miR-125b in mouse heart (Figure A-C). Here, we hypothesize that these two miRs might confer cardioprotection against ischemic injury. Using cultured cardiomyocyte (CM) and in vivo approaches, we show that these miRs are ischemic stress-responsive protectors against CM apoptosis. CMs lacking miR-125a or miR-125b have an increased sensitivity to stress-induced apoptosis, while CMs overexpressing miR-125a or miR-125b have increased phospho-AKT pro-survival signaling. Moreover, we demonstrate that loss-of-function of miR-125b in mouse heart causes abnormalities in cardiac structure and function after myocardial infarction. The cardioprotective roles of the two miRs during ischemic injury are in part attributed to direct repression of the pro-apoptotic genes Bak1 and Klf13 in CMs (Figure D). In conclusion, these findings reveal pivotal roles for miR-125a and miR-125b as important regulators of CM survival during cardiac injury.

scholarly journals An in vivo genetic screen in Drosophila identifies the orthologue of human cancer/testis gene SPO11 among a network of targets to inhibit lethal(3)malignant brain tumour growth

Open Biology ◽  
2017 ◽  
Vol 7 (8) ◽  
pp. 170156 ◽  
Author(s):  
Fabrizio Rossi ◽  
Cristina Molnar ◽  
Kazuya Hashiyama ◽  
Jan P. Heinen ◽  
Judit Pampalona ◽  
...  
Keyword(s):  
Brain Tumour ◽  
Target Genes ◽  
Human Cancer ◽  
Loss Of Function ◽  
Malignant Growth ◽  
Malignant Brain Tumour ◽  
Rnai Technology ◽  
Wide Range ◽  
Cancer Testis

Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo . We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is meiotic W68 ( mei-W68 ), the Drosophila orthologue of the human cancer/testis gene Sporulation-specific protein 11 ( SPO11 ), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that Drosophila mei-W68/SPO11 drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a SPO11 orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like Drosophila.

Vegf regulates embryonic erythroid development through Gata1 modulation

Blood ◽  
2010 ◽  
Vol 116 (12) ◽  
pp. 2141-2151 ◽  
Author(s):  
Benjamin Drogat ◽  
Joanna Kalucka ◽  
Laura Gutiérrez ◽  
Hamida Hammad ◽  
Steven Goossens ◽  
...  
Keyword(s):  
Target Genes ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Erythroid Progenitor ◽  
Expression Levels ◽  
Conditional Deletion ◽  
Differentiation Block ◽  
Erythroid Development

Abstract To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf164 isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.

scholarly journals miR-128-3p Is a Novel Regulator of Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Diseases

2020 ◽  
Vol 126 (12) ◽  
Author(s):  
Floriana Maria Farina ◽  
Ignacio Fernando Hall ◽  
Simone Serio ◽  
Stefania Zani ◽  
Montserrat Climent ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Target Genes ◽  
Methylation Status ◽  
Vascular Diseases ◽  
Loss Of Function ◽  
Vsmc Proliferation ◽  
Small Noncoding Rnas ◽  
Underlying Mechanisms

Rationale: MicroRNAs (miRNAs, miRs) are small noncoding RNAs that modulate gene expression by negatively regulating translation of target genes. Although the role of several miRNAs in vascular smooth muscle cells (VSMCs) has been extensively characterized, the function of miRNA-128-3p (miR-128) is still unknown. Objective: To determine if miR-128 modulates VSMC phenotype and to define the underlying mechanisms. Methods and Results: We screened for miRNAs whose expression is modulated by an altered DNA methylation status in VSMCs, and among the hits, we selected miR-128. We found that miR-128 was expressed in various tissues, primary murine cells, and pathological murine and human vascular specimens. Through gain- and loss-of-function approaches, we determined that miR-128 affects VSMC proliferation, migration, differentiation, and contractility. The alterations of those properties were dependent upon epigenetic regulation of key VSMC differentiation genes; notably, Kruppel-like factor 4 was found to be a direct target of miR-128 and able to modulate the methylation status of the pivotal VSMC gene myosin heavy chain 11 ( Myh11 ). Finally, in vivo lentiviral delivery of miR-128 prevented intimal hyperplasia in a mouse model of carotid restenosis without modifying vital cardiovascular parameters. Conclusion: miR-128 is a critical modulator of VSMCs and is regulated by epigenetic modifications upon stress. Its modulation in the context of disease could be exploited for therapeutic purposes.

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