Restricted patterning of vestigial expression in Drosophila wing imaginal discs requires synergistic activation by both Mad and the drifter POU domain transcription factor

The Drosophila Vestigial protein has been shown to play an essential role in the regulation of cell proliferation and differentiation within the developing wing imaginal disc. Cell-specific expression of vg is controlled by two separate transcriptional enhancers. The boundary enhancer controls expression in cells near the dorsoventral (DV) boundary and is regulated by the Notch signal transduction pathway, while the quadrant enhancer responds to the Decapentaplegic and Wingless morphogen gradients emanating from cells near the anteroposterior (AP) and DV boundaries, respectively. MAD-dependent activation of the vestigial quadrant enhancer results in broad expression throughout the wing pouch but is excluded from cells near the DV boundary. This has previously been thought to be due to direct repression by a signal from the DV boundary; however, we show that this exclusion of quadrant enhancer-dependent expression from the DV boundary is due to the absence of an additional essential activator in those cells. The Drosophila POU domain transcriptional regulator, Drifter, is expressed in all cells within the wing pouch expressing a vgQ-lacZ transgene and is also excluded from the DV boundary. Viable drifter hypomorphic mutations cause defects in cell proliferation and wing vein patterning correlated with decreased quadrant enhancer-dependent expression. Drifter misexpression at the DV boundary using the GAL4/UAS system causes ectopic outgrowths at the distal wing tip due to induction of aberrant Vestigial expression, while a dominant-negative Drifter isoform represses expression of vgQ-lacZ and causes severe notching of the adult wing. In addition, we have identified an essential evolutionarily conserved sequence element bound by the Drifter protein with high affinity and located adjacent to the MAD binding site within the quadrant enhancer. Our results demonstrate that Drifter functions along with MAD as a direct activator of Vestigial expression in the wing pouch.

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The vrille Gene of Drosophila Is a Maternal Enhancer of decapentaplegic and Encodes a New Member of the bZIP Family of Transcription Factors

Genetics ◽

10.1093/genetics/146.4.1345 ◽

1997 ◽

Vol 146 (4) ◽

pp. 1345-1363 ◽

Cited By ~ 3

Author(s):

Hélène George ◽

Régine Terracol

Keyword(s):

Transcription Factors ◽

Signal Transduction Pathway ◽

Dorsoventral Patterning ◽

Wing Vein ◽

Human T Cell ◽

Tadpole Tail ◽

Bzip Domain ◽

Bzip Family ◽

Mutation Allele

We report here the genetical and molecular characterization of a new Drosophila zygotic lethal locus, vrille (vri). vri alleles act not only as dominant maternal enhancers of embryonic dorsoventral patterning defects caused by easter and decapentaplegic (dpp) mutations, but also as dominant zygotic enhancers of dpp alleles for phenotypes in wing. The vri gene encodes a new member of the bZIP family of transcription factors closely related to gene 9 of Xenopus laevis, induced by thyroid hormone during the tadpole tail resorption program, and NF-IL3A, a human T cell transcription factor that transactivates the interleukin-3 promoter. NF-IL3A shares 93% similarity and 60% identity with Vri for a stretch of 68 amino acids that includes the bZIP domain. Although all the alleles tested behave like antimorphs, the dominant enhancement is also seen with a nonsense mutation allele that prevents translation of the bZIP domain. Because of the strong dominant enhancement of dpp phenotypes by vri alleles in both embryo and wing, and also the similarity between the wing vein phenotypes caused by the vri and shortvein dpp alleles, we postulate that vri interacts either directly or indirectly with certain components of the dpp (a TGFβ homologue) signal transduction pathway.

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Disrupting the LINC complex by AAV mediated gene transduction prevents progression of Lamin induced cardiomyopathy

Nature Communications ◽

10.1038/s41467-021-24849-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ruth Jinfen Chai ◽

Hendrikje Werner ◽

Peter Yiqing Li ◽

Yin Loon Lee ◽

Khaing Thet Nyein ◽

...

Keyword(s):

Cardiac Failure ◽

Viral Vector ◽

Nuclear Periphery ◽

Dominant Negative ◽

Gene Transduction ◽

Linc Complex ◽

Specific Expression ◽

Common Cause ◽

Complex Protein ◽

One Year

AbstractMutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3–4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1’s function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.

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Olfactory expression of trace amine-associated receptors requires cooperative cis-acting enhancers

Nature Communications ◽

10.1038/s41467-021-23824-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ami Shah ◽

Madison Ratkowski ◽

Alessandro Rosa ◽

Paul Feinstein ◽

Thomas Bozza

Keyword(s):

Gene Expression ◽

Large Family ◽

Sequence Motifs ◽

Specific Expression ◽

Cis Acting ◽

Conserved Sequence ◽

Trace Amine ◽

Sequence Elements ◽

Cell Type Specific Expression ◽

Cell Type Specific

AbstractOlfactory sensory neurons express a large family of odorant receptors (ORs) and a small family of trace amine-associated receptors (TAARs). While both families are subject to so-called singular expression (expression of one allele of one gene), the mechanisms underlying TAAR gene choice remain obscure. Here, we report the identification of two conserved sequence elements in the mouse TAAR cluster (T-elements) that are required for TAAR gene expression. We observed that cell-type-specific expression of a TAAR-derived transgene required either T-element. Moreover, deleting either element reduced or abolished expression of a subset of TAAR genes, while deleting both elements abolished olfactory expression of all TAARs in cis with the mutation. The T-elements exhibit several features of known OR enhancers but also contain highly conserved, unique sequence motifs. Our data demonstrate that TAAR gene expression requires two cooperative cis-acting enhancers and suggest that ORs and TAARs share similar mechanisms of singular expression.

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Insulin Stimulates Primary β-Cell Proliferation via Raf-1 Kinase

Endocrinology ◽

10.1210/en.2007-1557 ◽

2008 ◽

Vol 149 (5) ◽

pp. 2251-2260 ◽

Cited By ~ 56

Author(s):

Jennifer L. Beith ◽

Emilyn U. Alejandro ◽

James D. Johnson

Keyword(s):

Cell Proliferation ◽

Dominant Role ◽

Cell Mass ◽

Diabetes Type 2 ◽

Dominant Negative ◽

Cell Replication ◽

Β Cell ◽

Physiological Control ◽

Primary Mouse

A relative decrease in β-cell mass is key in the pathogenesis of type 1 diabetes, type 2 diabetes, and in the failure of transplanted islet grafts. It is now clear that β-cell duplication plays a dominant role in the regulation of adult β-cell mass. Therefore, knowledge of the endogenous regulators of β-cell replication is critical for understanding the physiological control of β-cell mass and for harnessing this process therapeutically. We have shown that concentrations of insulin known to exist in vivo act directly on β-cells to promote survival. Whether insulin stimulates adult β-cell proliferation remains unclear. We tested this hypothesis using dispersed primary mouse islet cells double labeled with 5-bromo-2-deoxyuridine and insulin antisera. Treating cells with 200-pm insulin significantly increased proliferation from a baseline rate of 0.15% per day. Elevating glucose from 5–15 mm did not significantly increase β-cell replication. β-Cell proliferation was inhibited by somatostatin as well as inhibitors of insulin signaling. Interestingly, inhibiting Raf-1 kinase blocked proliferation stimulated by low, but not high (superphysiological), insulin doses. Insulin-stimulated mouse insulinoma cell proliferation was dependent on both phosphatidylinositol 3-kinase/Akt and Raf-1/MAPK kinase pathways. Overexpression of Raf-1 was sufficient to increase proliferation in the absence of insulin, whereas a dominant-negative Raf-1 reduced proliferation in the presence of 200-pm insulin. Together, these results demonstrate for the first time that insulin, at levels that have been measured in vivo, can directly stimulate β-cell proliferation and that Raf-1 kinase is involved in this process. These findings have significant implications for the understanding of the regulation of β-cell mass in both the hyperinsulinemic and insulin-deficient states that occur in the various forms of diabetes.

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Intestinal Apolipoprotein A-IV Gene Transcription Is Controlled by Two Hormone-Responsive Elements: A Role for Hepatic Nuclear Factor-4 Isoforms

Molecular Endocrinology ◽

10.1210/me.2004-0462 ◽

2005 ◽

Vol 19 (9) ◽

pp. 2320-2334 ◽

Cited By ~ 22

Author(s):

Amena Archer ◽

Dominique Sauvaget ◽

Valérie Chauffeton ◽

Pierre-Etienne Bouchet ◽

Jean Chambaz ◽

...

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Distal Region ◽

Proximal Region ◽

Dominant Negative ◽

Specific Expression ◽

Nuclear Factors ◽

Dominant Negative Form ◽

Responsive Element ◽

Hormone Responsive Element

Abstract In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4α and γ. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4α repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4α and γ functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.

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Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2162 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2162-2171 ◽

Cited By ~ 66

Author(s):

C S Madsen ◽

S C Ghivizzani ◽

W W Hauswirth

Keyword(s):

Mitochondrial Dna ◽

Protein Binding ◽

Loop Formation ◽

Sequence Element ◽

Conserved Sequence ◽

Loop Region ◽

In Organello ◽

Close Proximity ◽

D Loop

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.

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Homology-Dependent Maternal Inhibition of Developmental Excision of Internal Eliminated Sequences inParamecium tetraurelia

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7075 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7075-7085 ◽

Cited By ~ 83

Author(s):

Sandra Duharcourt ◽

Anne-Marie Keller ◽

Eric Meyer

Keyword(s):

Inhibition Efficiency ◽

Germ Line ◽

Single Copy ◽

Sequence Specificity ◽

Direct Repeats ◽

Maternal Control ◽

Sequence Element ◽

Conserved Sequence ◽

Internal Eliminated Sequences ◽

Sexual Generation

ABSTRACT Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events.Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5′-TA-3′ direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.

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Abstract 160: Pim-1 Preserves Cardiac Stem Cell Proliferation with Age

Circulation Research ◽

10.1161/res.111.suppl_1.a160 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Michael McGregor ◽

Shabana Din ◽

Natalie Gude ◽

Mark A Sussman

Keyword(s):

Cell Proliferation ◽

Stem Cell ◽

Time Course ◽

Protein A ◽

Cell Types ◽

Tissue Homeostasis ◽

Dominant Negative ◽

Repair Capacity ◽

Tissue Samples ◽

The Impact

Rationale Cardiac stem cells (CSC) regulate cardiomyogenesis and support regenerative processes in the heart, but aging adversely affects stem cell repair capacity. Aging is a primary cause of impaired cardiac function characterized by accumulation of senescent cells. CSC senescence is associated with permanent growth arrest that decreases survival signaling and cellular replacement, inevitably diminishing the capacity of the heart to maintain tissue homeostasis. Therefore, promoting CSC growth may improve cardiac performance with age. Pim-1 kinase exhibits protective and proliferative effects in the myocardium but the role of Pim-1 in cardiac aging has not been thoroughly studied. Objective Demonstrate that Pim-1 promotes stem cell growth in the aged myocardium correlating with increased expression of centromere protein A (CENP-A), a kinetochore-associated protein known to support cell proliferation in numerous species and cell types. Methods & Results CENP-A expression levels were evaluated from murine myocardial tissue samples ranging in age from 11 days post coitum to 4 months of age with analysis by immunoblot as well as quantitative PCR. CENP-A expression was colocalized with c-kit as a marker of CSC by immunohistochemical labeling, revealing a decline in CENP-A expression over the time course of postnatal myocardial maturation. The impact of Pim-1 upon CENP-A level was assessed by comparative analysis of non-transgenic mice versus genetically modified transgenic mouse lines expressing either Pim-1 (wild type) or a dominant negative functionally dead Pim-1 mutant. Pim-1 overexpression increases persistence of CENP-A in CSCs with age, as well as the prevalence of cycling CSCs as marked by phosph-H3 expression, while the functionally dead mutant accelerates CENP-A diminution and decreases CSC proliferation. Conclusion CENP-A decline in c-kit positive cells with age provides intriguing evidence of a potential mechanism for the diminished capacity of CSCs to maintain tissue homeostasis. Pim-1 mitigates CENP-A diminution, demonstrating the promising potential of Pim-1 to promote cardiac growth and repair with age.

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Cdk5 mediates changes in morphology and promotes apoptosis of astrocytoma cells in response to heat shock

Journal of Cell Science ◽

10.1242/jcs.114.6.1145 ◽

2001 ◽

Vol 114 (6) ◽

pp. 1145-1153 ◽

Cited By ~ 1

Author(s):

C. Gao ◽

S. Negash ◽

H.S. Wang ◽

D. Ledee ◽

H. Guo ◽

...

Keyword(s):

Heat Shock ◽

Cell Line ◽

Fluorescent Protein ◽

Morphological Changes ◽

Normal Growth ◽

Dominant Negative ◽

Specific Expression ◽

Cell Matrix ◽

Dominant Negative Mutation ◽

U373 Cells

The cyclin-dependent kinase member, Cdk5, is expressed in a variety of cell types, but neuron-specific expression of its activator, p35, is thought to limit its activity to neurons. Here we demonstrate that both Cdk5 and p35 are expressed in the human astrocytoma cell line, U373. Cdk5 and p35 are present in the detergent-insoluble cytoskeletal fraction of this cell line and Cdk5 localizes to filopodia and vinculin-rich regions of cell-matrix contact in lamellopodia. When exposed to a 46(o)C heat shock, U373 cells change shape, lose cell-matrix contacts and show increased levels of apoptosis. To test whether Cdk5 activation might play a role in these events, U373 cells were stably transfected with histidine-tagged or green fluorescent protein-tagged constructs of Cdk5 or a dominant negative mutation, Cdk5T33. Under normal growth conditions, growth characteristics of the stably transfected lines were indistinguishable from untransfected U373 cells and Cdk5 localization was not changed. However, when subjected to heat shock, cells stably transfected with Cdk5-T33 remained flattened, showed little loss of cell-matrix adhesion, and exhibited significantly lower levels of apoptosis. In contrast, cells that overexpressed wild-type Cdk5 showed morphological changes similar to those seen in untransfected U373 cells in response to heat shock and had significantly higher levels of apoptosis. Heat-shocked cells showed changes in p35 mobility and stability of the Cdk5/p35 complex consistent with endogenous Cdk5 activity. Together these findings suggest that endogenous Cdk5 activity may play a key role in regulating morphology, attachment, and apoptosis in U373 cells, and raise the possibility that Cdk5 may be a general regulator of cytoskeletal organization and cell adhesion in both neuronal and non-neuronal cells.

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Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7685-7692.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7685-7692 ◽

Cited By ~ 86

Author(s):

Sylvie Urbé ◽

Ian G. Mills ◽

Harald Stenmark ◽

Naomi Kitamura ◽

Michael J. Clague

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Membrane Trafficking ◽

Signal Transduction Pathway ◽

Dominant Negative ◽

Kinase Substrate ◽

Hepatocyte Growth ◽

Tyrosine Kinase Substrate

ABSTRACT Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.

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