Tufted Is a Gain-of-Function Allele That Promotes Ectopic Expression of the Proneural Gene amos in Drosophila

Abstract The Tufted1 (Tft1) dominant mutation promotes the generation of ectopic bristles (macrochaetae) in the dorsal mesothorax of Drosophila. Here we show that Tft1 corresponds to a gain-of-function allele of the proneural gene amos that is associated with a chromosomal aberration at 36F-37A. This causes ectopic expression of amos in large domains of the lateral-dorsal embryonic ectoderm, which results in supernumerary neurons of the PNS, and in the notum region of the third instar imaginal wing, which gives rise to the mesothoracic extra bristles. Revertants of Tft1, which lack ectopic neurons and bristles, do not show ectopic expression of amos. One revertant is a loss-of-function allele of amos and has a recessive phenotype in the embryonic PNS. Our results suggest that both normal and ectopic Tft1 bristles are generated following similar rules, and both are subjected to Notch-mediated lateral inhibition. The ability of Tft1 bristles to appear close together may be due to amos having a stronger proneural capacity than that of other proneural genes like asense and scute. This ability might be related to the wild-type function of amos in promoting development of large clusters of closely spaced olfactory sensilla.

Download Full-text

Mutant p53 Disrupts MCF-10A Cell Polarity in Three-dimensional Culture via Epithelial-to-mesenchymal Transitions

Journal of Biological Chemistry ◽

10.1074/jbc.m110.214585 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16218-16228 ◽

Cited By ~ 60

Author(s):

Yanhong Zhang ◽

Wensheng Yan ◽

Xinbin Chen

Keyword(s):

Cell Polarity ◽

Expression Patterns ◽

Ectopic Expression ◽

Three Dimensional ◽

Mutant P53 ◽

Wild Type ◽

Gain Of Function ◽

Three Dimensional Culture ◽

Wild Type P53 ◽

E Cadherin

Mutant p53 is not only deficient in tumor suppression but also acquires additional activity, called gain of function. Mutant p53 gain of function is recapitulated in knock-in mice that carry one null allele and one mutant allele of the p53 gene. These knock-in mice develop aggressive tumors compared with p53-null mice. Recently, we and others showed that tumor cells carrying a mutant p53 are addicted to the mutant for cell survival and resistance to DNA damage. To further define mutant p53 gain of function, we used the MCF-10A three-dimensional model of mammary morphogenesis. MCF-10A cells in three-dimensional culture undergo a series of morphological changes and form polarized and growth-arrested spheroids with hollow lumen, which resembles normal glandular architectures in vivo. Here, we found that endogenous wild-type p53 in MCF-10A cells was not required for acinus formation, but knockdown of endogenous wild-type p53 (p53-KD) led to partial clearance of cells in the lumen due to decreased apoptosis. Consistent with this, p53-KD altered expression patterns of the cell adhesion molecule E-cadherin, the cytoskeletal marker β-catenin, and the extracellular matrix protein laminin V. We also found that ectopic expression of the mutant G245S led to a phenotype similar to p53-KD, whereas a combination of ectopic expression of siRNA-resistant G245S with p53-KD led to a less cleared lumen. In contrast, ectopic expression of mutant R248W, R175H, and R273H disrupted normal acinus architectures with filled lumen and led to formation of irregular and multiacinus structures regardless of p53-KD. In addition, these mutants altered normal expression patterns and/or levels of E-cadherin, β-catenin, laminin V, and tight junction marker ZO-1. Furthermore, epithelial-to-mesenchymal transitions (EMT) markers, Snail, Slug, and Twist, were highly induced by mutant p53 and/or p53-KD. Together, we postulate that EMT represents a mutant p53 gain of function and mutant p53 alters cell polarity via EMT.

Download Full-text

spiel ohne grenzen/pou2is required for zebrafish hindbrain segmentation

Development ◽

10.1242/dev.129.7.1645 ◽

2002 ◽

Vol 129 (7) ◽

pp. 1645-1655 ◽

Cited By ~ 1

Author(s):

Giselbert Hauptmann ◽

Heinz-Georg Belting ◽

Uta Wolke ◽

Karen Lunde ◽

Iris Söll ◽

...

Keyword(s):

Neuronal Differentiation ◽

Hox Genes ◽

Ectopic Expression ◽

Loss Of Function ◽

Wild Type ◽

Specific Expression ◽

Size And Shape ◽

Regulatory Cascade ◽

Normal Expression ◽

Segmental Identity

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.

Download Full-text

Suppression of EZH2 Accelerates MYC-Driven Lymphomagenesis By Inhibition of Apoptosis

Blood ◽

10.1182/blood.v124.21.3009.3009 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3009-3009 ◽

Cited By ~ 1

Author(s):

Iris Appelmann ◽

Claudio Scuoppo ◽

Vishal Thapar ◽

Daniela Ledezma ◽

Amaia Lujambio ◽

...

Keyword(s):

Catalytic Activity ◽

B Cell ◽

Conflicts Of Interest ◽

Human Cancer ◽

B Cell Lymphoma ◽

Metastatic Potential ◽

Loss Of Function ◽

Wild Type ◽

Proliferating Cells ◽

Gain Of Function

Abstract EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), which also contains the non-catalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). This complex methylates histone H3 at lysine 27 (H3K27) which, together with H3K4 methylation, generates a bivalent “code” that primes genes for either expression or silencing. EZH2 is highly expressed in stem cells and many proliferating cells, downregulated in differentiated cells and frequently altered in cancer in ways that point to a context dependent role for this gene. For instance, overexpression of EZH2 has been described in prostate and breast cancer, where this overexpression is associated with invasive growth metastatic potential and poor clinical outcome. More recently, both gain and loss of function mutations of EZH2 were identified in human cancer. In particular, activating mutations of EZH2 affecting a tyrosine at position 641 located within the SET domain (Y641) were observed in lymphoma. In striking contrast to wild type EZH2 which catalyzes the monomethylation of H3K27 very efficiently and shows less efficient catalytic activity in the subsequent di- and trimethylation reactions, the mutation generates a neomorphic protein with enhanced catalytic activity and efficiency for di- and tri-methylation, hinting toward a “cooperation” of both wild type and mutant EZH2 to increase total H3K27me3 levels and representing a functional equivalent of EZH2 overexpression in human lymphoma. Gain of function mutations in EZH2 are frequent in Diffuse Large B-cell Lymphoma (DLBCL; 22%) and in Follicular Lymphoma (FL; 7%) and recent data implicate EZH2 as an oncogene in DLBCL and FL lymphomas. In contrast to DLBCL and FL, EZH2 mutations have so far never been identified in MYC-driven B-cell Non-Hodgkin Lymphoma (B-NHL), and EZH2 expression is suppressed in Burkitt’s Lymphoma (BL), a lymphoma for which a MYC translocation is pathognomonic, suggesting that in this context EZH2 could have a role different from its role in DLBCL and FL. We probed the role of EZH2 in MYC-driven lymphomagenesis by mimicking the loss of function mutations by RNAi mediated suppression of EZH2 and the gain of function mutations by over-expression of the Y641 mutant. Our results show that suppression of EZH2, but not overexpression of the EZH2 Y641 mutant, accelerates MYC-driven lymphomagenesis by attenuating apoptosis. Our model recapitulates the transcriptional signature of a subset of B-NHLs driven by MYC overactivation and EZH2 suppression. Taken together, our data imply EZH2 as a tumor suppressor in the context of MYC activation and thus raise a warning for the use of EZH2-targeted therapies in some B-NHLs subtypes. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Localization of the mei-1 gene product of Caenorhaditis elegans, a meiotic-specific spindle component.

The Journal of Cell Biology ◽

10.1083/jcb.126.1.199 ◽

1994 ◽

Vol 126 (1) ◽

pp. 199-209 ◽

Cited By ~ 67

Author(s):

S Clark-Maguire ◽

P E Mains

Keyword(s):

Regulatory Network ◽

Meiotic Spindle ◽

Loss Of Function ◽

Wild Type ◽

Female Meiosis ◽

Gain Of Function ◽

Spindle Poles ◽

Meiotic Spindles ◽

Female Germline ◽

Meiosis I

Genetic evidence suggests that the product of the mei-1 gene of Caenorhabditis elegans is specifically required for meiosis in the female germline. Loss-of-function mei-1 mutations block meiotic spindle formation while a gain-of-function allele instead results in spindle defects during the early mitotic cleavages. In this report, we use immunocytochemistry to examine the localization of the mei-1 product in wild-type and mutant embryos. During metaphase of meiosis I in wild-type embryos, mei-1 protein was found throughout the spindle but was more concentrated toward the poles. At telophase I, mei-1 product colocalized with the chromatin at the spindle poles. The pattern was repeated during meiosis II but no mei-1 product was visible during the subsequent mitotic cleavages. The mei-1 gain-of-function allele resulted in ectopic mei-1 staining in the centers of the microtubule-organizing centers during interphase and in the spindles during the early cleavages. This aberrant localization is probably responsible for the poorly formed and misoriented cleavage spindles characteristic of the mutation. We also examined the localization of mei-1(+) product in the presence of mutations of genes that genetically interact with mei-1 alleles. mei-2 is apparently required to localize mei-1 product to the spindle during meiosis while mel-26 acts as a postmeiotic inhibitor. We conclude that mei-1 encodes a novel spindle component, one that is specialized for the acentriolar meiotic spindles unique to female meiosis. The genes mei-2 and mel-26 are part of a regulatory network that confines mei-1 activity to meiosis.

Download Full-text

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

AJP Cell Physiology ◽

10.1152/ajpcell.00054.2011 ◽

2011 ◽

Vol 301 (6) ◽

pp. C1325-C1343 ◽

Cited By ~ 17

Author(s):

Andrew K. Stewart ◽

Boris E. Shmukler ◽

David H. Vandorpe ◽

Alicia Rivera ◽

John F. Heneghan ◽

...

Keyword(s):

Steady State ◽

Membrane Potential ◽

Xenopus Oocytes ◽

Cellular Responses ◽

Transport Mechanisms ◽

Loss Of Function ◽

Wild Type ◽

Gain Of Function ◽

Cation Conductance ◽

Methylammonium Chloride

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li+and86Rb+, with secondarily increased86Rb+influx sensitive to ouabain and to bumetanide. Increased RhAG-associated14C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li+,86Rb+, and14C-MA were pharmacologically distinct, and Li+uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH4+and Gd3+. RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH3/NH4+, but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA+). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH4Cl, but MA/MA+elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li+substitution or bath addition of 5 mM NH4Cl or MA/MA+. These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH3/NH4+and MA/MA+; 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA+transport, and decreased NH3/NH4+-associated depolarization; and 3) RhAG transports NH3/NH4+and MA/MA+by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.

Download Full-text

EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region

Development ◽

10.1242/dev.128.2.181 ◽

2001 ◽

Vol 128 (2) ◽

pp. 181-191 ◽

Cited By ~ 5

Author(s):

A. Liu ◽

A.L. Joyner

Keyword(s):

Mutant Mouse ◽

Explant Culture ◽

Loss Of Function ◽

Wild Type ◽

Pax6 Expression ◽

Gain Of Function ◽

Genetic Pathways ◽

Regulated Expression ◽

Mutant Mouse Embryos ◽

Double Mutant Mouse

Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address through what genetic pathways FGF8 acts, we examined the epistatic relationships of mid/hindbrain genes that respond to FGF8, using a novel mouse brain explant culture system. We found that En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, we found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Previous Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone.

Download Full-text

Gain-of-Function Mutations of fem-3, a Sex-Determination Gene in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/115.1.107 ◽

1987 ◽

Vol 115 (1) ◽

pp. 107-119 ◽

Cited By ~ 1

Author(s):

M Kathryn Barton ◽

Timothy B Schedl ◽

Judith Kimble

Keyword(s):

Sex Determination ◽

Germ Line ◽

Critical Role ◽

Temperature Shift ◽

Sensitive Period ◽

Temperature Sensitive ◽

Loss Of Function ◽

Wild Type ◽

Gain Of Function ◽

In The Wild

ABSTRACT We have isolated nine gain-of-function (gf) alleles of the sex-determination gene fem-3 as suppressors of feminizing mutations in fem-1 and fem-2. The wild-type fem-3 gene is needed for spermatogenesis in XX self-fertilizing hermaphrodites and for male development in both soma and germ line of XO animals. Loss-of-function alleles of fem-3 transform XX and XO animals into females (spermless hermaphrodites). In contrast, fem-3(gf) alleles masculinize only one tissue, the hermaphrodite germ line. Thus, XX fem-3(gf) mutant animals have a normal hermaphrodite soma, but the germ line produces a vast excess of sperm and no oocytes. All nine fem-3(gf) alleles are temperature sensitive. The temperature-sensitive period is from late L4 to early adult, a period just preceding the first signs of oogenesis. The finding of gain-of-function alleles which confer a phenotype opposite to that of loss-of-function alleles supports the idea that fem-3 plays a critical role in germ-line sex determination. Furthermore, the germ-line specificity of the fem-3(gf) mutant phenotype and the late temperature-sensitive period suggest that, in the wild-type XX hermaphrodite, fem-3 is negatively regulated so that the hermaphrodite stops making sperm and starts making oocytes. Temperature shift experiments also show that, in the germ line, sexual commitment appears to be a continuing process. Spermatogenesis can resume even after oogenesis has begun, and oogenesis can be initiated much later than normal

Download Full-text

Superficial Dsg2 Expression Limits Epidermal Blister Formation Mediated by Pemphigus Foliaceus Antibodies and Exfoliative Toxins

Dermatology Research and Practice ◽

10.1155/2010/410278 ◽

2010 ◽

Vol 2010 ◽

pp. 1-10 ◽

Cited By ~ 9

Author(s):

Donna Brennan ◽

Ying Hu ◽

Walid Medhat ◽

Alicia Dowling ◽

Mỹ G. Mahoney

Keyword(s):

Cell Adhesion ◽

Ectopic Expression ◽

Pemphigus Foliaceus ◽

Loss Of Function ◽

Wild Type ◽

Adhesive Properties ◽

Epidermal Structure ◽

Blister Formation ◽

And Function ◽

Cell Cell

Cell-cell adhesion mediated by desmosomes is crucial for maintaining proper epidermal structure and function, as evidenced by several severe and potentially fatal skin disorders involving impairment of desmosomal proteins. Pemphigus foliaceus (PF) and staphylococcal scalded skin syndrome (SSSS) are subcorneal blistering diseases resulting from loss of function of the desmosomal cadherin, desmoglein 1 (Dsg1). To further study the pathomechanism of these diseases and to assess the adhesive properties of Dsg2, we employed a recently established transgenic (Tg) mouse model expressing Dsg2 in the superficial epidermis. Neonatal Tg and wild type (WT) mice were injected with purified ETA or PF Ig. We showed that ectopic expression of Dsg2 reduced the extent of blister formation in response to both ETA and PF Ig. In response to PF Ig, we observed either a dramatic loss or a reorganization of Dsg1-α, Dsg1-β, and, to a lesser extent, Dsg1-γ, in WT mice. The Inv-Dsg2 Tg mice showed enhanced retention of Dsg1 at the cell-cell border. Collectively, our data support the role for Dsg2 in cell adhesion and suggest that ectopic superficial expression of Dsg2 can increase membrane preservation of Dsg1 and limit epidermal blister formation mediated by PF antibodies and exfoliative toxins.

Download Full-text

The Sensitivity of Activated Cys Ret Mutants to Glial Cell Line-Derived Neurotrophic Factor Is Mandatory To Rescue Neuroectodermic Cells from Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.21.20.6719-6730.2001 ◽

2001 ◽

Vol 21 (20) ◽

pp. 6719-6730 ◽

Cited By ~ 13

Author(s):

Baharia Mograbi ◽

Renata Bocciardi ◽

Isabelle Bourget ◽

Thierry Juhel ◽

Dariush Farahi-Far ◽

...

Keyword(s):

Thyroid Carcinoma ◽

Tyrosine Kinase ◽

Glial Cell ◽

Receptor Tyrosine Kinase ◽

Neurotrophic Factor ◽

Developmental Defect ◽

Loss Of Function ◽

Wild Type ◽

Gain Of Function ◽

Activating Mutations

ABSTRACT Hirschsprung's disease (HSCR), a frequent developmental defect of the enteric nervous system is due to loss-of-function mutations of RET, a receptor tyrosine kinase essential for the mediation of glial cell-derived neurotrophic factor (GDNF)-induced cell survival. Instead, gain-of-function Cys mutations (e.g., Cys609, Cys620, and Cys634) of the same gene are responsible for thyroid carcinoma (MEN2A/familial medullary thyroid carcinoma) by causing a covalent Ret dimerization, leading to ligand-independent activation of its tyrosine kinase. In this context, the association of Cys609- or Cys620-activating mutations with HSCR is still an unresolved paradox. To address this issue, we have compared these two mutants with the Cys634 Ret variant, which has never been associated with HSCR, for their ability to rescue neuroectodermic cells (SK-N-MC cells) from apoptosis. We show here that despite their constitutively activated kinase, the mere expression of these three mutants does not allow cell rescue. Instead, we demonstrate that like the wild-type Ret, the Cys634 Ret variant can trigger antiapoptotic pathways only in response to GDNF. In contrast, Cys609 or Cys620 mutations, which impair the terminal Ret glycosylation required for its insertion at the plasma membrane, abrogate GDNF-induced cell rescue. Taken together, these data support the idea that sensitivity to GDNF is the mandatory condition, even for constitutively activated Ret mutants, to rescue neuroectodermic cells from apoptosis. These findings may help clarify how a gain-of-function mutation can be associated with a developmental defect.

Download Full-text

The let-60 locus controls the switch between vulval and nonvulval cell fates in Caenorhabditis elegans.

Genetics ◽

10.1093/genetics/126.4.899 ◽

1990 ◽

Vol 126 (4) ◽

pp. 899-913 ◽

Cited By ~ 9

Author(s):

M Han ◽

R V Aroian ◽

P W Sternberg

Keyword(s):

Caenorhabditis Elegans ◽

Genetic Interaction ◽

Dominant Negative ◽

Loss Of Function ◽

Wild Type ◽

Gain Of Function ◽

Recessive Lethal ◽

Lethal Mutations ◽

Anchor Cell ◽

Inductive Signal

Abstract During induction of the Caenorhabditis elegans hermaphrodite vulva by the anchor cell of the gonad, six multipotent vulval precursor cells (VPCs) have two distinct fates: three VPCs generate the vulva and the other three VPCs generate nonspecialized hypodermis. Genes that control the fates of the VPCs in response to the anchor cell signal are defined by mutations that cause all six VPCs to generate vulval tissue (Multivulva or Muv) or that cause all six VPCs to generate hypodermis (Vulvaless or Vul). Seven dominant Vul mutations were isolated as dominant suppressors of a lin-15 Muv mutation. These mutations are dominant alleles of the gene let-60, previously identified only by recessive lethal mutations. Our genetic studies of these dominant Vul recessive lethal mutations, recessive lethal mutations, intragenic revertants of the dominant Vul mutations, and the closely mapping semi-dominant multivulva lin-34 mutations suggest that: (1) loss-of-function mutations of let-60 are recessive lethal at a larval stage, but they also cause a Vul phenotype if the lethality is rescued maternally by a lin-34 gain-of-function mutation. (2) The dominant Vul alleles of let-60 are dominant negative mutations whose gene products compete with wild-type activity. (3) lin-34 semidominant Muv alleles are either gain-of-function mutations of let-60 or gain-of-function mutations of an intimately related gene that elevates let-60 activity. We propose that let-60 activity controls VPC fates. In a wild-type animal, reception by a VPC of inductive signal activates let-60, and it generates into a vulval cell type; in absence of inductive signal, let-60 activity is low and the VPC generates hypodermal cells. Our genetic interaction studies suggest that let-60 acts downstream of let-23 and lin-15 and upstream of lin-1 and lin-12 in the genetic pathway specifying the switch between vulval and nonvulval cell types.

Download Full-text