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1.
Mol Ther Nucleic Acids ; 25: 237-250, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34458008

RESUMO

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

2.
Aging (Albany NY) ; 13(15): 19127-19144, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34339392

RESUMO

The turnover of the epidermis beginning with the progenitor cells in the basal layer to the fully differentiated corneocytes is tightly regulated by calcium. Calcium more than anything else promotes the differentiation of keratinocytes which implies the need for a calcium gradient with low concentrations in the stratum basale and high concentrations in the stratum granulosum. One of the hallmarks of skin aging is a collapse of this gradient that has a direct impact on the epidermal fitness. The rise of calcium in the stratum basale reduces cell proliferation, whereas the drop of calcium in the stratum granulosum leads to a changed composition of the cornified envelope. We showed that keratinocytes respond to the calcium induced block of cell division by a large increase of the expression of several miRNAs (hsa-mir542-5p, hsa-mir125a, hsa-mir135a-5p, hsa-mir196a-5p, hsa-mir491-5p and hsa-mir552-5p). The pitfall of this rescue mechanism is a dramatic change in gene expression which causes a further impairment of the epidermal barrier. This effect is attenuated by a pseudogene (SPRR2C) that gives rise to a lncRNA. SPRR2C specifically resides in the stratum granulosum/corneum thus acting as a sponge for miRNAs.

3.
J Invest Dermatol ; 141(4): 883-893.e6, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32946877

RESUMO

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide-based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.


Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/terapia , Terapia Genética/métodos , Oligonucleotídeos Antissenso/administração & dosagem , Cicatrização/genética , Animais , Biópsia , Linhagem Celular , Modelos Animais de Doenças , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Éxons/genética , Fibroblastos , Fibrose , Humanos , Queratinócitos , Camundongos , Camundongos Transgênicos , Mutação , Oligonucleotídeos Antissenso/genética , Cultura Primária de Células , Pele/efeitos dos fármacos
4.
J Invest Dermatol ; 140(10): 1985-1993.e5, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32142798

RESUMO

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.


Assuntos
Sistemas CRISPR-Cas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Edição de Genes , Células Cultivadas , Reparo do DNA por Junção de Extremidades , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Queratinócitos/metabolismo , Mutação , Ribonucleoproteínas/farmacologia
5.
Geroscience ; 42(1): 19-38, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31676965

RESUMO

Originally Lipid droplets (LDs) were considered as being droplets for lipid storage only. Increasing evidence, however, demonstrates that LDs fulfill a pleiotropy of additional functions. Among them is the modulation of protein as well as lipid homeostasis. Under unfavorable pro-oxidative conditions, proteins can form aggregates which may exceed the overall proteolytic capacity of the proteasome. After stress termination LDs can adjust and support the removal of these aggregates. Additionally, LDs interact with mitochondria, specifically take over certain proteins and thus prevent apoptosis. LDs, which are loaded with these harmful proteins, are subsequently eliminated via lipophagy. Recently it was demonstrated that this autophagic process is a modulator of longevity. LDs do not only eliminate potentially dangerous proteins, but they are also able to prevent lipotoxicity by storing specific lipids. In the present study we used the model organism Saccharomyces cerevisiae to compare the proteome as well as lipidome of mitochondria and LDs under different conditions: replicative aging, stress and apoptosis. In this context we found an accumulation of proteins at LDs, supporting the role of LDs in proteostasis. Additionally, the composition of main lipid classes such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylglycerols, triacylglycerols, ceramides, phosphatidic acids and ergosterol of LDs and mitochondria changed during stress conditions and aging.


Assuntos
Gotículas Lipídicas , Saccharomyces cerevisiae , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Lipídeos , Mitocôndrias/metabolismo , Proteostase
6.
Cell Rep ; 27(3): 955-970.e7, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995488

RESUMO

Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, we identified 1,983 protein-protein interactions (PPIs), more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, we validated a subset of our interactions using MITOMI and bimolecular fluorescence complementation assays. We combined our interactome with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. Our analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. We further demonstrate the utility of our Drosophila interactome in shedding light on human TF-TF interactions. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation.


Assuntos
Drosophila melanogaster/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , DNA/química , DNA/metabolismo , Regulação da Expressão Gênica , Microscopia de Fluorescência , Mapas de Interação de Proteínas/genética , Elementos Reguladores de Transcrição , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
7.
Elife ; 72018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30247122

RESUMO

Transcription factors achieve specificity by establishing intricate interaction networks that will change depending on the cell context. Capturing these interactions in live condition is however a challenging issue that requires sensitive and non-invasive methods.We present a set of fly lines, called 'multicolor BiFC library', which covers most of the Drosophila transcription factors for performing Bimolecular Fluorescence Complementation (BiFC). The multicolor BiFC library can be used to probe two different binary interactions simultaneously and is compatible for large-scale interaction screens. The library can also be coupled with established Drosophila genetic resources to analyze interactions in the developmentally relevant expression domain of each protein partner. We provide proof of principle experiments of these various applications, using Hox proteins in the live Drosophila embryo as a case study. Overall this novel collection of ready-to-use fly lines constitutes an unprecedented genetic toolbox for the identification and analysis of protein-protein interactions in vivo.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Biblioteca Gênica , Mapeamento de Interação de Proteínas/métodos , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Cor , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Fluorescência , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Ligação Proteica , Fatores de Transcrição/metabolismo
8.
Yeast ; 35(2): 237-249, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29044689

RESUMO

In recent decades Saccharomyces cerevisiae has proven to be one of the most valuable model organisms of aging research. Pathways such as autophagy or the effect of substances like resveratrol and spermidine that prolong the replicative as well as chronological lifespan of cells were described for the first time in S. cerevisiae. In this study we describe the establishment of an aging reporter that allows a reliable and relative quick screening of substances and genes that have an impact on the replicative lifespan. A cDNA library of the flatworm Dugesia tigrina that can be immortalized by beheading was screened using this aging reporter. Of all the flatworm genes, only one could be identified that significantly increased the replicative lifespan of S.cerevisiae. This gene is the cysteine protease cathepsin L that was sequenced for the first time in this study. We were able to show that this protease has the capability to degrade such proteins as the yeast Sup35 protein or the human α-synuclein protein in yeast cells that are both capable of forming cytosolic toxic aggregates. The degradation of these proteins by cathepsin L prevents the formation of these unfolded protein aggregates and this seems to be responsible for the increase in replicative lifespan.


Assuntos
Catepsina L/metabolismo , Planárias/microbiologia , Saccharomyces cerevisiae/genética , Animais , Catepsina L/genética , DNA Complementar , DNA Fúngico , Regulação Fúngica da Expressão Gênica , Hydra , Longevidade , Saccharomyces cerevisiae/metabolismo
9.
Development ; 144(24): 4573-4587, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29084803

RESUMO

Cells in ectotherms function normally within an often wide temperature range. As temperature dependence is not uniform across all the distinct biological processes, acclimation presumably requires complex regulation. The molecular mechanisms that cope with the disruptive effects of temperature variation are still poorly understood. Interestingly, one of five different ß-tubulin paralogs, ßTub97EF, was among the genes upregulated at low temperature in cultured Drosophila cells. As microtubules are known to be cold sensitive, we analyzed whether ßTub97EF protects microtubules at low temperatures. During development at the optimal temperature (25°C), ßTub97EF was expressed in a tissue-specific pattern primarily in the gut. There, as well as in hemocytes, expression was increased at low temperature (14°C). Although ßTub97EF mutants were viable and fertile at 25°C, their sensitivity within the well-tolerated range was slightly enhanced during embryogenesis specifically at low temperatures. Changing ß-tubulin isoform ratios in hemocytes demonstrated that ß-Tubulin 97EF has a pronounced microtubule stabilizing effect. Moreover, ßTub97EF is required for normal microtubule stability in the gut. These results suggest that ßTub97EF upregulation at low temperature contributes to acclimation by stabilizing microtubules.


Assuntos
Temperatura Baixa , Drosophila melanogaster/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/biossíntese , Aclimatação , Animais , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Trato Gastrointestinal/metabolismo , Domínios Proteicos/fisiologia , Isoformas de Proteínas/metabolismo , Ativação Transcricional/genética , Tubulina (Proteína)/genética
10.
Front Oncol ; 7: 111, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28620580

RESUMO

NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase), where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors) could be studied as a new target for cancer treatment, in particular for inhibition of metastasis.

11.
Cell Death Discov ; 3: 17016, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28386457

RESUMO

In recent years it turned out that there is not only extensive communication between the nucleus and mitochondria but also between mitochondria and lipid droplets (LDs) as well. We were able to demonstrate that a number of proteins shuttle between LDs and mitochondria and it depends on the metabolic state of the cell on which organelle these proteins are predominantly localized. Responsible for the localization of the particular proteins is a protein domain consisting of two α-helices, which we termed V-domain according to the predicted structure. So far we have detected this domain in the following proteins: mammalian BAX, BCL-XL, TCTP and yeast Mmi1p and Erg6p. According to our experiments there are two functions of this domain: (1) shuttling of proteins to mitochondria in times of stress and apoptosis; (2) clearing the outer mitochondrial membrane from pro- as well as anti-apoptotic proteins by moving them to LDs after the stress ceases. In this way the LDs are used by the cell to modulate stress response.

12.
Biomolecules ; 5(2): 545-89, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25906193

RESUMO

Oxidative stress in skin plays a major role in the aging process. This is true for intrinsic aging and even more for extrinsic aging. Although the results are quite different in dermis and epidermis, extrinsic aging is driven to a large extent by oxidative stress caused by UV irradiation. In this review the overall effects of oxidative stress are discussed as well as the sources of ROS including the mitochondrial ETC, peroxisomal and ER localized proteins, the Fenton reaction, and such enzymes as cyclooxygenases, lipoxygenases, xanthine oxidases, and NADPH oxidases. Furthermore, the defense mechanisms against oxidative stress ranging from enzymes like superoxide dismutases, catalases, peroxiredoxins, and GSH peroxidases to organic compounds such as L-ascorbate, α-tocopherol, beta-carotene, uric acid, CoQ10, and glutathione are described in more detail. In addition the oxidative stress induced modifications caused to proteins, lipids and DNA are discussed. Finally age-related changes of the skin are also a topic of this review. They include a disruption of the epidermal calcium gradient in old skin with an accompanying change in the composition of the cornified envelope. This modified cornified envelope also leads to an altered anti-oxidative capacity and a reduced barrier function of the epidermis.


Assuntos
Estresse Oxidativo , Envelhecimento da Pele , Humanos , Pele/enzimologia , Pele/metabolismo , Pele/efeitos da radiação
13.
Genome Res ; 25(4): 514-23, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25568052

RESUMO

Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise ∼ 5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such "bivalent" chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue.


Assuntos
Padronização Corporal/genética , Drosophila melanogaster/embriologia , Discos Imaginais/embriologia , Fatores de Transcrição/genética , Asas de Animais/embriologia , Animais , Animais Geneticamente Modificados , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína/genética , Interferência de RNA , RNA Polimerase II/genética , RNA Interferente Pequeno
14.
Exp Gerontol ; 68: 59-65, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25262846

RESUMO

The human epidermis provides a very effective barrier function against chemical, physical and microbial insults from the environment. This is only possible as the epidermis renews itself constantly. Stem cells located at the basal lamina which forms the dermoepidermal junction provide an almost inexhaustible source of keratinocytes which differentiate and die during their journey to the surface where they are shed off as scales. Despite the continuous renewal of the epidermis it nevertheless succumbs to aging as the turnover rate of the keratinocytes is slowing down dramatically. Aging is associated with such hallmarks as thinning of the epidermis, elastosis, loss of melanocytes associated with an increased paleness and lucency of the skin and a decreased barrier function. As the differentiation of keratinocytes is strictly calcium dependent, calcium also plays an important role in the aging epidermis. Just recently it was shown that the epidermal calcium gradient in the skin that facilitates the proliferation of keratinocytes in the stratum basale and enables differentiation in the stratum granulosum is lost in the process of skin aging. In the course of this review we try to explain how this calcium gradient is built up on the one hand and is lost during aging on the other hand. How this disturbed calcium homeostasis is affecting the gene expression in aged skin and is leading to dramatic changes in the composition of the cornified envelope will also be discussed. This loss of the epidermal calcium gradient is not only specific for skin aging but can also be found in skin diseases such as Darier disease, Hailey-Hailey disease, psoriasis and atopic dermatitis, which might be very helpful to get a deeper insight in skin aging.


Assuntos
Cálcio/metabolismo , Epiderme/fisiologia , Expressão Gênica/genética , Envelhecimento da Pele/genética , Diferenciação Celular/fisiologia , Proteínas Ricas em Prolina do Estrato Córneo/fisiologia , Regulação para Baixo/fisiologia , Humanos , Queratinócitos/citologia , Dermatopatias/genética , Regulação para Cima/fisiologia
15.
Nat Protoc ; 9(7): 1607-20, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24922270

RESUMO

Overexpression screens can be used to explore gene function in Drosophila melanogaster, but to demonstrate their full potential, comprehensive and systematic collections of fly strains are required. Here we provide a protocol for high-throughput cloning of Drosophila open-reading frames (ORFs) that are regulated by upstream activation sequences (UAS sites); the resulting GAL4-inducible UAS-ORF plasmid library is then used to generate Drosophila strains by ΦC31 integrase-mediated site-specific integration. We also provide details for FLP/FRT-mediated in vivo exchange of epitope tags (or regulatory regions) in the ORF library strains, which further extends the potential applications of the library. These transgenic UAS-ORF strains are a useful resource to complement and validate genetic experiments performed with loss-of-function mutants and RNA interference (RNAi) lines. The duration of the complete protocol strongly depends on the number of ORFs required, but embryos can be injected and balanced fly stocks can be established within ∼7-8 weeks for a few genes.


Assuntos
Clonagem Molecular/métodos , Drosophila melanogaster/genética , Biblioteca Gênica , Fases de Leitura Aberta , Animais , Código de Barras de DNA Taxonômico , Embrião não Mamífero/citologia , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Plasmídeos/genética , Transgenes
16.
Development ; 140(11): 2434-42, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23637332

RESUMO

Overexpression screens are used to explore gene functions in Drosophila, but this strategy suffers from the lack of comprehensive and systematic fly strain collections and efficient methods for generating such collections. Here, we present a strategy that could be used efficiently to generate large numbers of transgenic Drosophila strains, and a collection of 1149 UAS-ORF fly lines that were created with the site-specific ΦC31 integrase method. For this collection, we used a set of 655 genes that were cloned as two variants, either as an open reading frame (ORF) with a native stop codon or with a C-terminal 3xHA tag. To streamline the procedure for transgenic fly generation, we demonstrate the utility of injecting pools of plasmids into embryos, each plasmid containing a randomised sequence (barcode) that serves as a unique identifier for plasmids and, subsequently, fly strains. We also developed a swapping technique that facilitates the rapid exchange of promoters and epitope tags in vivo, expanding the versatility of the ORF collection. The work described here serves as the basis of a systematic library of Gal4/UAS-regulated transgenes.


Assuntos
Drosophila/genética , Biblioteca Gênica , Técnicas Genéticas , Fases de Leitura Aberta , Animais , Código de Barras de DNA Taxonômico , Proteínas de Drosophila/genética , Epitopos/química , Plasmídeos/metabolismo , Transgenes
17.
Dev Cell ; 25(2): 207-19, 2013 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-23583758

RESUMO

We created a site-directed UAS-ORF library of 655 growth-regulating genes in Drosophila. This library represents a large collection of genes regulating cell cycle, cell size, and proliferation and will be a valuable resource for studying growth regulation in vivo. By using misexpression of genes, we prevent problems arising from genetic redundancy and can uncover novel gene functions. To validate the usefulness of this library, we screened for Wingless (Wg) pathway components. We used a combination of experimental and bioinformatic approaches to predict candidates and identified three serine/threonine kinases as regulators of Wg signaling. We show that one of these, Nek2, optimizes pathway response by direct phosphorylation of Dishevelled. In addition, we describe functional relations for roughly 5% of all Drosophila genes and identify a large number of genes that regulate cell size, proliferation, and final organ size upon misexpression.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Fases de Leitura Aberta/genética , Proteínas Wnt/genética , Proteína Wnt1/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biologia Computacional , Proteínas Desgrenhadas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteína Wnt1/metabolismo
18.
Exp Dermatol ; 22(5): 329-35, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23614739

RESUMO

The main function of the epidermis is to protect us against a multitude of hostile attacks from the environment. Its main cell type, the keratinocytes have a sophisticated system of different proteins and lipids available to form the cornified envelope, which is responsible for the barrier function of the skin. During ageing, dramatic changes are taking place. Some proteins of the SPRR-, S100- and LCE3-family are massively up-regulated, whereas others like loricrin, filaggrin and the LCE1&2 protein families are significantly down-regulated. The latter ones are known to be under control of calcium and/or 'calcium response elements'. We were able to show that the calcium peak specific for the stratum granulosum, which is the site where loricrin and the LCE1&2 families are synthesized, is reduced during ageing. The resulting cornified envelope in old skin has an extensively changed composition on the molecular level compared to young skin. This knowledge is of critical importance to understand chronic wound formation and ulcers in old age.


Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/genética , Epiderme/fisiologia , Queratinócitos/fisiologia , Envelhecimento da Pele/genética , Transcriptoma , Adolescente , Adulto , Idoso , Cálcio/metabolismo , Calgranulina B/genética , Criança , Pré-Escolar , Células Epidérmicas , Feminino , Prepúcio do Pênis/citologia , Prepúcio do Pênis/fisiologia , Humanos , Lactente , Recém-Nascido , Proteínas de Filamentos Intermediários/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas/genética , Úlcera Cutânea/genética , Adulto Jovem
19.
Genes Dev ; 24(9): 881-6, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20439429

RESUMO

Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. Here we uncover Coop (corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Finally, we show that Coop antagonizes the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt1/metabolismo , Animais , Proteínas do Domínio Armadillo/metabolismo , Proteínas Correpressoras/metabolismo , Drosophila melanogaster/genética , Fatores de Transcrição/metabolismo
20.
Methods Mol Biol ; 420: 175-95, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18641947

RESUMO

The site-specific recombinase FLP is used in Drosophila to precisely manipulate the genome, in particular, to eliminate gene function by mitotic recombination and to activate transgenes in discrete populations of cells. These approaches are already part of the standard tool kit for studying gene function. The number of applications for the FLP recombinase has increased over the years and further members of the large family of site-specific recombinases are being added to the arsenal of fly geneticists, most recently, the phiC31 integrase. This chapter will introduce these recombinases and describe how such instruments are utilized to accurately manipulate the Drosophila genome.


Assuntos
Drosophila melanogaster/genética , Inativação Gênica , Técnicas de Transferência de Genes , Mutagênese , Transgenes , Alelos , Animais , DNA Nucleotidiltransferases/metabolismo , Feminino , Deleção de Genes , Técnicas Genéticas , Masculino , Mitose , Modelos Genéticos , Recombinação Genética
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