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1.
Proc Natl Acad Sci U S A ; 115(42): 10684-10689, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30257938

RESUMO

The Hippo tumor-suppressor pathway regulates organ growth, cell proliferation, and stem cell biology. Defects in Hippo signaling and hyperactivation of its downstream effectors-Yorkie (Yki) in Drosophila and YAP/TAZ in mammals-result in progenitor cell expansion and overgrowth of multiple organs and contribute to cancer development. Deciphering the mechanisms that regulate the activity of the Hippo pathway is key to understanding its function and for therapeutic targeting. However, although the Hippo kinase cascade and several other upstream inputs have been identified, the mechanisms that regulate Yki/YAP/TAZ activity are still incompletely understood. To identify new regulators of Yki activity, we screened in Drosophila for suppressors of tissue overgrowth and Yki activation caused by overexpression of atypical protein kinase C (aPKC), a member of the apical cell polarity complex. In this screen, we identified mutations in the heterogeneous nuclear ribonucleoprotein Hrb27C that strongly suppressed the tissue defects induced by ectopic expression of aPKC. Hrb27C was required for aPKC-induced tissue growth and Yki target gene expression but did not affect general gene expression. Genetic and biochemical experiments showed that Hrb27C affects Yki phosphorylation. Other RNA-binding proteins known to interact with Hrb27C for mRNA transport in oocytes were also required for normal Yki activity, although they suppressed Yki output. Based on the known functions of Hrb27C, we conclude that Hrb27C-mediated control of mRNA splicing, localization, or translation is essential for coordinated activity of the Hippo pathway.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas Nucleares/genética , Proteína II de Ligação a Poli(A)/genética , Proteína II de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Transativadores/genética , Proteínas de Sinalização YAP
2.
Int J Mol Sci ; 21(13)2020 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-32605129

RESUMO

Can hyperactivation of a few key signaling effectors be the underlying reason for the majority of epithelial cancers despite different driver mutations? Here, to address this question, we use the Drosophila model, which allows analysis of gene expression from tumors with known initiating mutations. Furthermore, its simplified signaling pathways have numerous well characterized targets we can use as pathway readouts. In Drosophila tumor models, changes in the activities of three pathways, Jun N-terminal Kinase (JNK), Janus Kinase / Signal Transducer and Activator of Transcription (JAK/STAT), and Hippo, mediated by AP-1 factors, Stat92E, and Yorkie, are reported frequently. We hypothesized this may indicate that these three pathways are commonly deregulated in tumors. To assess this, we mined the available transcriptomic data and evaluated the activity levels of eight pathways in various tumor models. Indeed, at least two out of our three suspects contribute to tumor development in all Drosophila cancer models assessed, despite different initiating mutations or tissues of origin. Surprisingly, we found that Notch signaling is also globally activated in all models examined. We propose that these four pathways, JNK, JAK/STAT, Hippo, and Notch, are paid special attention and assayed for systematically in existing and newly developed models.


Assuntos
Carcinogênese/patologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição STAT/metabolismo , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Metanálise como Assunto , Proteínas Nucleares/genética , Fatores de Transcrição STAT/genética , Transdução de Sinais , Transativadores/genética , Fator de Transcrição AP-1/genética , Proteínas de Sinalização YAP
3.
PLoS Genet ; 11(2): e1004994, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25679813

RESUMO

Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes.


Assuntos
Carcinogênese/genética , Cromatina/genética , Proteínas de Drosophila/genética , Fatores de Transcrição STAT/genética , Fator de Transcrição AP-1/genética , Animais , Drosophila/genética , Elementos Facilitadores Genéticos , Olho/crescimento & desenvolvimento , Olho/metabolismo , Olho/patologia , Redes Reguladoras de Genes , Humanos , Elementos Isolantes/genética , Regiões Promotoras Genéticas
4.
PLoS Genet ; 9(8): e1003731, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24009524

RESUMO

Organ development is directed by selector gene networks. Eye development in the fruit fly Drosophila melanogaster is driven by the highly conserved selector gene network referred to as the "retinal determination gene network," composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. Here, we identify a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. We found that central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. Our results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. We conclude that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development.


Assuntos
Diferenciação Celular/genética , Drosophila melanogaster/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Organogênese/genética , Retina/crescimento & desenvolvimento , Animais , Sequência Conservada , Proteínas de Ligação a DNA , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Retinaldeído
5.
Elife ; 122023 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-37133250

RESUMO

Wound response programs are often activated during neoplastic growth in tumors. In both wound repair and tumor growth, cells respond to acute stress and balance the activation of multiple programs, including apoptosis, proliferation, and cell migration. Central to those responses are the activation of the JNK/MAPK and JAK/STAT signaling pathways. Yet, to what extent these signaling cascades interact at the cis-regulatory level and how they orchestrate different regulatory and phenotypic responses is still unclear. Here, we aim to characterize the regulatory states that emerge and cooperate in the wound response, using the Drosophila melanogaster wing disc as a model system, and compare these with cancer cell states induced by rasV12scrib-/- in the eye disc. We used single-cell multiome profiling to derive enhancer gene regulatory networks (eGRNs) by integrating chromatin accessibility and gene expression signals. We identify a 'proliferative' eGRN, active in the majority of wounded cells and controlled by AP-1 and STAT. In a smaller, but distinct population of wound cells, a 'senescent' eGRN is activated and driven by C/EBP-like transcription factors (Irbp18, Xrp1, Slow border, and Vrille) and Scalloped. These two eGRN signatures are found to be active in tumor cells at both gene expression and chromatin accessibility levels. Our single-cell multiome and eGRNs resource offers an in-depth characterization of the senescence markers, together with a new perspective on the shared gene regulatory programs acting during wound response and oncogenesis.


Assuntos
Proteínas de Drosophila , Neoplasias , Animais , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Redes Reguladoras de Genes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias/patologia , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo
6.
Dev Dyn ; 238(9): 2139-48, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19623613

RESUMO

The eye-antennal imaginal disc of Drosophila melanogaster has often been described as an epithelial monolayer with complex signaling events playing out in two dimensions. However, the imaginal disc actually comprises two opposing epithelia (the peripodial epithelium, or PE, and the disc proper, or DP) separated by a lumen to form a sac-like structure. Recent studies expose complex molecular interactions between the PE and the DP, and reveal dynamic communication between the two tissues. Further findings suggest the PE makes important contributions to DP development by acting as a source of signaling molecules as well as cells. Here we summarize those findings and highlight implications for further research.


Assuntos
Epitélio/embriologia , Olho/citologia , Olho/embriologia , Transdução de Sinais , Animais , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Epitélio/metabolismo , Olho/metabolismo , Humanos , Modelos Biológicos
7.
Methods Mol Biol ; 1893: 27-42, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30565122

RESUMO

Drosophila melanogaster has been a central player in the discovery of the Hippo pathway and in understanding its in vivo functions. From a technique standpoint, the Flp-FRT system for the generation of genetic mosaics has been a principle tool. It has broadly been used in the discovery of Hippo pathway members in mutagenesis screens, in the analysis of target gene expression, and in genetic epistasis. Here we briefly introduce this tool, summarize its use in the Hippo pathway field, and provide a protocol for the generation of Flp-FRT clones in imaginal discs with dissection and staining for reporter gene expression to characterize candidate Hippo pathway genes.


Assuntos
Drosophila/genética , Mosaicismo , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Expressão Gênica , Marcação de Genes , Genes Reporter , Discos Imaginais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva , Mitose/genética , Mutagênese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética , Transdução de Sinais
8.
J Hematol Oncol ; 11(1): 108, 2018 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-30144809

RESUMO

BACKGROUND: The CNOT3 protein is a subunit of the CCR4-NOT complex, which is involved in mRNA degradation. We recently identified CNOT3 loss-of-function mutations in patients with T-cell acute lymphoblastic leukemia (T-ALL). METHODS: Here, we use different Drosophila melanogaster eye cancer models to study the potential tumor suppressor function of Not3, the CNOT3 orthologue, and other members of the CCR4-NOT complex. RESULTS: Our data show that knockdown of Not3, the structural components Not1/Not2, and the deadenylases twin/Pop2 all result in increased tumor formation. In addition, overexpression of Not3 could reduce tumor formation. Not3 downregulation has a mild but broad effect on gene expression and leads to increased levels of genes involved in DNA replication and ribosome biogenesis. CycB upregulation also contributes to the Not3 tumor phenotype. Similar findings were obtained in human T-ALL cell lines, pointing out the conserved function of Not3. CONCLUSIONS: Together, our data establish a critical role for Not3 and the entire CCR4-NOT complex as tumor suppressor.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/patogenicidade , Neoplasias Oculares/genética , Genes Supressores de Tumor/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/metabolismo , Animais , Modelos Animais de Doenças , Neoplasias Oculares/metabolismo , Humanos , Ligação Proteica
9.
Nat Genet ; 50(7): 1011-1020, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29867222

RESUMO

Transcriptional enhancers function as docking platforms for combinations of transcription factors (TFs) to control gene expression. How enhancer sequences determine nucleosome occupancy, TF recruitment and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains, we found that SNPs affecting binding sites of the TF Grainy head (Grh) causally determine the accessibility of epithelial enhancers. We show that deletion and ectopic expression of Grh cause loss and gain of DNA accessibility, respectively. However, although Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs-GRHL1, GRHL2 and GRHL3-function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers but not for their activation. Our data support a model positing that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Nucleossomos/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Células Epiteliais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células MCF-7 , Polimorfismo de Nucleotídeo Único , Ativação Transcricional
10.
Curr Biol ; 26(16): 2101-13, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27476594

RESUMO

Cancer cells have abnormal gene expression profiles; however, to what degree these are chaotic or driven by structured gene regulatory networks is often not known. Here we studied a model of Ras-driven invasive tumorigenesis in Drosophila epithelial tissues and combined in vivo genetics with next-generation sequencing and computational modeling to decipher the regulatory logic of tumor cells. Surprisingly, we discovered that the bulk of the tumor-specific gene expression is controlled by an ectopic network of a few transcription factors that are overexpressed and/or hyperactivated in tumor cells. These factors are Stat, AP-1, the bHLH proteins Myc and AP-4, the nuclear hormone receptor Ftz-f1, the nuclear receptor coactivator Taiman/SRC3, and Mef2. Notably, many of these transcription factors also are hyperactivated in human tumors. Bioinformatic analysis predicted that these factors directly regulate the majority of the tumor-specific gene expression, that they are interconnected by extensive cross-regulation, and that they show a high degree of co-regulation of target genes. Indeed, the factors of this network were required in multiple epithelia for tumor growth and invasiveness, and knockdown of several factors caused a reversion of the tumor-specific expression profile but had no observable effect on normal tissues. We further found that the Hippo pathway effector Yorkie was strongly activated in tumor cells and initiated cellular reprogramming by activating several transcription factors of this network. Thus, modeling regulatory networks identified an ectopic and ordered network of master regulators that control a large part of tumor cell-specific gene expression.


Assuntos
Carcinogênese , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Redes Reguladoras de Genes , Transdução de Sinais , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas
11.
Curr Biol ; 23(3): 229-35, 2013 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-23333314

RESUMO

The Drosophila Yorkie (Yki) protein and its mammalian homolog Yes-associated protein (YAP) are potent growth promoters, and YAP overexpression is associated with multiple types of cancer. Yki and YAP are transcriptional coactivators and function as downstream effectors of the Hippo tumor suppressor pathway. The regulation of Yki and YAP by the Hippo signaling pathway has been extensively investigated; however, how they regulate gene expression is poorly understood. To identify additional regulators of Yki activity, we performed a genome-wide RNAi screen in Drosophila S2 cells. In this screen, we identified the conserved protein Mask (Multiple ankyrin repeats single KH domain) as a novel promoter of Yki activity in vitro and validated this function in vivo in Drosophila. We found that Mask is required downstream of the Hippo pathway for Yki to induce target-gene expression and that Mask forms complexes with Yki. The human Mask homolog MASK1 complexes with YAP and is required for the full activity of YAP. Additionally, elevated MASK1 expression is associated with worsened outcomes for breast cancer patients. We conclude that Mask is a novel cofactor for Yki/YAP required for optimal Yki/YAP activity during development and oncogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Drosophila/genética , Proteínas de Drosophila/genética , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição , Proteínas de Sinalização YAP
12.
Development ; 135(24): 4071-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19004852

RESUMO

Patterning of sensory organs requires precise regulation of neural induction and repression. The neurocrystalline pattern of the adult Drosophila compound eye is generated by ordered selection of single founder photoreceptors (R8s) for each unit eye or ommatidium. R8 selection requires mechanisms that restrict R8 potential to a single cell from within a group of cells expressing the proneural gene atonal (ato). One model of R8 selection suggests that R8 precursors are selected from a three-cell ;R8 equivalence group' through repression of ato by the homeodomain transcription factor Rough (Ro). A second model proposes that lateral inhibition is sufficient to select a single R8 from an equipotent group of cells called the intermediate group (IG). Here, we provide new evidence that lateral inhibition, but not ro, is required for the initial selection of a single R8 precursor. We show that in ro mutants, ectopic R8s develop from R2,5 photoreceptor precursors independently of ectopic Ato and hours after normal R8s are specified. We also show that Ro directly represses the R8 specific zinc-finger transcription factor senseless (sens) in the developing R2,5 precursors to block ectopic R8 differentiation. Our results support a new model for R8 selection in which lateral inhibition establishes a transient pattern of selected R8s that is permanently reinforced by a repressive bistable loop between sens and ro. This model provides new insight into the strategies that allow successful integration of a repressive patterning signal, such as lateral inhibition, with continued developmental plasticity during retinal differentiation.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/crescimento & desenvolvimento , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Células Fotorreceptoras de Invertebrados/citologia , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Padronização Corporal , Proteínas de Ciclo Celular/genética , Sondas de DNA/genética , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas Associadas aos Microtúbulos/genética , Modelos Neurológicos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Fatores de Transcrição/genética
13.
Development ; 132(12): 2895-905, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15930118

RESUMO

Drosophila eye development is controlled by a conserved network of retinal determination (RD) genes. The RD genes encode nuclear proteins that form complexes and function in concert with extracellular signal-regulated transcription factors. Identification of the genomic regulatory elements that govern the eye-specific expression of the RD genes will allow us to better understand how spatial and temporal control of gene expression occurs during early eye development. We compared conserved non-coding sequences (CNCSs) between five Drosophilids along the approximately 40 kb genomic locus of the RD gene dachshund (dac). Our analysis uncovers two separate eye enhancers in intron eight and the 3' non-coding regions of the dac locus defined by clusters of highly conserved sequences. Loss- and gain-of-function analyses suggest that the 3' eye enhancer is synergistically activated by a combination of eya, so and dpp signaling, and only indirectly activated by ey, whereas the 5' eye enhancer is primarily regulated by ey, acting in concert with eya and so. Disrupting conserved So-binding sites in the 3' eye enhancer prevents reporter expression in vivo. Our results suggest that the two eye enhancers act redundantly and in concert with each other to integrate distinct upstream inputs and direct the eye-specific expression of dac.


Assuntos
Proteínas de Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas Nucleares/genética , Retina/crescimento & desenvolvimento , Retina/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homozigoto , Íntrons/genética , Dados de Sequência Molecular , Mutação/genética , Especificidade de Órgãos , Elementos de Resposta/genética
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