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1.
PLoS Genet ; 12(7): e1006204, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27442438

RESUMO

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.


Assuntos
Olho Composto de Artrópodes/embriologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Proteínas Nucleares/fisiologia , Animais , Olho Composto de Artrópodes/metabolismo , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/fisiologia , Discos Imaginais/embriologia , Morfogênese , Transdução de Sinais
2.
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
3.
Science ; 381(6654): 198-204, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37440660

RESUMO

Gene expression is controlled by the precise activation and repression of transcription. Repression is mediated by specialized transcription factors (TFs) that recruit co-repressors (CoRs) to silence transcription, even in the presence of activating cues. However, whether CoRs can dominantly silence all enhancers or display distinct specificities is unclear. In this work, we report that most enhancers in Drosophila can be repressed by only a subset of CoRs, and enhancers classified by CoR sensitivity show distinct chromatin features, function, TF motifs, and binding. Distinct TF motifs render enhancers more resistant or sensitive to specific CoRs, as we demonstrate by motif mutagenesis and addition. These CoR-enhancer compatibilities constitute an additional layer of regulatory specificity that allows differential regulation at close genomic distances and is indicative of distinct mechanisms of transcriptional repression.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Elementos Facilitadores Genéticos , Proteínas Repressoras , Animais , Cromatina/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos
4.
Science ; 366(6468): 1029-1034, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31754005

RESUMO

The Hippo signaling pathway and its two downstream effectors, the YAP and TAZ transcriptional coactivators, are drivers of tumor growth in experimental models. Studying mouse models, we show that YAP and TAZ can also exert a tumor-suppressive function. We found that normal hepatocytes surrounding liver tumors displayed activation of YAP and TAZ and that deletion of Yap and Taz in these peritumoral hepatocytes accelerated tumor growth. Conversely, experimental hyperactivation of YAP in peritumoral hepatocytes triggered regression of primary liver tumors and melanoma-derived liver metastases. Furthermore, whereas tumor cells growing in wild-type livers required YAP and TAZ for their survival, those surrounded by Yap- and Taz-deficient hepatocytes were not dependent on YAP and TAZ. Tumor cell survival thus depends on the relative activity of YAP and TAZ in tumor cells and their surrounding tissue, suggesting that YAP and TAZ act through a mechanism of cell competition to eliminate tumor cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Colangiocarcinoma/metabolismo , Hepatócitos/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Colangiocarcinoma/patologia , Via de Sinalização Hippo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas Experimentais/patologia , Melanoma/metabolismo , Melanoma/secundário , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/economia , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Carga Tumoral , Proteínas de Sinalização YAP
5.
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
6.
Dev Cell ; 42(6): 667-680.e4, 2017 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-28950103

RESUMO

Hyperactivating mutations in Ras signaling are hallmarks of carcinomas. Ras signaling mediates cell fate decisions as well as proliferation during development. It is not known what dictates whether Ras signaling drives differentiation versus proliferation. Here we show that the Hippo pathway is critical for this decision. Loss of Hippo switches Ras activation from promoting cellular differentiation to aggressive cellular proliferation. Transcriptome analysis combined with genetic tests show that this excessive proliferation depends on the synergistic induction of Ras target genes. Using ChIP-nexus, we find that Hippo signaling keeps Ras targets in check by directly regulating the expression of two key downstream transcription factors of Ras signaling: the ETS-domain transcription factor Pointed and the repressor Capicua. Our results highlight how independent signaling pathways can impinge on each other at the level of transcription factors, thereby providing a safety mechanism to keep proliferation in check under normal developmental conditions.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Transdução de Sinais , Transcrição Gênica , Proteínas ras/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Receptores ErbB/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Modelos Biológicos , Mutação/genética , Pupa/metabolismo , Regulon/genética , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo
7.
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
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