RESUMO
We report the identification of yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, as an antagonist of the Wingless signalling pathway. We demonstrate that cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan(-/-) cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. We conclude that yan represents a new and unsuspected molecular link between the Wingless and DER pathways.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas do Olho/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína Wnt1/metabolismo , Animais , Proteínas do Domínio Armadillo/antagonistas & inibidores , Linhagem Celular , Drosophila/genética , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Olho/metabolismo , Proteínas do Olho/genética , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Ligação Proteica , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Fatores de Transcrição/antagonistas & inibidoresRESUMO
BACKGROUND: Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ss-catenin pathway. METHODOLOGY/PRINCIPAL FINDINGS: In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a beta-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/beta-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/beta-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation. CONCLUSIONS: These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/beta-catenin pathway.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Receptores de LDL/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Proteína Axina , Sequência de Bases , Células Cultivadas , Primers do DNA , Humanos , Imunoprecipitação , Hibridização In Situ , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Off-target effects have been demonstrated to be a major source of false-positives in RNA interference (RNAi) high-throughput screens. In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog signaling pathways using second generation double-stranded RNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization, which determine the efficacy of RNAi-knockdown of target genes.