RESUMO
Here we show that designed transcription activator-like effectors (TALEs) that bind to defined areas of the interferon beta promoter are capable to induce IFN-beta expression and signaling in human cells. Importantly, TALE-mediated IFN-beta signaling occurs independently of pathogen pattern recognition but effectively prohibits viral RNA replication as demonstrated with a hepatitis C virus replicon. TALEs were thus indicated to be valuable tools in various applications addressing, for example, virus-host interactions.
Assuntos
Interferon beta/biossíntese , Fatores de Transcrição/genética , Ativação Transcricional , Sequência de Bases , Linhagem Celular Tumoral , Hepacivirus/fisiologia , Humanos , Imunidade Inata , Interferon beta/genética , Regiões Promotoras Genéticas , Ligação Proteica , Engenharia de Proteínas , RNA Viral/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transdução de Sinais , Fatores de Transcrição/biossíntese , Replicação ViralRESUMO
We here identify protein kinase D1 (PKD1) as a major regulator of anchorage-dependent and -independent growth of cancer cells controlled via the transcription factor Snail1. Using FRET, we demonstrate that PKD1, but not PKD2, efficiently interacts with Snail1 in nuclei. PKD1 phosphorylates Snail1 at Ser-11. There was no change in the nucleocytoplasmic distribution of Snail1 using wild type Snail1 and Ser-11 phosphosite mutants in different tumor cells. Regardless of its phosphorylation status or following co-expression of constitutively active PKD, Snail1 was predominantly localized to cell nuclei. We also identify a novel mechanism of PKD1-mediated regulation of Snail1 transcriptional activity in tumor cells. The interaction of the co-repressors histone deacetylases 1 and 2 as well as lysyl oxidase-like protein 3 with Snail1 was impaired when Snail1 was not phosphorylated at Ser-11, which led to reduced Snail1-associated histone deacetylase activity. Additionally, lysyl oxidase-like protein 3 expression was up-regulated by ectopic PKD1 expression, implying a synergistic regulation of Snail1-driven transcription. Ectopic expression of PKD1 also up-regulated proliferation markers such as Cyclin D1 and Ajuba. Accordingly, Snail1 and its phosphorylation at Ser-11 were required and sufficient to control PKD1-mediated anchorage-independent growth and anchorage-dependent proliferation of different tumor cells. In conclusion, our data show that PKD1 is crucial to support growth of tumor cells via Snail1.