RESUMO
BACKGROUND: Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy based on barcoded randomly integrated reporters with Gal4-mediated tethering. We applied the assay to Drosophila heterochromatin protein 1a (HP1a), which is mostly known as a repressive protein but has also been linked to transcriptional activation. RESULTS: Recruitment to over 1000 genomic locations revealed that HP1a is a potent repressor able to silence even highly expressing reporter genes. However, the local chromatin context can modulate HP1a function. In pericentromeric regions, HP1a-induced repression was enhanced by twofold. In regions marked by a H3K36me3-rich chromatin signature, HP1a-dependent silencing was significantly decreased. We found no evidence for an activating function of HP1a in our experimental system. Furthermore, we did not observe stable transmission of repression over mitotic divisions after loss of targeted HP1a. CONCLUSIONS: The multiplexed tethered reporter assay should be applicable to a large number of chromatin proteins and will be a useful tool to dissect combinatorial regulatory interactions in chromatin.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inativação Gênica , Histonas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The efficacy and the mutation spectrum of genome editing methods can vary substantially depending on the targeted sequence. A simple, quick assay to accurately characterize and quantify the induced mutations is therefore needed. Here we present TIDE, a method for this purpose that requires only a pair of PCR reactions and two standard capillary sequencing runs. The sequence traces are then analyzed by a specially developed decomposition algorithm that identifies the major induced mutations in the projected editing site and accurately determines their frequency in a cell population. This method is cost-effective and quick, and it provides much more detailed information than current enzyme-based assays. An interactive web tool for automated decomposition of the sequence traces is available. TIDE greatly facilitates the testing and rational design of genome editing strategies.