Expression profiling by high throughput sequencing Methylation profiling by high throughput sequencing
Summary
DNA methylation is important for silencing genes and transposable elements. Changes in DNA methylation can be heritable and thus, the loss or gain of methylation can lead to the formation of stable epialleles. A well characterized example of a stable epiallele in plants is fwa-4, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing upregulation of FWA and a heritable late flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the Human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA upregulation, and a heritable late flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCAS9 based targeted demethylation system using the TET1cd and a modified SunTag system and demonstrate its ability to target demethylation at and activate the expression of FWA and CACTA1. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.
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