Monitoring Dynamic Changes in DNA and Histone Methylation at Repetitive Genomic Regions in Live Cells
- Conference: International Conference on Epigenetics and Bioengineering
- Year: 2017
- Proceeding: International Conference on Epigenetics and Bioengineering
- Group: Poster Session
- Time: Wednesday, December 13, 2017 - 4:45pm-6:30pm
Monitoring the interplay between epigenetic marks that regulate these repetitive regions in real time would further our understanding of centromere and telomere dynamics in diseases. The dynamic arrangement of telomeres and centromeres varies largely as the cell cycle progresses as well as in diseased vs. healthy cells. Various methods utilizing gene specific tools such as CRISPR and TALEs have been developed to map the positions of these chromatin structures in live cells. While these methods are helpful in understanding the dynamic intra-nuclear positioning of telomere and centromere foci, they do not capture the epigenetic features that regulate telomeres and centromeres. Present methodologies to measure the methylation levels at specific genes rely on a combination of antibodies against DNA and histone methylation coupled with FISH. These are fixed cells methods that do not allow tracking epigenetic changes in a dynamic manner.
In this work, we developed a live-cell compatible DNA and histone methylation probe that can capture the DNA methylation and H3K9me3 modification levels at specific genomic loci. The developed probe comprises of two regions: 1) an epigenetic “reader” domain and 2) a gene specific binding domain. Several proteins such as the methyl binding domain and the chromo-domain contain recognition motifs for DNA methylation and H3K9me3 respectively. The specific recognition motifs of these domains were tagged with fluorescent reporter proteins and successfully identified their target sites with a high degree of affinity and specificity in live cells. TALE and CRISPR proteins tagged with fluorescent proteins were used to selectively recognize tandem repeat regions. The binding of the two-part probe on the region of interest results in a colocalization and FRET interaction between the two fluorescent tags, providing readout of the DNA/histone methylation at the specific locus. The probe was also introduced into cancer cell lines to quantify the differences in centromere and telomere methylation levels as compared to healthy cells.