Identifying Distinct Heterochromatin Regions Using Combinatorial Epigenetic Probes in Live Cells
- Conference: International Conference on Epigenetics and Bioengineering
- Year: 2017
- Proceeding: International Conference on Epigenetics and Bioengineering
- Group: General Submissions
- Time: Thursday, December 14, 2017 - 1:15pm-1:40pm
The functional relevance of heterochromatin and differences in heterochromatin composition of normal vs. diseased cells make it a significant nuclear feature to identify and characterize. A common approach towards identifying heterochromatin is the use of simple stains such as DAPI and Hoescht that bind non-specifically to DNA dense regions. cHc and fHC are marked by specific epigenetic signatures dominated by 5mC, H3K9me3 and H3K27me3 and can be delineated by antibodies against these modifications. Other approaches to identify heterochromatin include chromosome banding and FISH. These are all largely fixed cell or non-specific techniques that cannot be applied to monitor dynamic changes in heterochromatin distribution or distinguish between cHC and fHC. A readout of the epigenetic state of the heterochromatin is also absent in most methods.
Our work bridges these gaps in existing technology by: 1) Identifying different sub-compartments of HC, such as constitutive and facultative heterochromatin 2) Quantifying the epigenetic modifications levels at these loci and 3) Monitoring these changes in real-time. We have developed live cell probes for monitoring epigenetic modifications that are commonly associated with different regions of heterochromatin (such as H3K9me3 and 5mC at cHC regions). The probes rely on native fluorescently-tagged epigenetic “reader” domains which have high affinity and selectivity towards their epigenetic target and have been used to monitor changes in the epigenome. We engineered probes based on chromodomain and methyl-binding-domain proteins that successfully bind to their to target sites in various human cells. The developed probes have the ability to quantitatively capture changes in heterochromatin epigenetic modification levels. Fluorescent colocalization and FRET interactions between the fluorescent tags attached to the probes can be used to provide additional spatial information about the distribution of these modifications at the heterochromatin. Lastly, the probes were introduced into a cancer cell line and can be used to distinguish the heterochromatin features of diseased cells in comparison to healthy cells.