Ever dream of knowing the single-cell details of chromatin regulators in real-time only to awake trapped in the static world of heterogenous cell populations? Well, dream no longer because thanks to the efforts of the Elowitz laboratory at Caltech researchers now have the ability to see just how some of our favorite epigenetic repressors (HDAC4, KRAB, DNMT3B and EED) repress gene expression at the single-cell level in real-time.
The Single-Cell Reporter System
The system takes place in a CHOK1 cell line that contains a synthetic human chromosome with insulators flanking a reporter gene: a citrine tagged H2B. The citrine tagged H2B also has reverse Tet repressor (rTetr) binding sites in its promoter, which allows for inducible binding. When the rTetr sites are not bound, this reporter gene produces a strong fluorescence from citrine that is localized to the nucleus. In their system, the authors also introduce a chromatin repressor (CR: HDAC4, KRAB, DNMT3B and EED). This CR complex is further fused to rTetr, which makes it the perfect inducible match for the citrine H2B reporter gene.
Once the CR is induced by Dox, it can bind the citrine H2B promoter and the resulting reduction in citrine H2B fluorescence levels in single cells can be tracked by time-lapse microscopy and flow cytometry. While visualizing single cells, the team made three sets of observations the dynamics of CRs:
All-or-None Single-Cell Chromatin Repression
- Silencing occurs in an all-or-none fashion with the repressors determining the fraction of cells silenced rather than fine-tuning the amount of gene expression.
- All the tested factors cause epigenetic repression at different rates.
- The faster repressors are KRAB (which associates with H3K9me3 readers) and histone deacetylase HDAC4.
Stochastic Reactivation of Single-Cells
- 5 days after the removal of Dox, they observed stochastic reactivation in cells silenced with EED, KRAB, and HDAC4.
- The fastest reactivation occurred with HDAC4 repression.
- Interestingly, there was no reactivation after 30 days from Dox removal when DNMT3B was involved in the repression.
- EED and KRAB cells have a hybrid memory with some reactivation occurring within 2-3 weeks after Dox removal and some silencing remaining even after 30 days.
Reversible and Irreversible Silenced States
Finally, the team shows that the longer KRAB and EED are recruited, the more cells stay irreversibly silenced. These findings establish a three state model that explains the kinetics of hybrid memory.
Ultimately, this system offers up a novel way to to understand the all-or-none dynamics of chromatin repression in single-cells as well as the resulting memory of these events.
Get your dynamic chromatin repressive game on in Science, February 2016.