Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody

Background

Posttranslational modifications of histone proteins play important roles in transcriptional regulation and genome integrity. While the one-dimensional epigenomic landscape has been revealed in many cell types by chromatin immunoprecipitation and sequencing, less is known about the dynamics of histone modifications due to technical limitations (Kimura et al., 2015). Recently, a few techniques for detecting protein modifications in living cells have been developed. One strategy uses sensors based on fluorescence/Förster resonance energy transfer (FRET) to monitor the balance between the modifying and demodifying enzymes. However, the dynamics of endogenous modifications cannot be monitored using FRET sensors. Another strategy that we have developed uses probes based on modification-specific antibodies. Fab-based live endogenous modification labeling (FabLEM) is a live-imaging system using fluorescently labeled antigen-binding fragments (Fabs). Fabs loaded into cells bind to the target modification without disturbing cell function as the binding time is very small (a second to tens of seconds). A genetically encoded system to express a modification-specific intracellular antibody (mintbody) can be applied for observation with a longer period of time or in living animals (Figure 1). Both Fabs and mintbodies are just small enough to pass through the nuclear pore by diffusion. When the level of the target modification increases, more probes become enriched in the nucleus. Therefore, by measuring the nuclear/cytoplasmic intensity ratio, changes of modification level in living cells can be monitored (Hayashi-Takanaka et al., 2011; Sato et al., 2013 and 2016). The live cell modification monitoring system using mintbodies will be particularly useful to evaluate the effects of small chemicals and protein depletion and overexpression.

Histone H4 Lys20 monomethylation (H4K20me1) is an essential modification in mammals, involved in chromosome condensation, segregation, replication and repair, as well as gene regulation (Beck et al., 2012; Jørgensen et al., 2013). The level of H4K20me1 increases during G2 to M phases and the inhibition of PR-Set7/Set8, a methyltransferase responsible for H4K20 monomethylation, causes mitotic defects. In female cells, the enrichment of H4K20me1 in inactive X chromosomes is microscopically observed. H4K20me1-specific mintbody has proven useful for monitoring the dynamic behavior of H4K20me1 in living cells (Sato et al., 2016). In addition, alteration of H4K20me1 level by ectopic expression of a methyltransferase has been evaluated. Among methyltransferases (PR-Set7/Set8, SUV420H1, and SUV420H2) and a demethylase (PHF8), involved in H4K20me1 metabolism, the expression of SUV420H1, which add methyl-groups to monomethylated H4K20 towards to trimethylation, caused a drastic effect. As an example of measuring relative H4K20me1 levels, we here describe the method to evaluate the effect of SUV420H1 on H4K20me1 in living cells.


Figure 1. Schematic diagram of mintbody expression and function. A genetically encoded mintbody, which reversibly binds to specific modification, can be expressed in cells and animals that harbors the expression vector.