Monitoring Histone Methylation (H3K9me3) Changes in Live Cells

Abstract

H3K9me3 (methylation of lysine 9 of histone H3) is an epigenetic modification that acts as a repressor mark. Several diseases, including cancers and neurological disorders, have been associated with aberrant changes in H3K9me3 levels. Different tools have been developed to enable detection and quantification of H3K9me3 levels in cells. Most techniques, however, lack live cell compatibility. To address this concern, we have engineered recombinant protein sensors for probing H3K9me3 in situ. A heterodimeric sensor containing a chromodomain and chromo shadow domain from HP1a was found to be optimal in recognizing H3K9me3 and exhibited similar spatial resolution to commercial antibodies. Our sensor offers similar quantitative accuracy in characterizing changes in H3K9me3 compared to antibodies but claims single cell resolution. The sensor was applied to evaluate changes in H3K9me3 responding to environmental chemical atrazine (ATZ). ATZ was found to result in significant reductions in H3K9me3 levels after 24 h of exposure. Its impact on the distribution of H3K9me3 among cell populations was also assessed and found to be distinctive. We foresee the application of our sensors in multiple toxicity and drug-screening applications.

Lead exposure induces dysregulation of constitutive heterochromatin hallmarks in live cells

“…We quantified changes in H3K9me3 by measuring the I ntegrated I ntensity per N uclei (IIN). We and others have previously demonstrated that IIN correlates with global epigenetic modification level per cell ( Sanchez et al, 2017 , Sánchez et al, 2019 , Sánchez et al, 2020 , Lungu et al, 2017 , Hayashi-Takanaka et al, 2011 ). IIN analysis between the control and Pb-treated groups shows that Pb exposure significantly ( p < 0.05, one-way ANOVA followed by Tukey’s HSD post-hoc test) lowers the global level of H3K9me3, by 16% and 36% for Pb concentrations of 100 and 500 ppb, respectively (see Fig.…”

“…Z-stack images of cells were collected using Nikon EZ-C1 software. Collected images were analyzed using Cellprofiler ( Carpenter et al, 2006 ) to get the I ntegrated I ntensity per N uclei (IIN) as we detailed in our previous work ( Sanchez et al, 2017 , Sánchez et al, 2019 , Sánchez et al, 2020 ).…”

“…Recently, a series of live-cell compatible epigenetic probes have been developed using engineered epigenetic reader domains including works from our group. These probes have enabled us to trace intracellular levels of histone PTMs, such as acetylation ( Sanchez et al, 2017 ) and methylation ( Sánchez et al, 2019 ), as well as methylated DNA ( Kim et al, 2014 , Sánchez et al, 2020 ). Most recently monitoring changes in DNA methylation or H3K9me3 at specific gene regions were also achieved via BiFC probes ( Lungu et al, 2017 ).…”

Molecular probes for cellular imaging of post-translational proteoforms

“…The sensor for histone H3K9me3 based on a chromodomain was also reported. 54 These sensors could be coupled to a DNA-binding zinc finger domain to monitor locusspecific H3K9me3 modification, 55 or to other histone reader domains to simultaneously report multiple histone modifications, 56,57 or with a methyl binding domain to simultaneously visualize 5-methylcytosine. 57 Further strategies to visualize histone proteoforms involve creation of FRET-based sandwich sensors through strategic fusion of the PTM recognition domain and the histone protein in between a fluorescent protein FRET pair (Fig.…”

Review: In vitro Cell Platform for Understanding Developmental Toxicity

“…The detection of epigenetic changes by protein-based fluorescent probes allows for real-time analysis of the changes to the global epigenome levels. So far protein probes have been developed for binding and visualization of both DNA methylation and histone modifications ( Hendrich and Bird, 1998 ; Lungu et al, 2017 ; Sanchez et al, 2017 ; Sanchez et al, 2019 ). Further development of nanobody fragments have provided an additional domain that allows for high specificity and affinity for selected epigenetic targets ( Hattori et al, 2016 ; Jullien et al, 2016 ).…”