Meghna Vinod and Hans Bjornsson
Introduction: Histone modifications are crucial in regulating chromatin structure, chromatin accessibility, and gene expression. The histone machinery is a dynamic regulatory system that remains active throughout an individual’s lifetime. Disruption of the normal activity of histone H3K4 methylation causes Kabuki syndrome (KS). We hypothesize that modifier genes exist that regulate the levels of histone methylation activity.
Methods: We have created a reporter system that, upon modification of the histone tail by H3K4 trimethylation, brings two halves of a GFP molecule together, leading to fluorescence that can be measured. We have performed genome-wide CRISPR/Cas9 knockout (GECKO) with 123,411 guide RNAs that target over 19,000 genes in cells containing our reporter. After mutagenesis, we sorted the most fluorescent cells (3-5%) using flow cytometry, followed by Illumina sequencing of guides in sorted and unsorted fractions. This allows us to calculate the enrichment of specific guides using the MAGeCK software and validate top candidate genes using various in vitro techniques.
Results: From the GECKO analysis, we observed an increase in the GFP fluorescence on genome-wide knockout samples, and 269 genes appear significantly enriched (-Log10(RRA) >2.5) indicating a possible repressive role on H3K4me3 activity. We have prioritized 10 genes for validation, some of which are also known to play a role in histone acetylation machinery.
Conclusions: We have successfully identified potential negative regulators of H3K4 trimethylation and believe some could have potential therapeutic targets to treat KS. In addition, these insights could also help us understand the known interdependence between histone methylation and acetylation systems.
