MECHANISTIC STUDIES INTO HETEROCHROMATIN PROPAGATION BY THE HISTONE LYSINE METHYLTRANSFERASE, G9A

Abstract

G9a (also known as EHMT2) is a lysine histone methyltransferase that deposits mono- and dimethylation at lysine 9 of histone H3 (H3K9me1/2). G9a activity has long been linked to transcriptional regulation, as H3K9me1/2 is a histone post-translational modification (PTM) that is considered a hallmark of facultative heterochromatin, i.e. transcriptionally-silenced genomic regions. The C-terminal SET domain of G9a is responsible for catalysis, while a series of embedded ankyrin repeats (ARs) close to the SET domain preferentially bind H3K9me1/2. Given the ability for G9a to recognize and deposit H3K9me1/2, we sought to construct chromatinized substrates to assess how G9a engages with nucleosome(s). In this study, we employed a DNA-barcoded PTM library to profile the substrate scope of G9a and detailed how pre-installed PTMs, such as H3K4me3 and H4 poly-acetylation, can regulate the activity of this enzyme. We also prepared chemically- and spatially-defined nucleosome arrays to define how H3K9me1/2 anchors and positions the enzyme. We further detailed the precise geometric and domain requirements for G9a-deposited H3K9me1/2 spreading using heterotypic tetra and mononucleosomes. As part of this work, we exploited several streamlined methodologies to construct site-specifically modified MegaDalton-sized chromatin complexes with high levels of chemical control. We further attempted to extend the DNA-barcoding platform to develop a high-throughput system to study such G9a spreading. Collectively, our results shed light into how G9a senses the larger chromatin PTM landscape, ultimately leading to the establishment of silenced chromatin states.