H3K9me regulates heterochromatin silencing through incoherent feedforward loops

Histone H3 lysine-9 methylation (H3K9me) is associated with condensed and transcriptionally inactive heterochromatin1. Although it has long been known that H3K9me silences transcription to control a wide variety of biological phenomena in many eukaryotic species2,3, how the silencing is regulated under the control of H3K9me is still largely unclear. Moreover, how cells delimit regions with H3K9me to avoid silencing essential genes remains unexplored. Here, using Arabidopsis genetic systems that induce H3K9me2 and its associated non-CG DNA methylation (mCH) in the transcribed region of genes de novo, we show that the accumulation of H3K9me2/mCH paradoxically also leads to the deposition of the euchromatic mark H3K36me3. This induction of H3K36me3 depends on a SET domain methyltransferase, ASHH3, and brings about anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2/mCH4. H3K9me2/mCH-driven antagonistic actions of ASHH3-H3K36me3 and LDL2-H3K4me1-loss also regulate the de novo silencing of reactivated transposable elements (TEs). These results demonstrate that H3K9me2 both facilitates and impedes silencing, and the incoherent feedforward loops fine-tune the fate of genes and TEs. Our results illuminate a novel elaborate mechanism for partitioning chromatin domains and provide insights into the molecular basis underlying natural epigenetic variation.