DNA Methyltransferases Are Required to Induce Heterochromatic Re-Replication in Arabidopsis

Abstract

The relationship between epigenetic marks on chromatin and the regulation of DNA replication is poorly understood. Mutations of the H3K27 methyltransferase genes, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 ( ATXR5) and ATXR6, result in re-replication (repeated origin firing within the same cell cycle). Here we show that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, RNA sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs). Hence our results suggest a complex relationship between ATXR5/6 and DNA methylation in the regulation of DNA replication and transcription of TEs.

Author Summary

Before cell division the genome is required to replicate once to ensure that each daughter cell inherits a full copy of genomic DNA. Eukaryotic DNA is wrapped around histones to form nucleosomes. Chemical modifications of DNA and histones are known to regulate gene expression. There is growing evidence that these modifications also regulate DNA replication, however very little is understood. Two histone methyltransferases, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, are required to prevent over-replication of normally silent regions of the genome called heterochromatin. Heterochromatin is enriched with transposable elements (TEs) that are silenced by modifications such as DNA methylation. We find that losses of DNA methylation suppress the over-replication defect in an atxr5 atxr6 mutant background. This suggests that DNA methylation positively regulates DNA replication in the absence of ATXR5/6. We further study the relationship between ATXR5/6 and DNA methylation in regulating the expression of TEs and find that they cooperatively silence TEs. Together these findings reveal relationships between DNA and histone modifications in regulating basic biological processes such as DNA replication and gene expression.