Abstract
Eukaryotic genomes are regulated by epigenetic marks that act to modulate transcriptional control as well as to regulate DNA replication and repair. In Arabidopsis thaliana, mutation of the ATXR5 and ATXR6 histone methyltransferases causes reduction in histone H3 lysine 27 monomethylation, transcriptional upregulation of transposons, and a genome instability defect in which there is an accumulation of excess DNA corresponding to pericentromeric heterochromatin. We designed a forward genetic screen to identify suppressors of the atxr5/6 phenotype that uncovered loss-of-function mutations in two components of the TREX-2 complex (AtTHP1, AtSAC3B), a SUMO-interacting E3 ubiquitin ligase (AtSTUbL2) and a methyl-binding domain protein (AtMBD9). Additionally, using a reverse genetic approach, we show that a mutation in a plant homolog of the tumor suppressor gene BRCA1 enhances the atxr5/6 phenotype. Through characterization of these mutations, our results suggest models for the production atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell, and suggest that the atxr5 atxr6 transcriptional defects may be the cause of the genome instability defects in the mutants. These findings highlight the critical intersection of transcriptional silencing and DNA replication in the maintenance of genome stability of heterochromatin.
Highlights
The genome represents a biological entity that is necessarily static yet retains a level of plasticity
We previously showed that mutations that strongly reduce DNA methylation in an atxr5/6 mutant background suppress the over-replication phenotype of atxr5/6 [6], suggesting that the heterochromatic nature of these regions is necessary to engender the gain in DNA copy number phenotype of the atxr5/6 mutant
Loss of transposon silencing in atxr5/6 mutants is tissue specific and correlates with extra DNA at pericentromeric heterochromatin
Summary
The genome represents a biological entity that is necessarily static yet retains a level of plasticity. In Arabidopsis thaliana, we previously identified two redundant histone methyltransferases, ATXR5 and ATXR6 (referred to in the aggregate as ATXR5/6), that are responsible for monomethylating lysine 27 of histone H3 (H3K27me1) [5]. Loss of these methyltransferases in the atxr5/6 double mutant leads to a severe loss of transcriptional silencing at repetitive transposable elements (TEs) [5,6]. The atxr5/6 mutants display an unusual phenotype, wherein heterochromatin regions of the Arabidopsis genome exhibit an aberrant gain in DNA copy number (here referred to as over-replication). The regions producing extra DNA are highly repetitive and carry epigenetic marks characteristic of silent chromatin such as DNA methylation and H3K27me, and largely overlap with the pericentromeric regions transcriptionally derepressed in the atxr5/6 mutant
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