Identification of Multiple Proteins Coupling Transcriptional Gene Silencing to Genome Stability in Arabidopsis thaliana

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.

Methyl-CpG-binding domain 9 (MBD9) is required for H2A.Z incorporation into chromatin at a subset of H2A.Z-enriched regions in the Arabidopsis genome

“…Importantly, these phenotypes in the double mutant plants were reverted back to those of each single mutant by introducing either the genomic ARP6 or MBD9 constructs into the double mutants ( Figure S6), indicating that these defects were truly the result of simultaneous loss of ARP6 and MBD9 functions. These findings further support the idea that MBD9 is not a core subunit of the ARP6-containing SWR1 complex and suggest that this protein has additional functions outside of H2A.Z incorporation (Hale et al, 2016).…”

Genetic and epigenetic variation of transposable elements in Arabidopsis

“…ATXR5 and ATXR6 likely associate with the replication fork and are only capable of modifying the replication dependent histone variant H3.1, due to an amino acid difference between H3.1 and H3.3 at residue 31 [48]. Reverse and forward genetic studies have identified a plethora of suppressors of TE over-replication in atxr5 atxr6 , which have differential effects on the suppression of loss transcriptional silencing [41,48,49] It remains to be seen whether H3K27me1 controls heterochromatic DNA replication directly or indirectly, and also if H3K27me1 plays a role in delaying heterochromatic replication to late S-phase.…”

Selective nucleolus organizer inactivation in Arabidopsis is a chromosome position-effect phenomenon

“…This line is deficient for histone H3 lysine 27 (H3K27) monomethylase activity as a result of insertional mutations in the Arabidopsis trithorax-related protein 5 (ATXR5) and Arabidopsis trithorax-related protein 6 (ATXR6) genes (22). In atxr5 atxr6 double mutants, transposable elements are derepressed and genome instability involving heterochromatin over-replication occurs (23,24). We show that, in the course of generating the atxr5 atxr6 double mutant line, an estimated ∼2-3 Mb of the northern tip of chromosome 4 was replaced by corresponding sequences of chromosome 2, thus translocating NOR2 rRNA genes to the position of NOR4.…”