Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jun 2;12(6):e1006092.
doi: 10.1371/journal.pgen.1006092. eCollection 2016 Jun.

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

Christopher J Hale  1   2 , Magdalena E Potok  1 , Jennifer Lopez  1 , Truman Do  1 , Ao Liu  1 , Javier Gallego-Bartolome  1 , Scott D Michaels  3 , Steven E Jacobsen  1   4
Affiliations

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

Christopher J Hale et al. PLoS Genet. .

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.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Transcriptional silencing defects in an atxr5/6 mutant are strongly correlated with over-replication defects and are not induced by DNA damage.
(A) Flow cytometry profiles of wild type plants (Col) and atxr5/6 mutants for flower and cotyledon tissue with ploidy levels labeled for Col profiles. Excess DNA production is seen as a broadening of the peaks in atxr5/6 cotyledons compared to wild type. (B) Chromosomal views of DNA sequencing read ratio of atxr5/6 mutants compared to Col for flower and cotyledon tissues with diagrammatic representations of the Arabidopsis chromosomes shown below with boxes identifying pericentromeric heterochromatin. Gaps in the plot represent areas of low coverage. (C) Boxplot of RNA-seq RPKM values for atxr5/6-induced TEs from cotyledon tissue in floral and cotyledon tissue. (D) Venn diagrams describing the relationship between genes de novo identified as up-regulated in the irradiation and atxr5/6 mutant transcriptomes. (E) Boxplots showing the behavior of atxr5/6- irradiation-induced protein coding genes (top) and TEs (bottom) for various radiation dosages, time points, and genotypes.
Fig 2
Fig 2. Loss of the Arabidopsis BRCA1 homolog enhances the atxr5/6 extra DNA and transcriptional silencing phenotypes.
(A) Flow cytometry of cotyledon tissue for Col, atxr5/6, and atbrca1-1 atxr5/6 lines. Col and atxr5/6 data is same as shown in Fig 1A and is shown here for comparison. (B) Chromosomal views as in Fig 1B comparing atbrca1-1 atxr5/6 sorted 16C DNA-seq to sorted 16C reads from atxr5/6. (C) Boxplot of RNA-seq RPKM values for atxr5/6-induced TEs in cotyledon tissue for genotypes derived from the listed genotype. (D) Venn diagram of TEs de novo identified from atxr5/6 and atbrca1-1 atxr5/6.
Fig 3
Fig 3. Mutations in Arabidopsis TREX-2 complex proteins suppress the transcriptional silencing and extra-DNA phenotypes of atxr5/6 mutants.
(A) Flow cytometry of M2 atxr5/6 plants containing RAD51pro::GFP from the EMS_2_37 and (B) EMS_2_300 lines for both GFP+ and GFP- plants. (C) Heatmap of RNA-seq RPKM values over cotyledon atxr5/6-induced TEs for F2 EMS_2_37 or EMS_2_300 GFP+/- as well as control Col and atxr5/6 GFP+ plants. All lines except for Col contain RAD51pro::GFP and are in atxr5/6 background. (D) Boxplot of RNA-seq RPKM values from cotyledon tissue for identified atxr5/6-induced TEs (S1 Table) and (E) irradiation-induced genes (S2 Table) in TREX-2 insertional mutants.
Fig 4
Fig 4. The ems_2_129 mutation which suppresses the transcriptional silencing and extra-DNA phenotypes of atxr5/6 mutants maps to the MBD9 gene.
(A) Pie chart and (B) chromosomal view as shown in S3A and S3B Fig showing the distribution of significantly enriched mutations in EMS_2_129 (GFP-) plants identified in DNA-seq data. (C) Gene structure of MBD9 showing the newly identified point mutation from EMS mutagenesis as well the insertional mutant (triangles) used for complementation and downstream analysis. Black boxes represent exons. (D) Flow cytometry showing that the mbd9-3 insertional allele suppresses the atxr5/6 extra-DNA phenotype and (E) fails to complement the ems_2_129 EMS allele. (F) Boxplots showing the mbd9-3 allele suppresses the atxr5/6-induced expression of TEs and irradiation-responsive genes.
Fig 5
Fig 5. Loss of AtSTUbL2 causes suppression of the atxr5/6 transcriptional silencing and extra-DNA phenotypes.
(A) Flow cytometry of M2 atxr5/6 plants containing RAD51pro::GFP from the EMS_2_325 line for both GFP+ and GFP- plants. (B) Heatmap for cotyledon RNA-seq, as in Fig 3C for EMS_2_325 F2 material with the same Col and atxr5/6 GFP+ data as in Fig 3C.
Fig 6
Fig 6. Newly identified suppressors of atxr5/6 phenotypes do not have strong effects on DNA methylation.
(A) Barplot giving the number of DMRs identified in new suppressors of atxr5/6 with met1 used as positive control for DMR identification (Sample 1 = atxr5/6, 2 = mbd9-3, 3 = atsac3b-3, 4 = atthp1-1, 5 = atstubl2-2, 6 = met1-3). (B) Chromosomal views of log2 ratio of % cytosine methylation in mutants compared to a Col control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ding Q, MacAlpine DM. Defining the replication program through the chromatin landscape. Crit Rev Biochem Mol Biol. 2011;46: 165–79. 10.3109/10409238.2011.560139 - DOI - PMC - PubMed
    1. Alabert C, Groth A. Chromatin replication and epigenome maintenance. Nat Rev Mol Cell Biol. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.; 2012;13: 153–67. 10.1038/nrm3288 - DOI - PubMed
    1. Yoshida K, Poveda A, Pasero P. Time to be versatile: regulation of the replication timing program in budding yeast. J Mol Biol. 2013;425: 4696–705. 10.1016/j.jmb.2013.09.020 - DOI - PubMed
    1. Brustel J, Tardat M, Kirsh O, Grimaud C, Julien E. Coupling mitosis to DNA replication: the emerging role of the histone H4-lysine 20 methyltransferase PR-Set7. Trends Cell Biol. 2011;21: 452–60. 10.1016/j.tcb.2011.04.006 - DOI - PubMed
    1. Jacob Y, Feng S, LeBlanc C a, Bernatavichute Y V, Stroud H, Cokus S, et al. ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat Struct Mol Biol. 2009;16: 763–8. 10.1038/nsmb.1611 - DOI - PMC - PubMed

Publication types

MeSH terms