Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: Eukaryotic genomes are heavily regulated by epigenetic marks that often act to modulate the transcriptional control of genetic elements. In Arabidopsis thaliana the ATXR5 and ATXR6 histone methyltransferases, and their cognate H3K27 monomethylation mark, act in transcriptional silencing while also maintaining genome stability by preventing generation of excess DNA corresponding to pericentromeric heterochromatin. In this study we characterize the atxr5 atxr6 transcriptome and its relationship to the DNA damage response which suggests that the atxr5 atxr6 transcriptional defects may be epistatic to the genome instability defects in the mutants. In addition we isolate several factors that modulate both the transcriptional and genomic instability phenotypes of atxr5 atxr6 mutants, which suggest a mechanism for atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell. PolyA RNA sequencing (RNA-seq), whole-genome resequencing (DNA-seq), and whole-genome bisulfite sequencing (methyl-seq) was performed on Arabidospsis thaliana mutant and wildtype plants. DNA-seq was used to characterize DNA copy number and map EMS-induced mutations, RNA-seq was used to quantify transcript abundance and map EMS-induced mutations, and methyl-seq was used to assess DNA methylation. Details of the relationship between samples in this series and figures in the associated manuscript can be found in Supplemental Table 4 of the associated manuscript. Unless otherwise noted in the description all lines are ecotype Columbia, and all genotypes should be assumed homozygous unless otherwise indicated with a '/'.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Christopher Hale

PROVIDER: E-GEOD-77735 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the epsilon-ammonium of K9 positioned adjacent to bound SAH. These structural insights complemented by in vivo functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation. Plants homozygous for null mutations in the KRYPTONITE H3K9 methyltransferase were stably transformed with transgenes encoding the wildtype KYP protein or transgenes carrying induced point mutations in the KYP active site. The resulting lines were assayed for DNA methylation by whole-genome bisulfite sequencing to learn the efficiency with which wildtype and mutant versions of the KYP protein could restore DNA methylation lost in a kyp mutant. Samples 7 and 8 were run as single Illumina lanes and as such were compared to a previous Col sample (GSM881756), this Col sample was realigned to the TAIR10 genome for this study and as such updated processed files are available with this submission. These samples were used to define kyp mutant CHG context DMRs that were complemented upon introduction of the wildtype KYP protein. Samples 1-6 were run as multiplexed samples and were used to assay the degree of complementation for various point mutants. All plants are in the Col ecotype background.

Project description:Epigenetic gene silencing is of central importance to maintain genome integrity and is mediated by an elaborate interplay between DNA methylation, histone posttranslational modifications and chromatin remodeling complexes. DNA methylation and repressive histone marks usually correlate with transcriptionally silent heterochromatin, however there are exceptions to this interdependence. In Arabidopsis, mutation of MORPHEUS MOLECULE 1 (MOM1) causes transcriptional derepression of heterochromatin independently of changes in DNA methylation. More recently, two Arabidopsis homologs of mouse Microrchidia (MORC) have also been implicated in gene silencing and heterochromatin condensation without altering genome-wide DNA methylation patterns. In this study, we show that AtMORC6 physically interacts with AtMORC1 and with its close homologue AtMORC2 in two mutually exclusive protein complexes. RNA-seq analysis of high-order mutants indicates that AtMORC1 and AtMORC2 act redundantly to repress a common set of loci. We also examined the genetic interactions between AtMORC6 and MOM1 pathways. Although AtMORC6 and MOM1 control the silencing of a very similar set of genomic loci, we observed synergistic transcriptional regulation in the mom1/atmorc6 double mutant, suggesting that these epigenetic regulators act mainly by independent silencing mechanisms. RNA-seq libraries were prepared for two suites of mutants to allow direct comparisons between mutants within each set. The two sets consisted of the following samples: Set_1) A wildtype (Col) control, the morc1 mutant, the morc2 mutant, the morc1 morc2 double mutant, the morc6 mutant, and the morc1 morc2 morc6 triple mutant ; Set_2) A wildtpe (Col) control, the morc6 mutant, the mom1 mutant, and the mom1 morc6 double mutant. For each sample, two biological replicates were performed (denoted "bio_replicate_1" or "bio_replicate_2"). Whole genome bisulifte libraries were sequenced from material grown in parallel.

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

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