Unknown,Transcriptomics,Genomics,Proteomics

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High-throughput sequencing of methylated cytosine enriched by modification-dependent restriction endonuclease MspJI

ABSTRACT: In this study, we combine MspJI digestion and electrophoretic band selection with next generation high-throughput sequencing technology to detect 5-methylcytosines in Arabidopsis genome. By developing a bioinformatics workflow to attribute the CNNR sites recognized by MspJI to the reference genome, we fulfilled the systematic assessment of this method. According to the assessment, here we provide the method for generating a detailed map of plant methylome that could be feasible, reliable and economical in methylation investigation. Extracting the MspJI digested fragments, constructing sequencing library according to the Illumina protocol and sequencing with Illumina HiSeq2000. Repeatability and reproducibility studies were performed between two samples from the same individual. Specificity and sensitivity of the method was examined by comparing our data with WGBS data downloaded from GEO (GSE15922: GSM399600). **The WGBS seq data ('aerial_tissues_BS_seq_CNNR.gff') was generated from the GSM399600 Sample supplementary files ('aerial_tissues_BS_seq_alignment_batch-*.gff.gz') and was utilized to do comparative study with our MspJI-seq data including sensitivity and specificity analysis.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Yudong Xia

PROVIDER: E-GEOD-46428 | 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.

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High-throughput sequencing of methylated cytosine enriched by modification-dependent restriction endonuclease MspJI

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