In-vitro evaluation of methyltransferase enzymes efficiency
Ontology highlight
ABSTRACT: In this experiment we methylated naked DNA with increasing non-saturating concentrations of the M.SssI and M.CviPI methyltransferase enzymes.
Project description:We used a high-throughput sequencing method known as CPD-seq to map the formation of UV-induced cyclobutane pyrimidine dimers (CPD) at single nucleotide resolution in UV-irradiated yeast genomic DNA (naked DNA) in the presence or absence of cytosine methylation at CpG sites by the methyltransferase M.SssI.
Project description:Analysis of nucleosome positioning and chromatin state by using CpG methyltransferase M.SssI to methylate nuclei. Unmethylated regions that gain methylation (low to high beta value) are known to be accessible and nucleosome depleted. Intact nuclei are harvested from cells and treated with M.SssI. DNA is then extracted, bisulfite converted and run on an Infinium methylation array, along with a no-enzyme control. Background subtracted beta values (listed below) are used to determine regions that have gained methylation on enzyme treatment compared to the control - and these are used to infer chromatin state
Project description:Active DNA demethylation in mammals involves TET-mediated iterative oxidation of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) and subsequent excision repair of highly oxidized cytosine bases 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) by Thymine DNA glycosylase (TDG). However, quantitative and high-resolution analysis of active DNA demethylation activity remains challenging. Here we describe M.SssI methylase-assisted bisulfite sequencing (MAB-seq), a method that directly maps 5fC/5caC at single-base resolution. Genome-wide MAB-seq allows systematic identification of 5fC/5caC in Tdg-depleted embryonic stem cells, thereby generating a base-resolution map of active DNA demethylome. A comparison of 5fC/5caC and 5hmC distribution maps indicates that catalytic processivity of TET enzymes correlates with local chromatin accessibility. MAB-seq also reveals strong strand asymmetry of active demethylation within palindromic CpGs. Integrating MAB-seq with other base-resolution mapping methods enables quantitative measurement of cytosine modification states at key transitioning steps of active demethylation pathway, and reveals a regulatory role of 5fC/5caC excision repair in active DNA demethylation cascade. Analysis of 5fC/5caC excision repair-dependent active DNA demethylome by MAB-seq in mouse embryonic stem cells.
Project description:Analysis of nucleosome positioning and chromatin state by using CpG methyltransferase M.SssI to methylate nuclei. Unmethylated regions that gain methylation (low to high beta value) are known to be accessible and nucleosome depleted.
Project description:For the AcceSssIble assay, nuclei preparation and M.SssI methyltransferase (New England BioLabs) treatment were performed. The subsequent Infinium DNA methylation assay was performed at the University of Southern California Molecular Genomics Core Facility according to the manufacturer’s specifications (Illumina).
Project description:What methylation changes are occurring in different parts of early maturation stage seed largely remains unknown. To uncover the possible role of DNA methylation in different parts of early maturation stage seed, we characterized the methylome of seed coats,cotyledons, and the embryonic seed axis using Illumina sequencing. seed coats, cotyledon, and axis
Project description:Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism. MECP2 encodes a methyl-DNA-binding protein that is proposed to function as a transcriptional repressor, but, despite numerous studies examining neuronal gene expression in MeCP2 mutants, no coherent model has emerged for how MeCP2 regulates transcription. Here we identify a genome-wide length-dependent increase in the expression of long genes in neurons lacking MeCP2. This gene misregulation occurs in human RTT brains and correlates with onset and severity of phenotypes in Mecp2 mutant mice, suggesting that the disruption of long gene expression contributes to RTT pathology. We present evidence that MeCP2 represses long genes by binding to brain-enriched, methylated CA dinucleotides within genes and show that loss of methylated CA in the brain recapitulates gene expression defects observed in MeCP2 mutants. We find that long genes encode proteins with neuronal functions, and overlap substantially with genes that have been implicated in autism and Fragile X syndrome. Reversing the overexpression of long genes in neurons lacking MeCP2 can improve some RTT-associated cellular deficits. These findings suggest that a function of MeCP2 in the mammalian brain is to temper the expression of genes in a length-dependent manner, and that mutations in MeCP2 and possibly other autism genes may cause neurological dysfunction by disrupting the expression of long genes in the brain. Bisulfite-seq from mouse cortex and cerebellum
Project description:What methylation changes are occurring during seed development largely remains unknown. To uncover the possible role of DNA methylation during the transition from seed differentiation to maturation and dormany in soybean, we characterized the methylome of whole seeds representing the differentiation (GLOB stage), maturation (early- (EM), mid- (B1) and late- (AA1) maturation stages), and dormancy (DRY stage) phases of soybean seed development using Illumina sequencing. In addition, we characterized the methylome of the mid-maturation stage embryonic axis (B1-AX) to examine methylation differences, if any, between an embryonic region compared to the whole seed. Illumina sequencing of bisulfite-converted genomic DNA from globular stage (GLOB), early-maturation stage (EM), mid-maturation stage (B1), and late-maturation stage (AA1) seeds, dormancy stage (DRY) and mid-maturation embryonic axis (B1-AX).