Project description:We have knocked-in mCherry-MBD-NLS probe, which recognizes methylated CpG, to ROSA26 locus and named this reporter MethylRO (methylation probe in ROSA26 locus). In this study, we showed that MethylRO can capture DNA methylation dynamics both in vitro and in vivo.
Project description:In order to gain insight into DNA methylation readout, we have established a controlled strategy for profiling genomic targeting of chromatin-interacting factors in vivo. With this approach we determined binding preferences for the methyl-CpG binding domain (MBD) family of proteins, including disease relevant mutants, deletions and isoforms. In vivo binding of MBD proteins occurs as a linear function of local methylation density, and is dependent on functional MBD domain – methyl-CpG interactions. This directs specificity of MBD proteins to methylated, CpG dense and inactive regulatory regions. In contrast, binding to unmethylated sites is MBD protein specific and mediated via alternative domains or protein-protein interactions. The latter is observed for NuRD complex-mediated MBD2 tethering to a subset of unmethylated, tissue-specific regulatory regions, similar to MBD3. These functional binding maps reveal methylation-dependent and -independent binding modes determined by distinct protein domains and revise current models of DNA methylation readout through MBD proteins.
Project description:In order to gain insight into DNA methylation readout, we have established a controlled strategy for profiling genomic targeting of chromatin-interacting factors in vivo. With this approach we determined binding preferences for the methyl-CpG binding domain (MBD) family of proteins, including disease relevant mutants, deletions and isoforms. In vivo binding of MBD proteins occurs as a linear function of local methylation density, and is dependent on functional MBD domain M-bM-^@M-^S methyl-CpG interactions. This directs specificity of MBD proteins to methylated, CpG dense and inactive regulatory regions. In contrast, binding to unmethylated sites is MBD protein specific and mediated via alternative domains or protein-protein interactions. The latter is observed for NuRD complex-mediated MBD2 tethering to a subset of unmethylated, tissue-specific regulatory regions, similar to MBD3. These functional binding maps reveal methylation-dependent and -independent binding modes determined by distinct protein domains and revise current models of DNA methylation readout through MBD proteins. Comparative binding analysis for the MBD family of proteins utilizing recombinase-assisted mapping of biotin-tagged proteins (RAMBiO). This set contains maps for 29 samples including wild type MBD proteins, mutants and domain variants in mouse ES cells, derived neurons (NP and TN) and Dnmt1/3a/3b triple-KO ES cells (TKO).
Project description:Elevated plasma homocysteine is an independent risk factor for cardiovascular disease and stroke, however the etiology remains poorly understood. Elevated homocysteine is known to inhibit methyltransferases including DNA methyltransferases, but no methylome-wide analysis of elevated homocysteine has been reported. Peripheral blood genomic DNA methylation in 8 Singaporean-Chinese ischemic stroke patients (4 male, 4 female) with varying homocysteine titer and hypertensive status were profiled using methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) on Illumina Genome Analyzer IIx. A methylome wide screen was undertaken for gender, total plasma homocysteine, hypertension and age. The data show considerable variability within the small cohort, including at genes which are related to one carbon metabolism and cardiovascular disease. Peripheral blood genomic DNA methylation in 8 Singaporean-Chinese ischemic stroke patients (4 male, 4 female) was profiled using methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) on Illumina Genome Analyzer IIx. Methylation parrterns were correlated with homocysteine levels, lypertensive status, gender and age.
Project description:We generated 12 Gb of high-quality sequencing data (~6 Gb per sample) to clarify the molecular mechanism of salt tolerance between wild tipe and transgenic DgWRKY5 chrysanthemum under normal condition. A total of 1078 differentially expressed genes (DEGs) (593 up-regulated and 485 down-regulated) were identified between CK and DgWRKY5, including genes encoding transcription factors and protein kinases. We identified numerous differentially expressed genes that exhibited distinct expression patterns, and stress-related genes that were highly differentiated in wild tipe and transgenic DgWRKY5 chrysanthemum. These genes have known or potential roles in stress tolerance relative and were enriched in functional gene categories potentially responsible for chrysanthemum resistance. Therefore, they are appealing candidates for further investigation of the gene expression and associated regulatory mechanisms related to stress response .
Project description:Chrysanthemum is a garden plant with good economic benefit and high ornamental value. Chrysanthemum in the key period of flowering in autumn and winter, vulnerable to cold damage, affecting the normal growth of the chrysanthemum plant and even death. little is known regarding the study of histone crotonylation in plant cold response. In this study, we first obtained reference chrysanthemum transcriptome data via RNA sequencing. Next, we quantitatively investigated the chrysanthemum proteome, crotonylation, and the association between them in chrysanthemum following low temperature. In total, 365669 unigenes, 6693 proteins and 2017 crotonylation sites were quantified under low temperature stress. There were 24631 up-regulated and 22648 down-regulated unigenes (absolute log2-fold change > 1 and P value<0.05), 393 up-regulated and 500 down-regulated proteins using a 1.2-fold threshold (P<0.05). The lysine crotonylation mainly influenced in photosynthesis, ribosome, antioxidant enzyme and ROS system. In the process of low temperature, 61 lysine crotonylation sites in 89 proteins were up-regulated and 87 lysine crotonylation sites in 72 proteins are down-regulated (1.2-fold threshold, P<0.05).
Project description:We performed affinity-based enrichment with methyl-CpG binding domain protein followed by high-throughput sequencing (MBD-seq) to assay DNA methylation in mouse liver tissue.
Project description:We performed affinity-based enrichment with methyl-CpG binding domain protein followed by high-throughput sequencing (MBD-seq) to assay DNA methylation in mouse liver tissue.
Project description:DNA methylation was assessed in genomic DNA obtained from the arcuate nucleus of heifers fed to gain body weight at high (HG, n = 4) and low (LG, n = 4) rates from 4.5 to 8.5 mo of age. A methyl-CpG binding domain-based (MBD) protein assay was performed to capture fragments of methylated DNA (methylated-enriched DNA). Input (total) and methylated-enriched DNA were labeled with two different dyes and co-hybridized to a custom-designed oligonucleotide array targeted to genes associated with nutritional inputs and the control of puberty. The ratio of the log2 (enriched/input) of the normalized intensities, were determined. Data was analyzed comparing values of HG versus LG heifers. Two nutritional schedules: HG (n=4 heifers) and LG (n=4 heifers); one array per heifer; methylated enriched DNA (enriched) and total DNA (input) co-hybridyzed into each array Methylated-enriched DNA obtained from a methyl-CpG binding domain-based (MBD) protein assay
Project description:We studied the effects of chemical exposure on global DNA methylation by using mCherry-methyl-CpG-binding domain (MBD)-nls human induced pluripotent stem cells (iPSCs). According to the fluorescent MBD parameters, we divided the effects of 135 chemical on global DNA methylation into 3 categories; reduction (hypomethylation), increase (hypermethylation) and little/no effect. We performed DNA methylome assay to examine the effects of 6 epigenotoxic chemicals on genome-wide DNA methylation patterns in human iPSCs. Our results indicated that hypermethylation agents increased DNA methylation and downregulated the expression of genes involved in cell cycle or development.