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Selective chemical tracking of Dnmt1 catalytic activity in live cells

ABSTRACT: Selective chemical tracking of Dnmt1 catalytic activity in live cells

PROVIDER: PRJNA756279 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15528945.fastq.gz	Fastqsanger.gz
	SRR15528946.fastq.gz	Fastqsanger.gz
	SRR15528947.fastq.gz	Fastqsanger.gz
	SRR15528948.fastq.gz	Fastqsanger.gz
	SRR15528949.fastq.gz	Fastqsanger.gz

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Selective chemical tracking of Dnmt1 catalytic activity in live cells

Project description:Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases (DNMTs), whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro . We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse- internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide-tagging of Dnmt1-specific genomic targets in cellula . The deposited covalent tags were exploited as ‘click’ handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells paving the way to selective studies of other methylation pathways in eukaryotic systems.

2022-02-04 | GSE182445 | GEO

Distinct catalytic-dependent or -independent activities of DNMT1 in mouse embryonic stem cells

Project description:DNMT1 maintains DNA methylation profile and regulates gene expression. Although DNMT1 interacts with other proteins that regulate gene expressions, it remains unclear how the protein-protein interaction or the catalytic activity of DNMT1 itself affect gene expression and DNA methylation pattern. In this study, immunofluorescence staining showed abnormal DNA methylation pattern in chromosome arms of DNMT1-deficient (1KO) mouse embryonic stem cells (ESCs), and we hypothesized that DNMT1 helps methylation activity of DNMT3. To validate this hypothesis, we generate transgenic 1KO ESCs expressing catalytic inactive DNMT1 (1KO+1CI). Here, we used RNA-seq to analyze the effect of catalytic-dependent or -independent DNMT1 activity on gene expression in ESCs or epiblast-like cells (EpiLCs) induction.

2022-01-01 | GSE179006 | GEO

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Project description:Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

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Live tracking of basal stem cells of the epidermis during growth, homeostasis, and injury response in zebrafish

Project description:Basal stem cells of the epidermis continuously differentiate into keratinocytes and replenish themselves via self-renewal to maintain skin homeostasis. Numerous studies have attempted to reveal how basal cells undergo differentiation or self-renewal; however, this has been hampered by a lack of robust basal cell markers and analytical platforms that allow single-cell tracking. Here, we report integrin b4 (itgb4) is a useful marker for basal cell labelling, irrespective of the body region, stage, and regenerative status. We employed Cre-loxP recombination in combination with live cell tracking of single basal clones in the caudal fin and investigated the embryonic origin and behaviour of basal cells during fish growth and homeostasis. Although most basal cells, including those in fins, became quiescent in the adult stage, genetic cell ablation showed that basal cells were reactivated to either self-renew or differentiate, depending on the injured cell type. Our findings provide a platform for quantitative in vivo imaging of basal stem cells applicable at wider stages and under various conditions and reveal unanticipated basal cell dynamism in response to distinct wound signals.

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Distinct catalytic-dependent or -independent activities of DNMT1 in mouse embryonic stem cells

Project description:Distinct catalytic-dependent or -independent activities of DNMT1 in mouse embryonic stem cells

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Project description:Tracking live-cell single-molecule dynamics enables measurements of heterochromatin-associated protein-protein interactions

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A mutant DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

Project description:Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in gain of DNMT1 function. However, earlier data suggested that RFTS is required for DNMT1 activity. Here, we determined cellular consequences of RFTS deletion from DNMT1 in immortalized human bronchial epithelial cells. Compared to full-length DNMT1, ectopic expression of DNMT1- ?RFTS caused greater malignant transformation and enhanced promoter methylation that silenced DAPK and DUOX1 gene expression and increased intensity of promoter methylation across the genome. Strikingly, DNMT1-?RFTS also produced genomic hypomethylation and demethylation of Satellite 2 repeat sequences. Because deletion of RFTS is sufficient to induce focal hypermethylation and global hypomethylation in parallel, evidence suggests that the RFTS domain is a target responsible for epigenetic changes in cancer. DNA samples from HBEC3 cells transfected with vector control and HBEC3 cells with full-length and RFTS-deleted DNMT1 were assessed for genome-wide methylation using the microarray-based high resolution HpaII tiny fragment enriched by ligation-mediated PCR (HELP) assay.

2014-09-30 | E-GEOD-57829 | biostudies-arrayexpress

Tracking live-cell single-molecule dynamics enables measurements of heterochromatin-associated protein-protein interactions

Project description:Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and each of their binding partners based on which we inferred their most likely interaction sites. Our results indicate that H3K9me promotes specific complex formation between HP1 proteins and their interactors in a spatially restricted manner, while attenuating their ability to form off-chromatin complexes. As opposed to being an inert platform or scaffold to direct HP1 binding, our studies propose a novel function for H3K9me as an active participant in enhancing HP1-associated complex formation in living cells.

2024-05-01 | GSE263472 | GEO

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer/promoter dynamics [ChIP-seq]

Project description:Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer/promoter dynamics [ChIP-seq]

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RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

Project description:Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in gain of DNMT1 function. However, earlier data suggested that RFTS is required for DNMT1 activity. Here, we determined cellular consequences of RFTS deletion from DNMT1 in immortalized human bronchial epithelial cells. Compared to full-length DNMT1, ectopic expression of DNMT1- ΔRFTS caused greater malignant transformation and enhanced promoter methylation that silenced DAPK and DUOX1 gene expression and increased intensity of promoter methylation across the genome. Strikingly, DNMT1-ΔRFTS also produced genomic hypomethylation and demethylation of Satellite 2 repeat sequences. Because deletion of RFTS is sufficient to induce focal hypermethylation and global hypomethylation in parallel, evidence suggests that the RFTS domain is a target responsible for epigenetic changes in cancer.

2014-09-30 | GSE57829 | GEO

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