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Homo sapiens

ABSTRACT: Identification of direct targets and modified bases of RNA cytosine methyltransferases

PROVIDER: PRJNA169525 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR516285.fastq.gz	Fastqsanger.gz
	SRR516286.fastq.gz	Fastqsanger.gz
	SRR516287.fastq.gz	Fastqsanger.gz
	SRR516288.fastq.gz	Fastqsanger.gz
	SRR727006.fastq.gz	Fastqsanger.gz

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Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

Project description:Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contribution of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity and may constitute another layer of epigenetic regulation.

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Project description:DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. In human genomes, an epigenetic modification at the fifth carbon of cytosine bases comprises one fundamental pathway by which genes can be silenced or activated . Methods widely used to detect epigenetic modifications at cytosine bases rely on either deamination of unmodified cytosines to read as thymine or borane reduction of the modified oxidised cytosine bases to read as thymine. As a result, such methods fail to capture common C-to-T mutations, and importantly also fail to distinguish 5-methylcytosine (5mC - mainly found in silenced parts of the genome) from 5-hydroxymethylcytosine (5hmC - enriched in active gene bodies and enhancers). Hence, existing methods are unable to read the complete information stored in our genomes in a single workflow. Here, we build on biomodal's duet multiomics solution +modC, which has the unique ability to read genetic and epigenetic cytosine modifications (modC) in a single workflow. By introducing a high-fidelity methyltransferase during the workflow, we are able to construct an expanded information table that can be used to deconvolute A,T,C,G, 5-mC and 5-hmC simultaneously in a single read within a DNA molecule . We use a mouse embryonic stem cell-line, ES-E14TG2A, to map for the first time a simultaneous reading of the genome and epigenome at high depth and show how these epigenetic modifications are segregated across the genome. In summary, using our modified approach we demonstrate simultaneous, phased reading of all six genetic and epigenetic bases. This tool provides a more complete picture of the information stored in genomes and will have applications throughout biology and medicine.

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