Single cell epigenetic visualization assay.
Single cell epigenetic visualization assay.
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DOI：
10.1093/nar/gkab009
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发表时间：
2021-05-07
影响因子：
14.9
通讯作者：
Denisenko O
中科院分区：
文献类型：
--
作者：
Kint S;Van Criekinge W;Vandekerckhove L;De Vos WH;Bomsztyk K;Krause DS;Denisenko O
Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles. Epigenetic mark visualization at a gene of interest. Alkaline phosphatase (AP) is recruited to the epigenetic mark (purple) as an antibody conjugate. Gene specific oligonucleotides anchor phosphorylated sensor oligo (red) annealed to a detector oligo (green). When the epigenetic mark is present at the gene, the AP-dephosphorylated oligo survives subsequent λ-exonuclease treatment. The presence of the epigenetic mark at the gene is quantitated using the ratio of detector/sensor signal intensities (green/red).
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DOI：
10.1016/j.bbagrm.2016.04.001
发表时间：
2016-07
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Denisenko O;Lucas ES;Sun C;Watkins AJ;Mar D;Bomsztyk K;Fleming TP
通讯作者：
Fleming TP
DOI：
10.1073/pnas.1703087114
发表时间：
2017-09-05
影响因子：
11.1
作者：
Jeziorska, Danuta M.;Murray, Robert J. S.;Tufarelli, Cristina
通讯作者：
Tufarelli, Cristina
影响因子：
64.5
作者：
Goldberg, Aaron D.;Allis, C. David;Bernstein, Emily
通讯作者：
Bernstein, Emily
影响因子：
6.7
作者：
Kauder SE;Bosque A;Lindqvist A;Planelles V;Verdin E
通讯作者：
Verdin E
影响因子：
7.8
作者：
Clemson, CM;McNeil, JA;Willard, HF;Lawrence, JB
通讯作者：
Lawrence, JB