Dataset Information

Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping in Nanochannel Arrays.

ABSTRACT: Proteins and enzymes in the cell nucleus require physical access to their DNA target sites in order to perform genomic tasks such as gene activation and transcription. Hence, chromatin accessibility is a central regulator of gene expression, and its genomic profile holds essential information on the cell type and state. We utilized the E. coli Dam methyltransferase in combination with a fluorescent cofactor analogue to generate fluorescent tags in accessible DNA regions within the cell nucleus. The accessible portions of the genome are then detected by single-molecule optical genome mapping in nanochannel arrays. This method allowed us to characterize long-range structural variations and their associated chromatin structure. We show the ability to create whole-genome, allele-specific chromatin accessibility maps composed of long DNA molecules extended in silicon nanochannels.

SUBMITTER: Nifker G

PROVIDER: S-EPMC10210529 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Publications

Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping in Nanochannel Arrays.

Nifker Gil G Grunwald Assaf A Margalit Sapir S Tulpova Zuzana Z Michaeli Yael Y Har-Gil Hagai H Maimon Noy N Roichman Elad E Schütz Leonie L Weinhold Elmar E Ebenstein Yuval Y

ACS nano 20230508 10

Proteins and enzymes in the cell nucleus require physical access to their DNA target sites in order to perform genomic tasks such as gene activation and transcription. Hence, chromatin accessibility is a central regulator of gene expression, and its genomic profile holds essential information on the cell type and state. We utilized the <i>E. coli</i> Dam methyltransferase in combination with a fluorescent cofactor analogue to generate fluorescent tags in accessible DNA regions within the cell nu ...[more]

PMID: 37154345

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Project description:BackgroundThe challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples.ResultsHere we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh.ConclusionThe TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of clinically relevant samples to allow demarcation of regulatory elements of considerable prognostic significance.

Dataset Information

Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping in Nanochannel Arrays.

Publications

Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping in Nanochannel Arrays.

Similar Datasets

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