An Optimized Chromatin Immunoprecipitation Protocol for Quantification of Protein-DNA Interactions

Jonge, Wim J. de; Brok, Mariël; Kemmeren, Patrick; Holstege, Frank C.P.

doi:10.1016/j.xpro.2020.100020

Cited by 21 publications

(14 citation statements)

References 20 publications

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“…ChIP was performed as described in detail in (de Jonge et al , 2000) using biological triplicates. To summarize: cells were diluted in the morning to an optical density (OD) of 0.11–0.15 (WPA Biowave CO8000 Cell Density Meter) in 100 ml of SC medium, and grown for at least 2 doublings to an OD 600 = 0.8, which corresponds to about 2 × 10 7 cells per ml.…”

Section: Methodsmentioning

confidence: 99%

Genome‐wide off‐rates reveal how DNA binding dynamics shape transcription factor function

Jonge

Brok

Lijnzaad

et al. 2020

Molecular Systems Biology

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Protein– DNA interactions are dynamic, and these dynamics are an important aspect of chromatin‐associated processes such as transcription or replication. Due to a lack of methods to study on‐ and off‐rates across entire genomes, protein– DNA interaction dynamics have not been studied extensively. Here, we determine in vivo off‐rates for the Saccharomyces cerevisiae chromatin organizing factor Abf1, at 191 sites simultaneously across the yeast genome. Average Abf1 residence times span a wide range, varying between 4.2 and 33 min. Sites with different off‐rates are associated with different functional characteristics. This includes their transcriptional dependency on Abf1, nucleosome positioning and the size of the nucleosome‐free region, as well as the ability to roadblock RNA polymerase II for termination. The results show how off‐rates contribute to transcription factor function and that DIVORSEQ (Determining In Vivo Off‐Rates by SEQ uencing) is a meaningful way of investigating protein– DNA binding dynamics genome‐wide.

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Section: Methodsmentioning

confidence: 99%

Genome‐wide off‐rates reveal how DNA binding dynamics shape transcription factor function

Jonge

Brok

Lijnzaad

et al. 2020

Molecular Systems Biology

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“…To our knowledge, this is the first report to describe the applicability of extracting circulating nucleosomal DNA from ELISA immune complexes. Even though our study does not include a direct comparison of this approach with ChIP, this method may be superior to ChIP in preventing background noise signals because protein A/G agarose beads used in ChIP assays non-specifically bind DNA and nontarget proteins [10][11][12] .…”

Section: Resultsmentioning

confidence: 99%

“…This resembles chromatin immunoprecipitation (ChIP) assays where, however, there are relevant differences between the two approaches. In ChIP assays, protein A/G agarose beads are usually employed to bind antibodies of interest, and this is often associated with significant non-specific binding of DNA or non-target proteins to beads [10][11][12] . In the approach used in the present study, we extracted H4K20me3-associated circulating DNA directly from H4K20me3-containing nucleosomes that bound to anti-H4K20me3 antibodies immobilized on assay strips.…”

Section: Introductionmentioning

confidence: 99%

Histon ELISA ve Nicel PCR Testlerinin Birleştirilerek Histon Metilasyonu ile İlişkili Dolaşımdaki DNA’nın İzolasyonu ve Ölçülmesi

Özgür¹,

Yörüker²,

Keskin³

et al. 2022

NKTD

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Aim: Liquid biopsy-based measurement of post-translational histone modifications (PTHMs) in bodily fluids of patients with cancer represents a novel area of biomarker research. Here, we tested the applicability of an approach combining enzyme-linked immunosorbent assays (ELISA)like measurement of histone methylation and quantitative polymerase chain reaction to extract and quantify pericentric histone 4 lysine 20 trimethylation (H4K20me3)-related circulating DNA. Materials and Methods:After adding plasma into ELISA wells and letting H4K20me3-related nucleosomes bind to the antibody, nucleosomal DNA was detached from immune complexes using a buffer including NaHCO 3 , SDS, and proteinase K and purified. A single copy-and a multiple copysequence from pericentric regions of chromosomes 16 and 1 were amplified to quantify H4K20me3-related DNA from patients with colon cancer or colonic polyps.Results: Relative plasma level of H4K20me3-related nucleosomal DNA was lower in the patients with colon cancer than in controls for both target sequences, with a statistically significant difference at the chr16-specific target (relative values were 0.037 vs. 0.073, respectively; p=0.008). Conclusion:That H4K20me3-related nucleosomal DNA is present in decreased levels in colon cancer confirms our previous findings attained using other techniques. In conclusion, our findings indicate the applicability of direct extraction of protein-bound DNA from ELISA immune complexes and it could provide a surrogate means in the assessment of PTHMs or other protein-DNA interactions.

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“…ChIP was performed as described in ( 79 ) using three biological replicates. Cells were diluted in the morning to OD 600 0.2 in 100 mL of synthetic complete media and grown for at least 2 doublings to an OD 600 0.8.…”

Section: Methodsmentioning

confidence: 99%

DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes

Patel

Coppola

Pomp

et al. 2022

Preprint

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DNA supercoiling has emerged as a major contributor to gene regulation in bacteria. The impact of DNA supercoiling on transcription dynamics in eukaryotes is less clear. Here, using single-molecule dual-color RNA imaging in budding yeast, we show that transcriptional bursting of the divergent and tandem GAL genes is coupled. Upon topoisomerase degradation, supercoils that buildup from transcription inhibit subsequent transcription at neighboring genes, thereby reducing their simultaneous bursting. GAL gene transcription is inhibited more by negative than by positive supercoiling accumulation. Unlike bacteria, wildtype yeast has sufficient topoisomerase levels to minimize inhibition from supercoils at adjacent genes. Overall, we discover fundamental differences in supercoiling-mediated gene regulation between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.One sentence summaryTranscription causes twisting of the DNA double helix, which can inhibit transcription of adjacent genes.

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An Optimized Chromatin Immunoprecipitation Protocol for Quantification of Protein-DNA Interactions

Cited by 21 publications

References 20 publications

Genome‐wide off‐rates reveal how DNA binding dynamics shape transcription factor function

Genome‐wide off‐rates reveal how DNA binding dynamics shape transcription factor function

Histon ELISA ve Nicel PCR Testlerinin Birleştirilerek Histon Metilasyonu ile İlişkili Dolaşımdaki DNA’nın İzolasyonu ve Ölçülmesi

DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes

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