Sensitive Automated Measurement of Histone-DNA Affinities in Nucleosomes
Summary: The DNA of eukaryotes is wrapped around histone octamers to form nucleosomes. Although it is well established that the DNA sequence significantly influences nucleosome formation, its precise contribution has remained controversial, partially owing to the lack of quantitative affinity data....
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2020-02-01
|
| Series: | iScience |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2589004220300079 |
| _version_ | 1818962518022815744 |
|---|---|
| author | Max Schnepf Claudia Ludwig Peter Bandilla Stefano Ceolin Ulrich Unnerstall Christophe Jung Ulrike Gaul |
| author_facet | Max Schnepf Claudia Ludwig Peter Bandilla Stefano Ceolin Ulrich Unnerstall Christophe Jung Ulrike Gaul |
| author_sort | Max Schnepf |
| collection | DOAJ |
| description | Summary: The DNA of eukaryotes is wrapped around histone octamers to form nucleosomes. Although it is well established that the DNA sequence significantly influences nucleosome formation, its precise contribution has remained controversial, partially owing to the lack of quantitative affinity data. Here, we present a method to measure DNA-histone binding free energies at medium throughput and with high sensitivity. Competitive nucleosome formation is achieved through automation, and a modified epifluorescence microscope is used to rapidly and accurately measure the fractions of bound/unbound DNA based on fluorescence anisotropy. The procedure allows us to obtain full titration curves with high reproducibility. We applied this technique to measure the histone-DNA affinities for 47 DNA sequences and analyzed how the affinities correlate with relevant DNA sequence features. We found that the GC content has a significant impact on nucleosome-forming preferences, but 10 bp dinucleotide periodicities and the presence of poly(dA:dT) stretches do not. : Molecular Biology; Molecular Interaction; Biological Sciences Research Methodologies Subject Areas: Molecular Biology, Molecular Interaction, Biological Sciences Research Methodologies |
| first_indexed | 2024-12-20T12:30:35Z |
| format | Article |
| id | doaj.art-bbd6d83328ff4c26acd7c9a7fad8a478 |
| institution | Directory Open Access Journal |
| issn | 2589-0042 |
| language | English |
| last_indexed | 2024-12-20T12:30:35Z |
| publishDate | 2020-02-01 |
| publisher | Elsevier |
| record_format | Article |
| series | iScience |
| spelling | doaj.art-bbd6d83328ff4c26acd7c9a7fad8a4782022-12-21T19:40:43ZengElsevieriScience2589-00422020-02-01232Sensitive Automated Measurement of Histone-DNA Affinities in NucleosomesMax Schnepf0Claudia Ludwig1Peter Bandilla2Stefano Ceolin3Ulrich Unnerstall4Christophe Jung5Ulrike Gaul6Gene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 München, GermanyGene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 München, GermanyGene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 München, GermanyGene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 München, GermanyGene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 München, GermanyGene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 München, Germany; Corresponding authorGene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 München, GermanySummary: The DNA of eukaryotes is wrapped around histone octamers to form nucleosomes. Although it is well established that the DNA sequence significantly influences nucleosome formation, its precise contribution has remained controversial, partially owing to the lack of quantitative affinity data. Here, we present a method to measure DNA-histone binding free energies at medium throughput and with high sensitivity. Competitive nucleosome formation is achieved through automation, and a modified epifluorescence microscope is used to rapidly and accurately measure the fractions of bound/unbound DNA based on fluorescence anisotropy. The procedure allows us to obtain full titration curves with high reproducibility. We applied this technique to measure the histone-DNA affinities for 47 DNA sequences and analyzed how the affinities correlate with relevant DNA sequence features. We found that the GC content has a significant impact on nucleosome-forming preferences, but 10 bp dinucleotide periodicities and the presence of poly(dA:dT) stretches do not. : Molecular Biology; Molecular Interaction; Biological Sciences Research Methodologies Subject Areas: Molecular Biology, Molecular Interaction, Biological Sciences Research Methodologieshttp://www.sciencedirect.com/science/article/pii/S2589004220300079 |
| spellingShingle | Max Schnepf Claudia Ludwig Peter Bandilla Stefano Ceolin Ulrich Unnerstall Christophe Jung Ulrike Gaul Sensitive Automated Measurement of Histone-DNA Affinities in Nucleosomes iScience |
| title | Sensitive Automated Measurement of Histone-DNA Affinities in Nucleosomes |
| title_full | Sensitive Automated Measurement of Histone-DNA Affinities in Nucleosomes |
| title_fullStr | Sensitive Automated Measurement of Histone-DNA Affinities in Nucleosomes |
| title_full_unstemmed | Sensitive Automated Measurement of Histone-DNA Affinities in Nucleosomes |
| title_short | Sensitive Automated Measurement of Histone-DNA Affinities in Nucleosomes |
| title_sort | sensitive automated measurement of histone dna affinities in nucleosomes |
| url | http://www.sciencedirect.com/science/article/pii/S2589004220300079 |
| work_keys_str_mv | AT maxschnepf sensitiveautomatedmeasurementofhistonednaaffinitiesinnucleosomes AT claudialudwig sensitiveautomatedmeasurementofhistonednaaffinitiesinnucleosomes AT peterbandilla sensitiveautomatedmeasurementofhistonednaaffinitiesinnucleosomes AT stefanoceolin sensitiveautomatedmeasurementofhistonednaaffinitiesinnucleosomes AT ulrichunnerstall sensitiveautomatedmeasurementofhistonednaaffinitiesinnucleosomes AT christophejung sensitiveautomatedmeasurementofhistonednaaffinitiesinnucleosomes AT ulrikegaul sensitiveautomatedmeasurementofhistonednaaffinitiesinnucleosomes |