Leiden University Student Repository

Hooijschuur, Twan (2020)

Nucleosome mapping in single long DNA molecules detected by nanopore methylation sequencing

Bachelor thesis | Natuurkunde (BSc)

Nucleosomes condense DNA into higher order chromatin structures and the position of nucleosomes play an important role in the regulating of transcription. Current methods can only map ensemble averaged nucleosome profiles. Here we make use of the property that nucleosomes protect the DNA against methylation, induced by methyltransferase enzymes. Nanopore sequencing is subsequently used to detect methylated bases on single DNA molecules. This makes a single molecule approach possible for detecting nucleosomes. We use the Nucleosome-Unmethylated-Score(NUS) to map nucleosomes on single DNA molecules. The NUS was tested by mapping nucleosome occupancy on high nucleosome affinity 601 arrays. The NUS was further used to analyse active and silent genes in Saccharomyces cerevisiae rDNA and to characterise nucleosome remodelling in reconstituted chromatin by whole cell extract (WCE) proteins.The found nucleosome occupancy was comparable with in vivo positions observed from ensemble averaged...Show moreNucleosomes condense DNA into higher order chromatin structures and the position of nucleosomes play an important role in the regulating of transcription. Current methods can only map ensemble averaged nucleosome profiles. Here we make use of the property that nucleosomes protect the DNA against methylation, induced by methyltransferase enzymes. Nanopore sequencing is subsequently used to detect methylated bases on single DNA molecules. This makes a single molecule approach possible for detecting nucleosomes. We use the Nucleosome-Unmethylated-Score(NUS) to map nucleosomes on single DNA molecules. The NUS was tested by mapping nucleosome occupancy on high nucleosome affinity 601 arrays. The NUS was further used to analyse active and silent genes in Saccharomyces cerevisiae rDNA and to characterise nucleosome remodelling in reconstituted chromatin by whole cell extract (WCE) proteins.The found nucleosome occupancy was comparable with in vivo positions observed from ensemble averaged data. Nucleosome detection on single molecules advances our understanding of chromatin structure and its role in transcription regulation.Show less

Nucleosome

Methylation

Biophysics

Author

Hooijschuur, Twan

Faculty

Faculty of Science

Specialisation

Physics and Astronomy

Supervisors

Noort, John van

ECTS Credits

3

Language

en