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| Status |
Public on Nov 24, 2023 |
| Title |
In vitro reconstitution of chromatin domains (Mnase-Seq) |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
A key step towards defining the structure-function relationship of the genome is to identify the molecular mechanisms that drive higher-order genome folding. To this end, we reconstituted five S. cerevisiae chromosomes in vitro and developed a high-resolution MNase-based chromosome conformation capture assay to measure their 3D organization. We show that the formation of regularly spaced and phased nucleosome arrays is sufficient to drive higher-order genome folding into domains that resemble in vivo genome organization and thereby demonstrate that neither loop extrusion nor transcription are required for domain formation. The domain boundaries correspond to nucleosome-free regions and insulation strength scales with their width. Integrated molecular dynamics simulations show that domain compaction is dependent on nucleosome linker length, with longer linkers forming more compact structures. Together, our work demonstrates that fundamental properties of chromatin fibers are important determinants of higher-order genome folding and provides a proof-of-principle for bottom-up 3D genome studies.
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| Overall design |
We used a genome-wide in vitro reconstitution system (Oberbeckmann et al. 2021) to study how nucleosome positioning affects higher-order genome folding. To this end, we established a method to map 3D nucleosome contacts in vitro and named it in vitro Micro-C. DNA sequence of 5 chromosomes (V-IX) from S. cerevisiae was reconstituted into chromatin by salt gradient dialysis and then incubated with transcription factors only or additionally with various remodeler. Nucleosome positioning was confirmed with MNase-seq, while higher-order nucleoosme contacts were mapped with in vitro Micro-C.
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| Contributor(s) |
Quililan K, Oberbeckmann E, Cramer P, Oudelaar AM |
| Citation missing |
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| Submission date |
Dec 09, 2022 |
| Last update date |
Nov 25, 2023 |
| Contact name |
Elisa Oberbeckmann |
| E-mail(s) |
elisa.oberbeckmann@mpinat.mpg.de
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| Phone |
+49 551 2012809
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| Organization name |
Max-Planck Institute for Multidisciplinary Sciences
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| Department |
Molecular Biology
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| Lab |
Cramer
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| Street address |
Am Faßberg 11
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| City |
Göttingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
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| Platforms (1) |
| GPL26302 |
NextSeq 550 (Saccharomyces cerevisiae) |
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| Samples (26)
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| This SubSeries is part of SuperSeries: |
| GSE220647 |
In vitro reconstitution of chromatin domains shows a role for nucleosome positioning in 3D genome organization |
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| Relations |
| BioProject |
PRJNA910580 |
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