|Entry||Database: EMDB / ID: EMD-30070|
|Title||Nucleosome with LIN28B distal enhancer DNA sequence|
|Sample||Nucleosome with LIN28B distal enhancer DNA sequence|
|Biological species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.6 Å|
|Authors||Echigoya K / Koyama M / Takizawa Y / Kurumizaka H|
|Funding support|| Japan, 6 items |
|Citation||Journal: Sci Rep / Year: 2020|
Title: Nucleosome binding by the pioneer transcription factor OCT4.
Authors: Kenta Echigoya / Masako Koyama / Lumi Negishi / Yoshimasa Takizawa / Yuka Mizukami / Hideki Shimabayashi / Akari Kuroda / Hitoshi Kurumizaka /
Abstract: Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors ...Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors efficiently bind their target DNA sequences within the nucleosome. OCT4 has been identified as a pioneer transcription factor required for stem cell pluripotency. To study the nucleosome binding by OCT4, we prepared human OCT4 as a recombinant protein, and biochemically analyzed its interactions with the nucleosome containing a natural OCT4 target, the LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target sequences. By a combination of chemical mapping and cryo-electron microscopy single-particle analysis, we mapped the positions of the three target sequences within the nucleosome. A mutational analysis revealed that OCT4 preferentially binds its target DNA sequence located near the entry/exit site of the nucleosome. Crosslinking mass spectrometry consistently showed that OCT4 binds the nucleosome in the proximity of the histone H3 N-terminal region, which is close to the entry/exit site of the nucleosome. We also found that the linker histone H1 competes with OCT4 for the nucleosome binding. These findings provide important information for understanding the molecular mechanism by which OCT4 binds its target DNA in chromatin.
|Validation Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_30070.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.32 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Nucleosome with LIN28B distal enhancer DNA sequence
|Entire||Name: Nucleosome with LIN28B distal enhancer DNA sequence / Number of components: 1|
-Component #1: protein, Nucleosome with LIN28B distal enhancer DNA sequence
|Protein||Name: Nucleosome with LIN28B distal enhancer DNA sequence / Recombinant expression: No|
|Mass||Theoretical: 200 kDa|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||pH: 7.5|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %|
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Imaging||Microscope: FEI TALOS ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 QUANTUM (4k x 4k)|
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