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 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.
Highlights
Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer
To test the OCT4 binding to its target DNA sequence in the nucleosome, we reconstituted the nucleosome with a 162 base-pair DNA fragment containing the human LIN28B distal enhancer region (LIN28B nucleosome)
The target sites for pioneer Transcription factors (TFs) are considered to be buried in the closed chromatin, which is generally inaccessible to DNA binding proteins
Summary
Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. Pioneer transcription factors efficiently bind their target DNA 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. At the initial stage of the reprogramming process, OCT4, SOX2, and KLF4 may function as pioneer factors and bind to the “closed chromatin” state, inducing the conversion to the “open chromatin” s tate[9]. OCT4, SOX2, and KLF4 efficiently bind to reconstituted nucleosomes in vitro, and preferentially accumulate at genomic DNA regions occupied by nucleosomes in vivo[10]. We reconstituted the nucleosome with the native LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target DNA sequences.
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