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.
Subject(s)
DNA/chemistry , Heterochromatin/metabolism , Histones/chemistry , Nucleosomes/metabolism , Octamer Transcription Factor-3/chemistry , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Cell-Free System , Cloning, Molecular , Cryoelectron Microscopy , DNA/genetics , DNA/metabolism , Enhancer Elements, Genetic , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Heterochromatin/chemistry , Heterochromatin/ultrastructure , Histones/genetics , Histones/metabolism , Humans , Nucleic Acid Conformation , Nucleosomes/chemistry , Nucleosomes/ultrastructure , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Protein Binding , Protein Conformation , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Nucleosomes are dynamic entities that are repositioned along DNA by chromatin remodeling processes. A nucleosome repositioned by the switch-sucrose nonfermentable (SWI/SNF) remodeler collides with a neighbor and forms the intermediate "overlapping dinucleosome." Here, we report the crystal structure of the overlapping dinucleosome, in which two nucleosomes are associated, at 3.14-angstrom resolution. In the overlapping dinucleosome structure, the unusual "hexasome" nucleosome, composed of the histone hexamer lacking one H2A-H2B dimer from the conventional histone octamer, contacts the canonical "octasome" nucleosome, and they intimately associate. Consequently, about 250 base pairs of DNA are left-handedly wrapped in three turns, without a linker DNA segment between the hexasome and octasome moieties. The overlapping dinucleosome structure may provide important information to understand how nucleosome repositioning occurs during the chromatin remodeling process.
