Abstract
Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.
Highlights
In eukaryotic genomes, nucleosome is the primary units of chromatin responsible for the restriction of DNA accessibility and the storage of epigenetic information
The charge states of the mainly observed peak are labeled above the peak. (H) electrophoretic mobility shift assays (EMSAs) show the complexes of the 112-bp octasome with the Facilitates chromatin transcription (FACT) proteins treated by APase, detected by SYBR Gold nucleic acid gel stain
Our study reveals that FACT causes the partial replacement of the nucleosomal DNA end by the densely phosphorylated acidic intrinsically disordered (AID) segment, without alteration of the remaining octasome structure
Summary
Nucleosome is the primary units of chromatin responsible for the restriction of DNA accessibility and the storage of epigenetic information. The modular nature of nucleosome provides functional complexity through the structural variations, which regulate nuclear architecture and processes, including DNA transcription, replication, and repair. Both C-terminal acidic intrinsically disordered (AID) segments, located in Spt[16] and Pob[3] (Figs 1A and S1A), are important for the H2A-H2B binding[6]. FACT is involved in a broad range of processes, such as DNA transcription, replication, and repair[2,16,17,18,19] In spite of this diverse functional scope, every. Eukaryote contains only one FACT ortholog, implying that FACT should conduct universal behaviors in terms of nucleosome reorganization
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