Structural insights into histone binding and nucleosome assembly by chromatin assembly factor-1

Chromatin inheritance entails de novo nucleosome assembly after DNA replication by chromatin assembly factor-1 (CAF-1). Yet direct knowledge about CAF-1’s histone binding mode and nucleosome assembly process is lacking. In this work, we report the crystal structure of human CAF-1 in the absence of histones and the cryo–electron microscopy structure of CAF-1 in complex with histones H3 and H4. One histone H3-H4 heterodimer is bound by one CAF-1 complex mainly through the p60 subunit and the acidic domain of the p150 subunit. We also observed a dimeric CAF-1–H3-H4 supercomplex in which two H3-H4 heterodimers are poised for tetramer assembly and discovered that CAF-1 facilitates right-handed DNA wrapping of H3-H4 tetramers. These findings signify the involvement of DNA in H3-H4 tetramer formation and suggest a right-handed nucleosome precursor in chromatin replication. Description Editor’s summary Chromosomal duplication involves the replication of DNA and the assembly of chromatin onto the replicated DNA. Chromatin assembly factor-1 (CAF-1) is a heterotrimeric protein complex responsible for the deposition of newly synthesized histones H3 and H4 onto DNA in replication-coupled nucleosome assembly. Liu et al. revealed that in the presence of histones H3 and H4, the two otherwise flexibly tethered globular domains of CAF-1 attach to opposite ends of an elongated H3-H4 heterodimer, and a flexible loop of the p150 subunit becomes ordered to secure H3-H4 binding. Formation of an H3-H4 tetramer is a prerequisite for nucleosome assembly, and DNA induces the juxtaposition of two CAF-1–H3-H4 complexes poised for H3-H4 tetramer formation. CAF-1 promotes right-handed DNA wrapping of H3-H4 tetramers in vitro, in contrast to the left-handed DNA wrapping in the nucleosome. —Di Jiang CAF-1 binding primes histone H3-H4 heterodimers for tetramer assembly and assembles a right-handed nucleosome precursor. INTRODUCTION Inheritance of chromatin structure during cell division is essential for genome integrity and epigenetic inheritance. DNA replication disrupts parental nucleosomes, and nascent nucleosomes must be assembled on replicated DNA. Half of the histones for the replication-coupled nucleosome assembly are of parental origin, and the other half are newly synthesized. Chromatin assembly factor-1 (CAF-1) is an evolutionarily conserved heterotrimeric protein complex responsible for the deposition of newly synthesized histones H3 and H4 onto DNA. A mechanistic understanding of the structure and function of CAF-1 is needed to fully comprehend the principles of chromatin inheritance. RATIONALE We determined the crystal structure of the core domain of human CAF-1 in the absence of histones and used cryo–electron microscopy (cryo-EM) to solve the structure of CAF-1 bound to histones H3 and H4. Structural findings were corroborated with in vitro supercoiling assays and in vivo nascent nucleosome mapping analyses. We also investigated the handedness of the nucleosome precursor assembled by CAF-1 using a single-molecule freely orbiting magnetic tweezer (FOMT) method. RESULTS The crystal structure of the CAF-1 core domain shows that, in the absence of histones, it adopts a bilobal structure, with the p48 and p60 subunits freely tethered by the middle domain of the p150 subunit. Upon the binding of an H3-H4 heterodimer, p48 and p60 hug the opposite ends of the elongated H3-H4 heterodimer, whereas the negatively charged ED loop of p150 secures the binding by traversing the hunched positively charged surface of the H3-H4 dimer. The C-terminal portion of the ED loop plays a particularly important role in CAF-1’s histone binding and nucleosome assembly activities, as shown by in vitro histone binding and plasmid supercoiling assays, as well as in vivo nucleosome assembly analysis. The observed histone binding mode ensures that only one H3-H4 heterodimer is bound by a CAF-1 complex because the joining of a second H3-H4 dimer in the manner of a H3-H4 tetramer would be sterically prohibited by p60, which suggests that formation of an H3-H4 tetramer is regulated. A p60 cancer mutation located at the interface with H4 is shown to have a detrimental effect in cell proliferation and cause a global change of gene expression. These effects likely reflect CAF-1’s functions in chromatin accessibility and heterochromatin integrity. We demonstrate that the addition of short DNA oligomers promotes the dimerization of the CAF-1–H3-H4 complex. A cryo-EM structure of a 2:2 CAF-1–H3-H4 complex shows that the two H3-H4 heterodimers are placed nearby, with the H3 dimerization interface poised for interaction but not quite in the geometric configuration of an H3-H4 tetramer. Given that longer DNA fragments wrap H3-H4 as tetramers, the length of DNA may be a key factor in the CAF-1–mediated formation of H3-H4 tetramers. With a 147–base pair (bp) nucleosome-positioning Widom 601 DNA, we found that a right-handed CAF-1–bound ditetrasome was assembled through salt dialysis. This discovery was confirmed by single-molecule FOMT at a physiological salt concentration, indicating a possible unusual nucleosome precursor in chromatin replication. CONCLUSION Our study reveals the histone binding mode of CAF-1. It also elucidates the role of DNA in the dimerization of the CAF-1–H3-H4 complex and the assembly of H3-H4 tetramers and suggests the involvement of a right-handed nucleosome precursor in replication-coupled nucleosome assembly. Structures of CAF-1 with and without histones H3 and H4 bound. (Top middle) A 3.5-Å crystal structure of the core domain of CAF-1. Distinct subunits are labeled and color coded as indicated. The light pink segment with a dashed outline indicates disordered ED loop of p150. (Top left) A 3.5-Å cryo-EM structure of CAF-1 bound to a heterodimer of H3 (blue) and H4 (green). The light blue segment with a dashed outline indicates the disordered H3 loop that connects a short N-terminal segment and the body of H3. (Bottom left) A 4.6-Å cryo-EM map of a 2:2 CAF-1–H3-H4 complex in which CAF-1 subunits are colored gray. The graphic to the right of the arrow is a top view of the 2:2 dimer with CAF-1 omitted for viewing clarity. It shows the positioning of two H3-H4 heterodimers, shown in densities, in the 2:2 complex, compared with those in an H3-H4 tetramer, which is modeled by placing a fictitious H3-H4 dimer (ribbon representation) next to an observed one. (Top right) A 3.8-Å cryo-EM map of the CAF-1–bound right-handed ditetrasome compared with the left-handed DNA wrapping of a nucleosome core particle (NCP) schematically drawn at the bottom right corner.