Abstract
Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.
Highlights
The structural organization of the CAF-1 complex is only beginning to emerge[19,20], and the direct contribution of each subunit to CAF-1 functions remain unclear
Previous data showed that the three CAF-1 subunits, Cac[1], Cac[2] and Cac[3], assemble in a 1:1:1 stoichiometry[34], which was confirmed by our analysis with size exclusion chromatography in line with multi-angle light scattering (SEC-MALS) (Fig. 1d)
We have shown that the trimeric tCAF-1 complex stabilizes the histone fold core of WTH3-H4, but does not interfere with the tetramerization of the histones, as no significant changes are seen in the C-terminal part of H337
Summary
The structural organization of the CAF-1 complex is only beginning to emerge[19,20], and the direct contribution of each subunit to CAF-1 functions remain unclear. Other studies have attributed this function to the small subunit (Cac[3] in yeast), as the human and fly homologues bind histones H3 and H4 when tested in isolation[21,22,23,24]. This subunit is shared with other chromatin-related complexes, such as histone deacetylase complexes, NuRD, NURF and PRC225–29. We focus on the yeast CAF-1 complex and identify the regions that mediate complex formation between the three CAF-1 subunits (Cac[1], Cac[2] and Cac[3]) We show that these interfaces are relevant for CAF-1 function www.nature.com/scientificreports/. The Cac[1] acidic region contributes to histone binding and together, these shape a composite interface on CAF-1 for H3-H4 binding
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