The packaging of the eukaryotic genome into chromatin is essential for normal growth, development, and differentiation. The repeating unit of chromatin is the nucleosome core particle, which comprises 147 bp of DNA wound around a histone octamer (Luger et al., 1997
). Chromatin is a dynamic structure that tightly regulates transcription, replication, repair, and recombination. In the context of these processes, the most severe alteration of chromatin structure is the removal of histone proteins from DNA (chromatin disassembly) or the deposition of histone proteins onto naked DNA (chromatin assembly). The ordered packaging of DNA into chromatin is thought to involve the initial deposition of a heterotetramer of histones H3/H4 followed by two heterodimers of histones H2A/H2B to form the nucleosome. This process is mediated by histone chaperone proteins that regulate the association of the basic histone proteins with the DNA, which permits the nucleosome to form in an ordered and controlled manner (Akey and Luger, 2003
; Loyola and Almouzni, 2004
The histone chaperone Anti-silencing function 1 (Asf1) is the only histone chaperone that is implicated in both replication-dependent and replication-independent chromatin assembly (Nakatani et al., 2004
). Asf1 is also a critical factor in multiple other cellular processes. For example, Asf1 is a histone H3/H4 chaperone that assists Chromatin Assembly Factor 1 (CAF-1) during the assembly of newly-synthesized DNA into chromatin in vitro
(Mello et al., 2002
; Smith and Stillman, 1991
; Tyler et al., 1999
) and is required for replication-independent chromatin assembly together with the Hir histone chaperone (Green et al., 2005
; Tagami et al., 2004
). Asf1 also mediates chromatin disassembly from promoters in budding yeast during transcriptional activation (Adkins et al., 2004
) and chromatin disassembly and reassembly during transcriptional elongation (Schwabish and Struhl, 2006
). In fact, all non DNA-bound histones are bound to Asf1 (Groth et al., 2005
; Tagami et al., 2004
), underscoring its fundamental role as a central histone chaperone in eukaryotes.
The function and structure of Asf1 are highly conserved among eukaryotes. The N-terminal 155 residues of Asf1 form a globular core that consists of an immunoglobulin-like fold (Daganzo et al., 2003
; Mousson et al., 2005
) with highly conserved acidic patches that are predicted to mediate interactions with histone H3. The structures of histones H3 and H4 have also been determined as components of the nucleosome core particle and histone octamer (Arents et al., 1991
; Luger et al., 1997
). The yeast and Xenopus laevis
nucleosome structures underscore the structural and functional conservation of this complex throughout evolution (White et al., 2001
). Thus, it is well known how the individual histones interact with each other and with DNA, but there is little information about the interaction of the histones with each other or with chaperones in the absence of DNA. As a result, the mechanisms by which the nucleosomes are assembled and disassembled are not well understood.
Recently, we biophysically characterized the Asf1-H3/H4 complex and demonstrated that Asf1 binds to a heterodimer of histones H3 and H4 (English et al., 2005
). In addition, yeast two-hybrid analysis suggests that the C-terminus of histone H3 interacts with Asf1 (Munakata et al., 2000
), and NMR chemical-shift mapping revealed interactions of an H3 peptide (residues 122-135) with a highly conserved and acidic patch on the concave surface of Asf1 (Mousson et al., 2005
). This histone H3 peptide corresponds to helix 3, which is a crucial part of a four-helix bundle that forms the H3:H3 dimerization interface in the nucleosome (Luger et al., 1997
). Furthermore, a disruptive mutation in the middle of this H3-interacting region of Asf1, V94R, abolished the interaction with histone H3 (Mousson et al., 2005
). These data raised the possibility that the region that mediates H3:H3 dimerization in the H3/H4 heterotetramer may also be the region of H3 that binds to Asf1.
In this study, we have determined the crystal structure of Asf1 bound to histones H3/H4, revealing how a histone chaperone binds histones and how the structures of H3 and H4 differ outside of the histone octamer. We have confirmed that Asf1 binds to a heterodimer of histones H3/H4 and that Asf1 not only interacts with histone H3 but also with the C-terminal tail of histone H4 in vivo and in vitro. Of interest, the C-terminal tail of H4 undergoes a major conformational change upon binding to Asf1, suggesting a mechanism by which the H4 tail may act as a handle for the assembly and disassembly of the H3/H4 heterotetramers by Asf1.