The assembly of core histones onto eukaryotic DNA is modulated by several histone chaperone complexes, including Asf1, CAF-1, and HIRA. Asf1 is a unique histone chaperone that participates in both the replication-dependent and replication-independent pathways. Here we report the crystal structures of the apo-form of fission yeast Asf1/Cia1 ( Eukaryotic genomic DNA forms hierarchical nucleoprotein complex structures in the nucleus. The nucleosome core particle is the basal repeating unit of the complex, which is composed of ϳ147 bp of DNA wrapped around a core histone particle, comprising a tetramer of H3 and H4 and two dimers of H2A and H2B (1, 2). The precise and regular arraying of nucleosomes is supposed to be a key determinant for the formation of upper hierarchical structures, such as the 30-nm fibers and chromatin fibers. The assembly of the nucleosome is regulated by several groups of chromatin-associated factors involving histone chaperones, chromatin remodeling factors, and histone modification enzymes, which are tightly linked to the regulation of DNA metabolism (2).Histone chaperones are factors that bind to core histones and facilitate their deposition onto nucleosomes (3). Among the variety of histone chaperones, Asf1 4 is the most evolutionarily conserved in its primary structure (4 -8), and its function as a histone chaperone is conserved throughout the eukaryotes (5, 6, 9 -11). Asf1 associates with a variety of chromatin-associated factors, including the histone chaperones CAF-1 (12, 13) and HIR (10, 14, 15), and stimulates both the assembly and disassembly of chromatin (5,6,9,16,17). Consequently, Asf1 affects most DNA-mediated events, including gene expression (15,16,18,19) and silencing (4, 20 -22) as well as DNA repair (5, 13, 23, 24), replication (5) and recombination (25).Asf1 interacts with a heterodimer of histones H3/H4 through the C-terminal region of H3 (26, 27). Importantly, two human family members of Asf1 (ASF1A and ASF1B) are involved in both the major S-phase histone H3.1-and histone variant H3.3 complexes, whereas the Asf1-interactive histone chaperones CAF-1 and HIRA are detected only in the histone H3.1 and H3.3 complexes, respectively (28). Consistently, Asf1 facilitates both DNA replication-dependent and -independent histone depositions cooperatively with the CAF-1 and HIR complexes, respectively (5,28,29), indicating the central role of Asf1 in controlling the state of histone deposition in the nucleus. Recently, the complex structure of human Asf1 with the B-domain of HIRA was reported (30). In addition, biochemical studies suggested that human Cac2, the second largest subunit of CAF-1, interacts with Asf1 at the HIRA-binding region of Asf1 through its B-domain-like motif at the C terminus (30). Hence, human Asf1 is thought to interact mutually exclusively with the histone chaperones HIRA and CAF-1. However, structural evidence for the interaction between Asf1 and CAF-1 has yet to be obtained. In addition, it is not clear how Asf1 recognizes HIRA and CAF-1 for histone assem...