Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1-and HIRA-independent chromatin-modulating process for cell proliferation.During S phase, newly synthesized histones H3-H4 are assembled behind the replication fork, followed by loading of the H2A-H2B dimer to complete de novo nucleosome formation on newly replicated DNA (1-3). Besides such a replication-coupled chromatin assembly process, a DNA synthesis-independent chromatin assembly process also exists to operate histone deposition during transcription and DNA repair (4). These processes are mediated by several specialized histone chaperones (1). CAF-1 (chromatin assembly factor-1), a trimeric protein complex consisting of p150, p60, and p48 subunits, is the most characterized chaperone responsible for loading of histones H3 and H4 onto replicating DNA through an interaction with proliferating cell nuclear antigen (PCNA), 2

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