Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells.
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.
Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Fumiyuki
F
Sanematsu
Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Yasunari
Y
Takami
Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Hirak Kumar
HK
Barman
Department of Molecular Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
Tatsuo
T
Fukagawa
Department of Integrated Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
Tatsuya
T
Ono
Department of Integrated Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
Kei-Ichi
KI
Shibahara
Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan; Department of Life Science, Frontier Science Research Center, University of Miyazaki, 5200, Kihara, Kiyotake, Miyazaki 889-1692, Japan. Electronic address: tnakayam@med.miyazaki-u.ac.jp.
Tatsuo
T
Nakayama
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death; D002470:Cell Survival; D002645:Chickens; D002843:Chromatin / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D008938:Mitosis / Q000502:physiology; D009154:Mutation; D017434:Protein Structure, Tertiary
D018797:Cell Cycle Proteins; D002843:Chromatin