Newly synthesized DNA is rapidly assembled into mature nucleosomes by the deposition of preexisting and nascent histones, and some parts of this process are facilitated by chromatin assembly factor 1 (CAF-1). Loss-of-function mutants of CAF-1 in Arabidopsis , fasciata ( fas ), show a variety of morphological abnormalities and unique defects in gene expression in the meristems. In order to clarify the implications of CAF-1 in the maintenance of chromatin states in higher eukaryotes, we investigated transcriptional gene silencing (TGS) of various genes in fas mutants. Here, we show that TGS of endogenous CACTA transposons was released in a stochastic manner in fas . Other endogenous silent genes, a transposon AtMu1 and a hypothetical gene T5L23.26 at a heterochromatin knob, were also transcriptionally activated, and the activation of the three different silent loci at different chromosomal sites occurred non-concomitantly with each other. Furthermore, TGS of the silent β β β β -glucuronidase ( GUS ) transgene was also de-repressed randomly in fas . We conclude that CAF-1 ensures the stable inheritance of epigenetic states through growth and development in Arabidopsis .

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G 2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-␥ are required for cell viability. These observations highlighted the essential role of CAF-1-dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

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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ABSTRACT. The cleavage cycle, which is initiated by fertilization, consists of only S and M phases, and the gap phases (G1 and G2) appear after the midblastula transition (MBT) in the African clawed frog, Xenopus laevis. During early development in Xenopus, we examined the E2F activity, which controls transition from the G1 to S phase in the somatic cell cycle. Gel retardation and transactivation assays revealed that, although the E2F protein was constantly present throughout early development, the E2F transactivation activity was induced in a stagespecific manner, that is, low before MBT and rapidly increased after MBT. Introduction of the recombinant dominant negative E2F (dnE2F), but not the control, protein into the 2-cell stage embryos specifically suppressed E2F activation after MBT. Cells in dnE2F-injected embryos appeared normal before MBT, but ceased to proliferate and eventually died at the gastrula. These cells contained decreased cdk activity with enhanced inhibitory phosphorylation of Cdc2 at Tyr15. Thus, E2F activity is required for cell cycle progression and cell viability after MBT, but not essential for MBT transition and developmental progression during the cleavage stage.