scholarly journals The p150N domain of Chromatin Assembly Factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer

2016 ◽  
Author(s):  
Timothy D. Matheson ◽  
Paul D. Kaufman
Keyword(s):  
Dna Synthesis ◽  
Chromatin Assembly ◽  
Nucleolar Localization ◽  
Ki 67 ◽  
Fundamental Component ◽  
Terminal Domain ◽  
Chromatin Assembly Factor ◽  
Assembly Factor ◽  
Nuclear Structures ◽  
Histone Deposition

AbstractChromatin Assembly Factor 1 (CAF-1) deposits histones during DNA synthesis. The p150 subunit of human CAF-1 contains an N-terminal domain (p150N) that is dispensable for histone deposition but which promotes the localization of specific loci (Nucleolar-Associated Domains, or “NADs”) and proteins to the nucleolus during interphase. One of the p150N-regulated proteins is proliferation antigen Ki-67, whose depletion also decreases the nucleolar association of NADs. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins surrounding condensed chromosomes during mitosis. We show here that a subset of p150 localizes to the PCL during mitosis, and that p150N is required for normal levels of Ki-67 accumulation on the PCL. This activity requires the Sumoylation Interacting Motif (SIM) within p150N, which is also required for the nucleolar localization of NADs and Ki-67 during interphase. In this manner, p150N coordinates both interphase and mitotic nuclear structures via Ki67.

scholarly journals The p150N domain of chromatin assembly factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer

2017 ◽  
Vol 28 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Timothy D. Matheson ◽  
Paul D. Kaufman
Keyword(s):  
Dna Synthesis ◽  
Chromatin Assembly ◽  
Nucleolar Localization ◽  
Ki 67 ◽  
Fundamental Component ◽  
Terminal Domain ◽  
Chromatin Assembly Factor ◽  
Assembly Factor ◽  
Nuclear Structures ◽  
Histone Deposition

Chromatin assembly factor 1 (CAF-1) deposits histones during DNA synthesis. The p150 subunit of human CAF-1 contains an N-terminal domain (p150N) that is dispensable for histone deposition but promotes the localization of specific loci (nucleolar-associated domains [NADs]) and proteins to the nucleolus during interphase. One of the p150N-regulated proteins is proliferation antigen Ki-67, whose depletion also decreases the nucleolar association of NADs. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins surrounding condensed chromosomes during mitosis. We show here that a subset of p150 localizes to the PCL during mitosis and that p150N is required for normal levels of Ki-67 accumulation on the PCL. This activity requires the sumoylation-interacting motif within p150N, which is also required for the nucleolar localization of NADs and Ki-67 during interphase. In this manner, p150N coordinates both interphase and mitotic nuclear structures via Ki67.

scholarly journals Silencing of Chromatin Assembly Factor 1 in Human Cells Leads to Cell Death and Loss of Chromatin Assembly during DNA Synthesis

2004 ◽  
Vol 24 (7) ◽  
pp. 2853-2862 ◽  
Author(s):  
Arman Nabatiyan ◽  
Torsten Krude
Keyword(s):  
Cell Death ◽  
Dna Synthesis ◽  
Physiological Role ◽  
Human Cells ◽  
Chromatin Assembly ◽  
Cell Nuclei ◽  
Proliferating Cells ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

ABSTRACT In eukaryotic cells, chromatin serves as the physiological template for gene transcription, DNA replication, and repair. Chromatin assembly factor 1 (CAF-1) is the prime candidate protein to mediate assembly of newly replicated DNA into chromatin. To investigate the physiological role of CAF-1 in vivo, we used RNA interference (RNAi) to silence the 60-kDa subunit of CAF-1 (p60) in human cells. Transfection of a small interfering RNA (siRNA) directed against p60 resulted in efficient silencing of p60 expression within 24 h. This silencing led to an induction of programmed cell death in proliferating but not in quiescent human cells. Concomitantly, proliferating cells lacking p60 accumulated DNA double-strand breaks and increased levels of the phosphorylated histone H2A.X. Nuclear extracts from cells lacking p60 exhibited a 10-fold reduction of nucleosome assembly activity during DNA synthesis, which was restored upon addition of recombinant p60 protein. Nascent chromatin in cell nuclei lacking p60 showed significantly increased nuclease sensitivity, indicating chromatin assembly defects during DNA synthesis in vivo. Collectively, these data identify CAF-1 as an essential factor not only for S-phase-specific chromatin assembly but also for proliferating cell viability.

scholarly journals DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Francesca Mattiroli ◽  
Yajie Gu ◽  
Tejas Yadav ◽  
Jeremy L Balsbaugh ◽  
Michael R Harris ◽  
...  
Keyword(s):  
Dna Replication ◽  
Binding Mode ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Nucleosome Assembly ◽  
Domain Interaction ◽  
Chromatin Assembly Factor ◽  
Assembly Factor ◽  
Histone Deposition ◽  
Winged Helix Domain

Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4)2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.

scholarly journals Mechanistic insights into histone deposition and nucleosome assembly by the chromatin assembly factor-1

2018 ◽  
Vol 46 (19) ◽  
pp. 9907-9917 ◽  
Author(s):  
Paul V Sauer ◽  
Yajie Gu ◽  
Wallace H Liu ◽  
Francesca Mattiroli ◽  
Daniel Panne ◽  
...  
Keyword(s):  
Chromatin Assembly ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor ◽  
Histone Deposition

scholarly journals Chromatin Assembly Factor I Mutants Defective for PCNA Binding Require Asf1/Hir Proteins for Silencing

2002 ◽  
Vol 22 (2) ◽  
pp. 614-625 ◽  
Author(s):  
Denise C. Krawitz ◽  
Tamar Kama ◽  
Paul D. Kaufman
Keyword(s):  
Histone H3 ◽  
Chromatin Assembly ◽  
Nucleosome Assembly ◽  
Factor I ◽  
Chromatin Assembly Factor ◽  
Assembly Factor ◽  
Histone Deposition ◽  
Assembly Activity ◽  
Impaired Binding

ABSTRACT Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway. Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.

scholarly journals A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

2014 ◽  
Vol 25 (18) ◽  
pp. 2866-2881 ◽  
Author(s):  
Corey L. Smith ◽  
Timothy D. Matheson ◽  
Daniel J. Trombly ◽  
Xiaoming Sun ◽  
Eric Campeau ◽  
...  
Keyword(s):  
Repetitive Element ◽  
Chromatin Assembly ◽  
The Other ◽  
Dna Interactions ◽  
Interaction Sites ◽  
Chromatin Assembly Factor ◽  
Nucleolar Proteins ◽  
Nucleolar Chromosome ◽  
Assembly Factor ◽  
Chromosome Associations

Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element–containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

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/G2 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-γ 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.

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