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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.22.2.614-625.2002 ◽

2002 ◽

Vol 22 (2) ◽

pp. 614-625 ◽

Cited By ~ 124

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0426 ◽

2007 ◽

Vol 18 (1) ◽

pp. 129-141 ◽

Cited By ~ 53

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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Chromatin Assembly Factor 1 (CAF-1) facilitates the establishment of facultative heterochromatin during pluripotency exit

Nucleic Acids Research ◽

10.1093/nar/gkz858 ◽

2019 ◽

Vol 47 (21) ◽

pp. 11114-11131 ◽

Cited By ~ 10

Author(s):

Liang Cheng ◽

Xu Zhang ◽

Yan Wang ◽

Haiyun Gan ◽

Xiaowei Xu ◽

...

Keyword(s):

Cell Fate ◽

Embryonic Stem ◽

Chromatin Assembly ◽

Nuclear Antigen ◽

Gene Promoters ◽

Cell Fate Determination ◽

Nucleosome Assembly ◽

Fate Determination ◽

Chromatin Assembly Factor ◽

Assembly Factor

Abstract Establishment and subsequent maintenance of distinct chromatin domains during embryonic stem cell (ESC) differentiation are crucial for lineage specification and cell fate determination. Here we show that the histone chaperone Chromatin Assembly Factor 1 (CAF-1), which is recruited to DNA replication forks through its interaction with proliferating cell nuclear antigen (PCNA) for nucleosome assembly, participates in the establishment of H3K27me3-mediated silencing during differentiation. Deletion of CAF-1 p150 subunit impairs the silencing of many genes including Oct4, Sox2 and Nanog as well as the establishment of H3K27me3 at these gene promoters during ESC differentiation. Mutations of PCNA residues involved in recruiting CAF-1 to the chromatin also result in defects in differentiation in vitro and impair early embryonic development as p150 deletion. Together, these results reveal that the CAF-1-PCNA nucleosome assembly pathway plays an important role in the establishment of H3K27me3-mediated silencing during cell fate determination.

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The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1- mediated nucleosome assembly onto replicated DNA in vitro

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.97.14.7766 ◽

2000 ◽

Vol 97 (14) ◽

pp. 7766-7771 ◽

Cited By ~ 59

Author(s):

K.-i. Shibahara ◽

A. Verreault ◽

B. Stillman

Keyword(s):

Chromatin Assembly ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor ◽

Terminal Domains

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Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1

eLife ◽

10.7554/elife.23474 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 24

Author(s):

Paul Victor Sauer ◽

Jennifer Timm ◽

Danni Liu ◽

David Sitbon ◽

Elisabetta Boeri-Erba ◽

...

Keyword(s):

Dna Binding ◽

Histone H3 ◽

Chromatin Assembly ◽

Molecular Architecture ◽

Dna Molecules ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor ◽

Chaperone Complex

How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3–H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization.

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MUTATION ON WD DIPEPTIDE MOTIFS OF THE p48 SUBUNIT OF CHROMATIN ASSEMBLY FACTOR-1 CAUSING VIABILITY AND GROWTH OF DT40 CHICKEN B CELL LINE

Indonesian Journal of Chemistry ◽

10.22146/ijc.21468 ◽

2010 ◽

Vol 10 (2) ◽

pp. 245-250

Author(s):

Ahyar Ahmad ◽

Harningsih Karim

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Line ◽

Protein Complex ◽

Protein Family ◽

Chromatin Assembly ◽

Histone H4 ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor

Chromatin assembly factor-1 (CAF-1), a protein complex consisting of three subunits, p150, p60, and p48, is highly conserved from yeast to humans and facilitated nucleosome assembly of newly replicated DNA. The p48 subunit, CAF-1p48 (p48), with seven WD (Trp-Asp) repeat motifs, is a member of the WD protein family. The immunoprecipitation experiment revealed that ß-propeller structure of p48 was less stringent for it's binding to HDAC-1, but more stringent for its binding to both histones H4 and CAF-1p60 but not to ASF-1, indicating that the proper ß-propeller structure of p48 is essential for the binding to these two proteins histone H4 and CAF-1p60. Complementation experiments, involving missense and truncated mutants of FLAG-tagged p48, revealed that mutations of every of seven WD dipeptide motifs, like both the N-terminal and C-terminal truncated mutations, could not rescue for the tet-induced lethality. These results indicate not only that p48 is essential for the viability of vertebrate cells, although the yeast p48 homolog is nonessential, but also that all the seven WD dipeptide motifs are necessary for the maintenance of the proper structure of p48 that is fundamentally important for cell viability. Keywords: Chromatin assembly factor-1, complementation experiments, viability

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Two Fundamentally Distinct PCNA Interaction Peptides Contribute to Chromatin Assembly Factor 1 Function

Molecular and Cellular Biology ◽

10.1128/mcb.01051-09 ◽

2009 ◽

Vol 29 (24) ◽

pp. 6353-6365 ◽

Cited By ~ 44

Author(s):

Tom Rolef Ben-Shahar ◽

Araceli G. Castillo ◽

Michael J. Osborne ◽

Katherine L. B. Borden ◽

Jack Kornblatt ◽

...

Keyword(s):

Dna Replication ◽

Large Subunit ◽

Chromatin Assembly ◽

Nuclear Antigen ◽

Replication Forks ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor

ABSTRACT Chromatin assembly factor 1 (CAF-1) deposits histones H3 and H4 rapidly behind replication forks through an interaction with the proliferating cell nuclear antigen (PCNA), a DNA polymerase processivity factor that also binds to a number of replication enzymes and other proteins that act on nascent DNA. The mechanisms that enable CAF-1 and other PCNA-binding proteins to function harmoniously at the replication fork are poorly understood. Here we report that the large subunit of human CAF-1 (p150) contains two distinct PCNA interaction peptides (PIPs). The N-terminal PIP binds strongly to PCNA in vitro but, surprisingly, is dispensable for nucleosome assembly and only makes a modest contribution to targeting p150 to DNA replication foci in vivo. In contrast, the internal PIP (PIP2) lacks one of the highly conserved residues of canonical PIPs and binds weakly to PCNA. Surprisingly, PIP2 is essential for nucleosome assembly during DNA replication in vitro and plays a major role in targeting p150 to sites of DNA replication. Unlike canonical PIPs, such as that of p21, the two p150 PIPs are capable of preferentially inhibiting nucleosome assembly, rather than DNA synthesis, suggesting that intrinsic features of these peptides are part of the mechanism that enables CAF-1 to function behind replication forks without interfering with other PCNA-mediated processes.

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Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells

The Journal of Cell Biology ◽

10.1083/jcb.143.3.563 ◽

1998 ◽

Vol 143 (3) ◽

pp. 563-575 ◽

Cited By ~ 120

Author(s):

Emmanuelle Martini ◽

Danièle M.J. Roche ◽

Kathrin Marheineke ◽

Alain Verreault ◽

Geneviève Almouzni

Keyword(s):

Uv Irradiation ◽

Physiological Role ◽

Human Cells ◽

Chromatin Assembly ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor ◽

G2 Cells

The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.

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

10.1101/082339 ◽

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.

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