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

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

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 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.

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

2009 ◽  
Vol 29 (24) ◽  
pp. 6353-6365 ◽  
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.

scholarly journals In Vivo Study of the Nucleosome Assembly Functions of ASF1 Histone Chaperones in Human Cells

2008 ◽  
Vol 28 (11) ◽  
pp. 3672-3685 ◽  
Author(s):  
Angélique Galvani ◽  
Régis Courbeyrette ◽  
Morgane Agez ◽  
Françoise Ochsenbein ◽  
Carl Mann ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Histone H3 ◽  
Direct Determination ◽  
Human Cells ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Nucleosome Assembly ◽  
Assembly Factor

ABSTRACT Histone chaperones have been implicated in nucleosome assembly and disassembly as well as histone modification. ASF1 is a highly conserved histone H3/H4 chaperone that synergizes in vitro with two other histone chaperones, chromatin assembly factor 1 (CAF-1) and histone repression A factor (HIRA), in DNA synthesis-coupled and DNA synthesis-independent nucleosome assembly. Here, we identify mutants of histones H3.1 and H3.3 that are unable to interact with human ASF1A and ASF1B isoforms but that are still competent to bind CAF-1 and HIRA, respectively. We show that these mutant histones are inefficiently deposited into chromatin in vivo. Furthermore, we found that both ASF1A and ASF1B participate in the DNA synthesis-independent deposition of H3.3 in HeLa cells, thus highlighting an unexpected role for ASF1B in this pathway. This pathway does not require interaction of ASF1 with HIRA. We provide the first direct determination that ASF1A and ASF1B play a role in the efficiency of nucleosome assembly in vivo in human cells.

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

1998 ◽  
Vol 143 (3) ◽  
pp. 563-575 ◽  
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.

scholarly journals HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore

2011 ◽  
Vol 194 (2) ◽  
pp. 229-243 ◽  
Author(s):  
Meghan C. Barnhart ◽  
P. Henning J. L. Kuich ◽  
Madison E. Stellfox ◽  
Jared A. Ward ◽  
Emily A. Bassett ◽  
...  
Keyword(s):  
De Novo ◽  
Lac Repressor ◽  
Chromatin Assembly ◽  
Epigenetic Mark ◽  
Lac Operon ◽  
Nucleosome Assembly ◽  
Amino Terminal ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Centromeres of higher eukaryotes are epigenetically marked by the centromere-specific CENP-A nucleosome. New CENP-A recruitment requires the CENP-A histone chaperone HJURP. In this paper, we show that a LacI (Lac repressor) fusion of HJURP drove the stable recruitment of CENP-A to a LacO (Lac operon) array at a noncentromeric locus. Ectopically targeted CENP-A chromatin at the LacO array was sufficient to direct the assembly of a functional centromere as indicated by the recruitment of the constitutive centromere-associated network proteins, the microtubule-binding protein NDC80, and the formation of stable kinetochore–microtubule attachments. An amino-terminal fragment of HJURP was able to assemble CENP-A nucleosomes in vitro, demonstrating that HJURP is a chromatin assembly factor. Furthermore, HJURP recruitment to endogenous centromeres required the Mis18 complex. Together, these data suggest that the role of the Mis18 complex in CENP-A deposition is to recruit HJURP and that the CENP-A nucleosome assembly activity of HJURP is responsible for centromeric chromatin assembly to maintain the epigenetic mark.

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