scholarly journals Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1

2001 ◽  
Vol 29 (3) ◽  
pp. 629-637 ◽  
Author(s):  
A. Ahmad
Keyword(s):  
Leucine Zipper ◽  
Histone Acetyltransferase ◽  
Chromatin Assembly ◽  
Leucine Zipper Motif ◽  
Chromatin Assembly Factor ◽  
Assembly Factor ◽  
In Vitro Interactions

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 MOLECULAR CLONING, EXPRESSION AND FUNCTIONAL INTERACTION OF p48 SUBUNIT OF CHICKEN CHROMATIN ASSEMBLY FACTOR 1 WITH HISTONE DEACETYLASE 2 AND HISTONE ACETYLTRANSFERASE 1

2012 ◽  
Vol 12 (1) ◽  
pp. 77-83
Author(s):  
Ahyar Ahmad
Keyword(s):  
Amino Acids ◽  
Histone Deacetylase ◽  
Leucine Zipper ◽  
Histone Acetyltransferase ◽  
Leucine Zipper Motif ◽  
Histone Deacetylase 2 ◽  
Pulldown Assay ◽  
Assembly Factor ◽  
In Vitro Interaction

We cloned and sequenced cDNA encoding p48 subunit of the chicken CAF-1, chCAF-1p48, and histone acetyltransferase-1, chHAT-1 from chicken DT40 cell lines. We showed that the p48 subunit of CAF-1 tightly binds to two regions of chicken histone deacetylase 2, chHDAC-2, located between amino acid residues 82-180 and 245-314, respectively. We also established that two N-terminal, two C-terminal, or one N-terminal and one C-terminal WD repeat motif of chCAF-1p48 are required for this interaction. The GST pulldown assay, involving truncated and missense mutants of chCAF-1p48, revealed not only that a region containing the seventh WD dipeptide motif of chCAF-1p48, comprising amino acids 376-405, binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. We also established that the region, which is located between amino acids 380-408 of chHAT-1 and contains a leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. Mutation on each of four Leu residues in the leucine zipper motif of chHAT-1 causes the disappearance of the interaction with chCAF-1p48. These results should be useful information for understanding the participation of chCAF-1p48 protein as histones chaperone in DNA-utilizing processes, such as replication, recombination, repair and gene expression in DT40 chicken B cell.

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 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 Interaction between the DrosophilaCAF-1 and ASF1 Chromatin Assembly Factors

2001 ◽  
Vol 21 (19) ◽  
pp. 6574-6584 ◽  
Author(s):  
Jessica K. Tyler ◽  
Kimberly A. Collins ◽  
Jayashree Prasad-Sinha ◽  
Elizabeth Amiott ◽  
Michael Bulger ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Polytene Chromosomes ◽  
Normal Growth ◽  
Chromatin Assembly ◽  
Truncated Form ◽  
Assembly Factor ◽  
Development And Differentiation ◽  
Growth Development

ABSTRACT The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the 180- and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and theDrosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we observed that dASF1 and dCAF-1 p105 colocalized in vivo inDrosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin.

scholarly journals The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation

2013 ◽  
Vol 12 (5) ◽  
pp. 654-664 ◽  
Author(s):  
Ernest Radovani ◽  
Matthew Cadorin ◽  
Tahireh Shams ◽  
Suzan El-Rass ◽  
Abdel R. Karsou ◽  
...  
Keyword(s):  
Histone Acetyltransferase ◽  
Histone H3 ◽  
Chromatin Assembly ◽  
Carboxyl Terminus ◽  
Histone Chaperones ◽  
Content Type ◽  
Histone H3 Acetylation ◽  
H3 Acetylation

ABSTRACT Rtt109 is a fungal histone acetyltransferase (HAT) that catalyzes histone H3 acetylation functionally associated with chromatin assembly. Rtt109-mediated H3 acetylation involves two histone chaperones, Asf1 and Vps75. In vivo , Rtt109 requires both chaperones for histone H3 lysine 9 acetylation (H3K9ac) but only Asf1 for full H3K56ac. In vitro , Rtt109-Vps75 catalyzes both H3K9ac and H3K56ac, whereas Rtt109-Asf1 catalyzes only H3K56ac. In this study, we extend the in vitro chaperone-associated substrate specificity of Rtt109 by showing that it acetylates vertebrate linker histone in the presence of Vps75 but not Asf1. In addition, we demonstrate that in Saccharomyces cerevisiae a short basic sequence at the carboxyl terminus of Rtt109 (Rtt109C) is required for H3K9ac in vivo . Furthermore, through in vitro and in vivo studies, we demonstrate that Rtt109C is required for optimal H3K56ac by the HAT in the presence of full-length Asf1. When Rtt109C is absent, Vps75 becomes important for H3K56ac by Rtt109 in vivo . In addition, we show that lysine 290 (K290) in Rtt109 is required in vivo for Vps75 to enhance the activity of the HAT. This is the first in vivo evidence for a role for Vps75 in H3K56ac. Taken together, our results contribute to a better understanding of chaperone control of Rtt109-mediated H3 acetylation.

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