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. 2005 Sep;4(9):1583-90.
doi: 10.1128/EC.4.9.1583-1590.2005.

Functional Conservation and Specialization Among Eukaryotic Anti-Silencing Function 1 Histone Chaperones

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Free PMC article

Functional Conservation and Specialization Among Eukaryotic Anti-Silencing Function 1 Histone Chaperones

Beth A Tamburini et al. Eukaryot Cell. .
Free PMC article

Abstract

Chromatin disassembly and reassembly, mediated by histone chaperones such as anti-silencing function 1 (Asf1), are likely to accompany all nuclear processes that occur on the DNA template. In order to gain insight into the functional conservation of Asf1 across eukaryotes, we have replaced the budding yeast Asf1 protein with Drosophila Asf1 (dAsf1) or either of the two human Asf1 (hAsf1a and hAsf1b) counterparts. We found that hAsf1b is best able to rescue the growth defect of Saccharomyces cerevisiae lacking Asf1. Moreover, dAsf1 and hAsf1b but not hAsf1a can replace the role of yeast Asf1 in protecting against replicational stress and activating the PHO5 gene, while only hAsf1a can replace the role of Asf1 in protecting against double-stranded-DNA-damaging agents. Furthermore, it appears that the interaction between Asf1 and the DNA damage checkpoint protein Rad53 is not required for Asf1's role in maintaining genomic integrity. In addition to indicating the functional conservation of the Asf1 proteins across species, these studies suggest distinct roles for the two human Asf1 proteins.

Figures

FIG. 1.
FIG. 1.
A. Alignment of yeast, Drosophila, and human Asf1 proteins, showing conserved and nonconserved regions of the Asf1 gene, where black represents identical residues and gray represents conserved residues. B. Schematic of insertion of Asf1 homologs into yeast. PCR fragments carrying the open reading frames of human and Drosophila Asf1 were inserted by homologous recombination downstream of the endogenous yeast ASF1 promoter. Yeast DNA is represented by gray boxes and homolog DNA is represented by white boxes. A C-terminal 13-Myc epitope tag and kanamycin resistance gene were included for detection and selection on medium, respectively. C. Equivalent expression of the yeast, Drosophila, and human Asf1 proteins in yeast; 6 μg of total protein extracts from strains ACN026 (yAsf1), ROY1172 (no tag), BAT014 (dAsf1), BAT016 (hAsf1a), and KDY006 (hAsf1b) were loaded on a 7.5% SDS gel and Western blotted using a Myc antibody.
FIG. 2.
FIG. 2.
hAsf1b best complements the growth defect of yeast deleted for ASF1. A. Exponentially growing cultures of strains JKT049 (yAsf1), BAT015 (hAsf1a), BAT011 (hAsf1b), BAT013 (dAsf1), and ROY1169 (asf1Δ) were diluted to an OD of 0.1, and OD readings were taken over time and plotted. B. The same strains used in panel A were plated and grown for 3 days before images were photographed.
FIG. 3.
FIG. 3.
Ability of homologs of Asf1 to provide resistance to DNA-damaging agents. Exponentially growing cultures of yeast strains ROY1172 (yAsf1), BAT016 (hAsf1a), KDY006 (hAsf1b), BAT014 (dAsf1), and ROY1170 (asf1Δ) were plated in 10-fold serial dilutions on rich medium (control), rich medium plus 1.5 mU bleomycin, 100 mM hydroxyurea, 10 μM camptothecin, and 0.01% methyl methane sulfonate and grown for 2 to 3 days at 30°C.
FIG. 4.
FIG. 4.
Ability of homologs of Asf1 to mediate transcriptional silencing. Yeast strains ROY1171 (yAsf1), BAT015 (hAsf1a), BAT011 (hAsf1b), BAT013 (dAsf1), and ROY1169 (asf1Δ) were plated in 10-fold serial dilutions onto rich medium, medium containing the indicated amounts of 5′FOA per 250 ml, or medium containing low adenine and grown for 2 to 3 days at 30°C.
FIG. 5.
FIG. 5.
Ability of homologs of Asf1 to activate PHO5. Yeast strains JKT010 (yAsf1), BAT014 (dAsf1), BAT016 (hAsf1a), KDY006 (hAsf1b), and MPY0042 (asf1Δ) were grown overnight, diluted in phosphate-depleted medium to OD600 of 0.25, and phosphatase activity was measured at the time points indicated.
FIG. 6.
FIG. 6.
Asf1 homologs bind differentially to yeast histone H3 and Rad53. Yeast strains ROY1172 (no tag), ACN026 (yAsf1), BAT014 (dAsf1), BAT016 (hAsf1a), and KDY006 (hAsf1b) were grown to OD600 of ∼1.0, lysed, and immunoprecipitated with a Myc antibody. Equal amounts of protein were loaded onto an SDS-PAGE gel and Western blotted for either Myc, acetylated lysine 9/14 of H3 or Rad53. All soluble histones are modified on H3 lysine 9, and therefore the signal with the acetylated lysine 9/14 antibody reflects the amount of soluble histones. The presence (+) or absence (−) of treatment with the DSP cross-linker is indicated. Equal amounts of Asf1, H3K9/14, and Rad53 were detected in the input samples from all strains (data not shown).

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