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CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.

Liu WH, Roemer SC, Port AM, Churchill ME - Nucleic Acids Res. (2012)

Bottom Line: Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/H4 or H3/H4-DNA interactions.Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)(2) tetramers on DNA.Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Colorado School of Medicine, Mail Stop 8303, PO Box 6511, Aurora, CO 80045, USA.

ABSTRACT
Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/H4 or H3/H4-DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)(2) tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.

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CAF-1 induces the formation of H3/H4 oligomers. (a) A FRET approach to report H3/H4 tetramerization. Positioning of fluorophores FM and CPM used for FRET experiments on the cysteine 71 residue of H4. H3/H4 are presented here as tetramers. A mixed labeled population of H3/H4, composed of H3/H4 with FM at Cys71 on H4 and CPM at Cys71 on H4, reports FRET when CPM is excited at 385 nm and when the CPM–FM donor–acceptor pair is within close range. (b) 10 nM H3/H4CPM/FM under dimerizing conditions (20°C, Buffer = 10 mM Tris pH 7.5, 150 mM KCl, 2 mM MgCl, 1% glycerol, 0.5 mM TCEP, 0.05% BRIJ-35) display FRET in the presence of 100 nM 80 bp 601 DNA. (c) 100 nM of unlabeled Asf1 bound to 10 nM H3/H4CPM/FM does not result in FRET. (d) 100 nM CAF-1 bound to 10 nM H3/H4CPM/FM results in CPM–FM FRET to the same degree as H3/H4 tetramerization on 601 DNA. (e) Using the FRET system, the binding affinity of CAF-1 to H3/H4CPM/FM or H3110E/H4CPM/FM was determined by monitoring the FRET ratio (F517-519 nm/F464-466 nm) with each CAF-1 titration. These data were fitted using Equation (6) and show the mean and standard deviation from at least three independent experiments.
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gks906-F4: CAF-1 induces the formation of H3/H4 oligomers. (a) A FRET approach to report H3/H4 tetramerization. Positioning of fluorophores FM and CPM used for FRET experiments on the cysteine 71 residue of H4. H3/H4 are presented here as tetramers. A mixed labeled population of H3/H4, composed of H3/H4 with FM at Cys71 on H4 and CPM at Cys71 on H4, reports FRET when CPM is excited at 385 nm and when the CPM–FM donor–acceptor pair is within close range. (b) 10 nM H3/H4CPM/FM under dimerizing conditions (20°C, Buffer = 10 mM Tris pH 7.5, 150 mM KCl, 2 mM MgCl, 1% glycerol, 0.5 mM TCEP, 0.05% BRIJ-35) display FRET in the presence of 100 nM 80 bp 601 DNA. (c) 100 nM of unlabeled Asf1 bound to 10 nM H3/H4CPM/FM does not result in FRET. (d) 100 nM CAF-1 bound to 10 nM H3/H4CPM/FM results in CPM–FM FRET to the same degree as H3/H4 tetramerization on 601 DNA. (e) Using the FRET system, the binding affinity of CAF-1 to H3/H4CPM/FM or H3110E/H4CPM/FM was determined by monitoring the FRET ratio (F517-519 nm/F464-466 nm) with each CAF-1 titration. These data were fitted using Equation (6) and show the mean and standard deviation from at least three independent experiments.

Mentions: To gain quantitative insights into the nature of the CAF-1–H3/H4 interaction, EMSA and FRETexperiments were developed and used to measure binding affinity. The FRET approach examines a mixed labeled population of H3/H4, in which one half of the H4 protein was labeled with the FRET acceptor FM at Cys 71, whereas the other half was labeled with the FRET donor 7-Diethylamino-3-(4′-Maleimidylphenyl)-4-Methylcoumarin (CPM) at the same site (Figure 4a). This approach predicts FRET to be observed between CPM and FM upon CPM excitation, but only when the FRET donor and acceptor are close together.Figure 4.


CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.

Liu WH, Roemer SC, Port AM, Churchill ME - Nucleic Acids Res. (2012)

CAF-1 induces the formation of H3/H4 oligomers. (a) A FRET approach to report H3/H4 tetramerization. Positioning of fluorophores FM and CPM used for FRET experiments on the cysteine 71 residue of H4. H3/H4 are presented here as tetramers. A mixed labeled population of H3/H4, composed of H3/H4 with FM at Cys71 on H4 and CPM at Cys71 on H4, reports FRET when CPM is excited at 385 nm and when the CPM–FM donor–acceptor pair is within close range. (b) 10 nM H3/H4CPM/FM under dimerizing conditions (20°C, Buffer = 10 mM Tris pH 7.5, 150 mM KCl, 2 mM MgCl, 1% glycerol, 0.5 mM TCEP, 0.05% BRIJ-35) display FRET in the presence of 100 nM 80 bp 601 DNA. (c) 100 nM of unlabeled Asf1 bound to 10 nM H3/H4CPM/FM does not result in FRET. (d) 100 nM CAF-1 bound to 10 nM H3/H4CPM/FM results in CPM–FM FRET to the same degree as H3/H4 tetramerization on 601 DNA. (e) Using the FRET system, the binding affinity of CAF-1 to H3/H4CPM/FM or H3110E/H4CPM/FM was determined by monitoring the FRET ratio (F517-519 nm/F464-466 nm) with each CAF-1 titration. These data were fitted using Equation (6) and show the mean and standard deviation from at least three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526290&req=5

gks906-F4: CAF-1 induces the formation of H3/H4 oligomers. (a) A FRET approach to report H3/H4 tetramerization. Positioning of fluorophores FM and CPM used for FRET experiments on the cysteine 71 residue of H4. H3/H4 are presented here as tetramers. A mixed labeled population of H3/H4, composed of H3/H4 with FM at Cys71 on H4 and CPM at Cys71 on H4, reports FRET when CPM is excited at 385 nm and when the CPM–FM donor–acceptor pair is within close range. (b) 10 nM H3/H4CPM/FM under dimerizing conditions (20°C, Buffer = 10 mM Tris pH 7.5, 150 mM KCl, 2 mM MgCl, 1% glycerol, 0.5 mM TCEP, 0.05% BRIJ-35) display FRET in the presence of 100 nM 80 bp 601 DNA. (c) 100 nM of unlabeled Asf1 bound to 10 nM H3/H4CPM/FM does not result in FRET. (d) 100 nM CAF-1 bound to 10 nM H3/H4CPM/FM results in CPM–FM FRET to the same degree as H3/H4 tetramerization on 601 DNA. (e) Using the FRET system, the binding affinity of CAF-1 to H3/H4CPM/FM or H3110E/H4CPM/FM was determined by monitoring the FRET ratio (F517-519 nm/F464-466 nm) with each CAF-1 titration. These data were fitted using Equation (6) and show the mean and standard deviation from at least three independent experiments.
Mentions: To gain quantitative insights into the nature of the CAF-1–H3/H4 interaction, EMSA and FRETexperiments were developed and used to measure binding affinity. The FRET approach examines a mixed labeled population of H3/H4, in which one half of the H4 protein was labeled with the FRET acceptor FM at Cys 71, whereas the other half was labeled with the FRET donor 7-Diethylamino-3-(4′-Maleimidylphenyl)-4-Methylcoumarin (CPM) at the same site (Figure 4a). This approach predicts FRET to be observed between CPM and FM upon CPM excitation, but only when the FRET donor and acceptor are close together.Figure 4.

Bottom Line: Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/H4 or H3/H4-DNA interactions.Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)(2) tetramers on DNA.Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Colorado School of Medicine, Mail Stop 8303, PO Box 6511, Aurora, CO 80045, USA.

ABSTRACT
Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/H4 or H3/H4-DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)(2) tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.

Show MeSH
Related in: MedlinePlus