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Acetyltransferase p300/CBP associated Factor (PCAF) regulates crosstalk-dependent acetylation of histone H3 by distal site recognition.

Kornacki JR, Stuparu AD, Mrksich M - ACS Chem. Biol. (2014)

Bottom Line: We show that p300/CBP associated factor (PCAF)/GCN5 activity depends on the presence of a distal arginine residue of its histone H3 substrate.Modifications to H3 Arg8 decrease PCAF acetylation of H3 Lys14, and kinetic data indicate that arginine citrullination has the strongest effect in decreasing acetylation.PCAF discriminates modified peptides as well as semisynthetic proteins and reconstituted nucleosomes bearing Arg8 modifications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Department of Cell & Molecular Biology, and Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.

ABSTRACT
Epigenetic regulation is directed, in part, by the correlated placement of histone post-translational modifications, but the mechanisms controlling correlated modifications are incompletely understood. Correlations arise from crosstalk among modifications and are frequently attributed to protein-protein interactions that recruit enzymes to existing histone modifications. Here we report the use of a peptide array to discover acetyltransferase-mediated crosstalks. We show that p300/CBP associated factor (PCAF)/GCN5 activity depends on the presence of a distal arginine residue of its histone H3 substrate. Modifications to H3 Arg8 decrease PCAF acetylation of H3 Lys14, and kinetic data indicate that arginine citrullination has the strongest effect in decreasing acetylation. Mutagenesis experiments demonstrate that PCAF specifically interprets H3 Arg8 modifications through interaction with residue Tyr640 on the surface of its catalytic domain, and this interaction regulates Lys14 acetylation by substrate discrimination. PCAF discriminates modified peptides as well as semisynthetic proteins and reconstituted nucleosomes bearing Arg8 modifications. Together, this work describes a method for systematically mapping crosstalks and illustrates its application to the discovery and elucidation of novel PCAF crosstalks.

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Schematic of the SAMDIacetylation assay. (A) Self-assembled monolayerspresenting maleimide against a background of tri(ethylene glycol)are appended with the H3 substrate TARK9acSTGGK14APC and treated with PCAF/AcCoA to generate the enzymaticallyacetylated product. (B) SDS-PAGE of expressed acetyltransferases PCAF,GCN5, and p300. (C) A SAMDI spectrum of the initial monolayer hasa peak at m/z = 2110 correspondingto the peptide–alkyldisulfide conjugate. Following treatmentwith PCAF, the acetylated product peptide is observed at m/z = 2152.
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fig1: Schematic of the SAMDIacetylation assay. (A) Self-assembled monolayerspresenting maleimide against a background of tri(ethylene glycol)are appended with the H3 substrate TARK9acSTGGK14APC and treated with PCAF/AcCoA to generate the enzymaticallyacetylated product. (B) SDS-PAGE of expressed acetyltransferases PCAF,GCN5, and p300. (C) A SAMDI spectrum of the initial monolayer hasa peak at m/z = 2110 correspondingto the peptide–alkyldisulfide conjugate. Following treatmentwith PCAF, the acetylated product peptide is observed at m/z = 2152.

Mentions: To assayPCAF activity, we applied a solution containing the enzyme and cofactor(1 μM PCAF, 100 μM AcCoA, 50 mM Tris pH 8.0, 0.1 mM EDTA)to a monolayer presenting the H3 peptide substrate. The reaction wasincubated for 1 h. The monolayer was then rinsed, treated with matrix,and analyzed by mass spectrometry (Figure 1). The SAMDI spectrum of the original monolayer has a peak at m/z = 2110 that corresponds to the peptide–alkanethiolateconjugate. Following the reaction, a spectrum revealed a new peakat m/z = 2152 that corresponds tothe actetylated form of the peptide. Integration of peak areas showedthat acetylation proceeded in 70% yield.


Acetyltransferase p300/CBP associated Factor (PCAF) regulates crosstalk-dependent acetylation of histone H3 by distal site recognition.

Kornacki JR, Stuparu AD, Mrksich M - ACS Chem. Biol. (2014)

Schematic of the SAMDIacetylation assay. (A) Self-assembled monolayerspresenting maleimide against a background of tri(ethylene glycol)are appended with the H3 substrate TARK9acSTGGK14APC and treated with PCAF/AcCoA to generate the enzymaticallyacetylated product. (B) SDS-PAGE of expressed acetyltransferases PCAF,GCN5, and p300. (C) A SAMDI spectrum of the initial monolayer hasa peak at m/z = 2110 correspondingto the peptide–alkyldisulfide conjugate. Following treatmentwith PCAF, the acetylated product peptide is observed at m/z = 2152.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4301089&req=5

fig1: Schematic of the SAMDIacetylation assay. (A) Self-assembled monolayerspresenting maleimide against a background of tri(ethylene glycol)are appended with the H3 substrate TARK9acSTGGK14APC and treated with PCAF/AcCoA to generate the enzymaticallyacetylated product. (B) SDS-PAGE of expressed acetyltransferases PCAF,GCN5, and p300. (C) A SAMDI spectrum of the initial monolayer hasa peak at m/z = 2110 correspondingto the peptide–alkyldisulfide conjugate. Following treatmentwith PCAF, the acetylated product peptide is observed at m/z = 2152.
Mentions: To assayPCAF activity, we applied a solution containing the enzyme and cofactor(1 μM PCAF, 100 μM AcCoA, 50 mM Tris pH 8.0, 0.1 mM EDTA)to a monolayer presenting the H3 peptide substrate. The reaction wasincubated for 1 h. The monolayer was then rinsed, treated with matrix,and analyzed by mass spectrometry (Figure 1). The SAMDI spectrum of the original monolayer has a peak at m/z = 2110 that corresponds to the peptide–alkanethiolateconjugate. Following the reaction, a spectrum revealed a new peakat m/z = 2152 that corresponds tothe actetylated form of the peptide. Integration of peak areas showedthat acetylation proceeded in 70% yield.

Bottom Line: We show that p300/CBP associated factor (PCAF)/GCN5 activity depends on the presence of a distal arginine residue of its histone H3 substrate.Modifications to H3 Arg8 decrease PCAF acetylation of H3 Lys14, and kinetic data indicate that arginine citrullination has the strongest effect in decreasing acetylation.PCAF discriminates modified peptides as well as semisynthetic proteins and reconstituted nucleosomes bearing Arg8 modifications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Department of Cell & Molecular Biology, and Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.

ABSTRACT
Epigenetic regulation is directed, in part, by the correlated placement of histone post-translational modifications, but the mechanisms controlling correlated modifications are incompletely understood. Correlations arise from crosstalk among modifications and are frequently attributed to protein-protein interactions that recruit enzymes to existing histone modifications. Here we report the use of a peptide array to discover acetyltransferase-mediated crosstalks. We show that p300/CBP associated factor (PCAF)/GCN5 activity depends on the presence of a distal arginine residue of its histone H3 substrate. Modifications to H3 Arg8 decrease PCAF acetylation of H3 Lys14, and kinetic data indicate that arginine citrullination has the strongest effect in decreasing acetylation. Mutagenesis experiments demonstrate that PCAF specifically interprets H3 Arg8 modifications through interaction with residue Tyr640 on the surface of its catalytic domain, and this interaction regulates Lys14 acetylation by substrate discrimination. PCAF discriminates modified peptides as well as semisynthetic proteins and reconstituted nucleosomes bearing Arg8 modifications. Together, this work describes a method for systematically mapping crosstalks and illustrates its application to the discovery and elucidation of novel PCAF crosstalks.

Show MeSH
Related in: MedlinePlus