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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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Arginine PTMs and kinetic data for PCAF on Arg8-modified H3 peptides.(A) Structures of the arginine side chain (R) with modifications includingmethyl (Rme), symmetric dimethyl (Rme2s), asymmetric dimethyl (Rme2a),and citrulline (cit) forms. (B) Kinetic plot of PCAF on modified H3substrates by a solution-phase kinetic assay. Substrate concentrationwas varied while holding PCAF and AcCoA concentrations constant. Eachspot represents an average of initial reaction velocities performedin triplicate.
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fig3: Arginine PTMs and kinetic data for PCAF on Arg8-modified H3 peptides.(A) Structures of the arginine side chain (R) with modifications includingmethyl (Rme), symmetric dimethyl (Rme2s), asymmetric dimethyl (Rme2a),and citrulline (cit) forms. (B) Kinetic plot of PCAF on modified H3substrates by a solution-phase kinetic assay. Substrate concentrationwas varied while holding PCAF and AcCoA concentrations constant. Eachspot represents an average of initial reaction velocities performedin triplicate.

Mentions: Known PTMsto H3 Arg8 include methylation17 and deimination,18 and we therefore determined whether those modificationsto the peptide substrates had an impact on acetylation by PCAF. Wesynthesized peptides having each of the four possible modified forms,monomethyl (Rme), symmetric dimethyl (Rme2s),asymmetric dimethyl (Rme2a), and the deiminated form havingthe amino acid citrulline (cit), and we characterized their activitytoward PCAF (Figure 3). Determining Michaelis–Mentenkinetics in cases where the enzyme is soluble and the substrate isimmobilized can be challenging.19 For thisreason, we employed a solution-phase spectrophotometric assay developedby Denu and co-workers.20 We determinedkinetic profiles for the reaction across varying substrate concentrations(10–1300 μM), while maintaining PCAF and AcCoA concentrationat 1 and 100 μM, respectively. The initial velocities displayeda stepwise increase in KM as the nativeArg8 was mutated to Rme, Rme2s, Rme2a, and finally cit. Generally, kcat remainedconstant between the mutants, apart from the citrulline mutant, whichwas near the limit of resolution. The dimethyl and citrullinated peptideshad a 10-fold reduction in catalytic efficiency (kcat/KM) owing primarily toan increase in KM (Table 1).


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)

Arginine PTMs and kinetic data for PCAF on Arg8-modified H3 peptides.(A) Structures of the arginine side chain (R) with modifications includingmethyl (Rme), symmetric dimethyl (Rme2s), asymmetric dimethyl (Rme2a),and citrulline (cit) forms. (B) Kinetic plot of PCAF on modified H3substrates by a solution-phase kinetic assay. Substrate concentrationwas varied while holding PCAF and AcCoA concentrations constant. Eachspot represents an average of initial reaction velocities performedin triplicate.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Arginine PTMs and kinetic data for PCAF on Arg8-modified H3 peptides.(A) Structures of the arginine side chain (R) with modifications includingmethyl (Rme), symmetric dimethyl (Rme2s), asymmetric dimethyl (Rme2a),and citrulline (cit) forms. (B) Kinetic plot of PCAF on modified H3substrates by a solution-phase kinetic assay. Substrate concentrationwas varied while holding PCAF and AcCoA concentrations constant. Eachspot represents an average of initial reaction velocities performedin triplicate.
Mentions: Known PTMsto H3 Arg8 include methylation17 and deimination,18 and we therefore determined whether those modificationsto the peptide substrates had an impact on acetylation by PCAF. Wesynthesized peptides having each of the four possible modified forms,monomethyl (Rme), symmetric dimethyl (Rme2s),asymmetric dimethyl (Rme2a), and the deiminated form havingthe amino acid citrulline (cit), and we characterized their activitytoward PCAF (Figure 3). Determining Michaelis–Mentenkinetics in cases where the enzyme is soluble and the substrate isimmobilized can be challenging.19 For thisreason, we employed a solution-phase spectrophotometric assay developedby Denu and co-workers.20 We determinedkinetic profiles for the reaction across varying substrate concentrations(10–1300 μM), while maintaining PCAF and AcCoA concentrationat 1 and 100 μM, respectively. The initial velocities displayeda stepwise increase in KM as the nativeArg8 was mutated to Rme, Rme2s, Rme2a, and finally cit. Generally, kcat remainedconstant between the mutants, apart from the citrulline mutant, whichwas near the limit of resolution. The dimethyl and citrullinated peptideshad a 10-fold reduction in catalytic efficiency (kcat/KM) owing primarily toan increase in KM (Table 1).

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