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Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4.

Henry RA, Kuo YM, Andrews AJ - Biochemistry (2013)

Bottom Line: With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300.With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP.This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States.

ABSTRACT
Although p300 and CBP lysine acetyltransferases are often treated interchangeably, the inability of one enzyme to compensate for the loss of the other suggests unique roles for each. As these deficiencies coincide with aberrant levels of histone acetylation, we hypothesized that the key difference between p300 and CBP activity is differences in their specificity/selectivity for lysines within the histones. Utilizing a label-free, quantitative mass spectrometry based technique, we determined the kinetic parameters of both CBP and p300 at each lysine of H3 and H4, under conditions we would expect to encounter in the cell (either limiting acetyl-CoA or histone). Our results show that while p300 and CBP acetylate many common residues on H3 and H4, they do in fact possess very different specificities, and these specificities are dependent on whether histone or acetyl-CoA is limiting. Steady-state experiments with limiting H3 demonstrate that both CBP and p300 acetylate H3K14, H3K18, H3K23, with p300 having specificities up to 10¹⁰-fold higher than CBP. Utilizing tetramer as a substrate, both enzymes also acetylate H4K5, H4K8, H4K12, and H4K16. With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300. With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP. This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

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Determination of steady-state kinetic parameters of CBP- and p300-mediatedacetylation of histone H3/H4 when titrating acetyl-CoA. Experimentswere performed at 37 °C in 100 mM ammonium bicarbonate and 50mM HEPES buffer (pH 7.8) at 37 °C. Assays for p300 contained50 nM p300, 7.5 μM H3/H4, and varying concentrations of acetyl-CoA(1–200 μM). Assays for CBP contained 20 nM CBP, 10 μMH3/H4, and varying concentrations of acetyl-CoA (1–200 μM).Experiments were quenched with 4 volumes of TCA and boiled at 95 °Cfor 5 min. Sites displaying the highest specificity (kcat/KmnH) for either CBP or p300 where chosen for representative graphs.(A) Nonlinear fit of CBP acetylation of histone H4K16. (B) Nonlinearfit of p300 acetylation of histone H4K16. (C) Comparison of the specificityconstants (kcat/KmnH) of CBP (black) and p300 (gray)on H3K9, H3K14, H3K18, and H3K23 and H4K5, H4K8, H4K12, and H4K16.(D) The log of the ratio of specificity (CBP/p300) between CBP andp300 at each site of H3 and H4. All quantified sites can be foundin Supplemental Figures 5 and 6. The apparentkinetic parameters are summarized in Table 5.
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fig4: Determination of steady-state kinetic parameters of CBP- and p300-mediatedacetylation of histone H3/H4 when titrating acetyl-CoA. Experimentswere performed at 37 °C in 100 mM ammonium bicarbonate and 50mM HEPES buffer (pH 7.8) at 37 °C. Assays for p300 contained50 nM p300, 7.5 μM H3/H4, and varying concentrations of acetyl-CoA(1–200 μM). Assays for CBP contained 20 nM CBP, 10 μMH3/H4, and varying concentrations of acetyl-CoA (1–200 μM).Experiments were quenched with 4 volumes of TCA and boiled at 95 °Cfor 5 min. Sites displaying the highest specificity (kcat/KmnH) for either CBP or p300 where chosen for representative graphs.(A) Nonlinear fit of CBP acetylation of histone H4K16. (B) Nonlinearfit of p300 acetylation of histone H4K16. (C) Comparison of the specificityconstants (kcat/KmnH) of CBP (black) and p300 (gray)on H3K9, H3K14, H3K18, and H3K23 and H4K5, H4K8, H4K12, and H4K16.(D) The log of the ratio of specificity (CBP/p300) between CBP andp300 at each site of H3 and H4. All quantified sites can be foundin Supplemental Figures 5 and 6. The apparentkinetic parameters are summarized in Table 5.

Mentions: As with H3,we also performed acetyl-CoA titrations where H3/H4tetramer concentrations and enzyme concentrations were kept constant(Figure 4A,B, Figures S5and S6). Under these conditions, the order of specificity,not taking into consideration cooperativity, for CBP is K18 > K14> K23, which is the same as the H3/H4 substrate titration. Despitethe order being the same, the preference for K18 acetylation is notas pronounced when titrating acetyl-CoA, with only an ∼3-foldpreference over the second highest site, K14. When titrating acetyl-CoA,p300 displays the same order of specificity as CBP (but with the additionof K9), K18 > K14 > K23 > K9. However, under these conditionswe observea stronger preference of p300 for H3K18, with the kcat/k1/2 being over 2.5-foldhigher than second highest site, K14, instead of being approximatelyequal.


Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4.

Henry RA, Kuo YM, Andrews AJ - Biochemistry (2013)

Determination of steady-state kinetic parameters of CBP- and p300-mediatedacetylation of histone H3/H4 when titrating acetyl-CoA. Experimentswere performed at 37 °C in 100 mM ammonium bicarbonate and 50mM HEPES buffer (pH 7.8) at 37 °C. Assays for p300 contained50 nM p300, 7.5 μM H3/H4, and varying concentrations of acetyl-CoA(1–200 μM). Assays for CBP contained 20 nM CBP, 10 μMH3/H4, and varying concentrations of acetyl-CoA (1–200 μM).Experiments were quenched with 4 volumes of TCA and boiled at 95 °Cfor 5 min. Sites displaying the highest specificity (kcat/KmnH) for either CBP or p300 where chosen for representative graphs.(A) Nonlinear fit of CBP acetylation of histone H4K16. (B) Nonlinearfit of p300 acetylation of histone H4K16. (C) Comparison of the specificityconstants (kcat/KmnH) of CBP (black) and p300 (gray)on H3K9, H3K14, H3K18, and H3K23 and H4K5, H4K8, H4K12, and H4K16.(D) The log of the ratio of specificity (CBP/p300) between CBP andp300 at each site of H3 and H4. All quantified sites can be foundin Supplemental Figures 5 and 6. The apparentkinetic parameters are summarized in Table 5.
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fig4: Determination of steady-state kinetic parameters of CBP- and p300-mediatedacetylation of histone H3/H4 when titrating acetyl-CoA. Experimentswere performed at 37 °C in 100 mM ammonium bicarbonate and 50mM HEPES buffer (pH 7.8) at 37 °C. Assays for p300 contained50 nM p300, 7.5 μM H3/H4, and varying concentrations of acetyl-CoA(1–200 μM). Assays for CBP contained 20 nM CBP, 10 μMH3/H4, and varying concentrations of acetyl-CoA (1–200 μM).Experiments were quenched with 4 volumes of TCA and boiled at 95 °Cfor 5 min. Sites displaying the highest specificity (kcat/KmnH) for either CBP or p300 where chosen for representative graphs.(A) Nonlinear fit of CBP acetylation of histone H4K16. (B) Nonlinearfit of p300 acetylation of histone H4K16. (C) Comparison of the specificityconstants (kcat/KmnH) of CBP (black) and p300 (gray)on H3K9, H3K14, H3K18, and H3K23 and H4K5, H4K8, H4K12, and H4K16.(D) The log of the ratio of specificity (CBP/p300) between CBP andp300 at each site of H3 and H4. All quantified sites can be foundin Supplemental Figures 5 and 6. The apparentkinetic parameters are summarized in Table 5.
Mentions: As with H3,we also performed acetyl-CoA titrations where H3/H4tetramer concentrations and enzyme concentrations were kept constant(Figure 4A,B, Figures S5and S6). Under these conditions, the order of specificity,not taking into consideration cooperativity, for CBP is K18 > K14> K23, which is the same as the H3/H4 substrate titration. Despitethe order being the same, the preference for K18 acetylation is notas pronounced when titrating acetyl-CoA, with only an ∼3-foldpreference over the second highest site, K14. When titrating acetyl-CoA,p300 displays the same order of specificity as CBP (but with the additionof K9), K18 > K14 > K23 > K9. However, under these conditionswe observea stronger preference of p300 for H3K18, with the kcat/k1/2 being over 2.5-foldhigher than second highest site, K14, instead of being approximatelyequal.

Bottom Line: With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300.With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP.This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States.

ABSTRACT
Although p300 and CBP lysine acetyltransferases are often treated interchangeably, the inability of one enzyme to compensate for the loss of the other suggests unique roles for each. As these deficiencies coincide with aberrant levels of histone acetylation, we hypothesized that the key difference between p300 and CBP activity is differences in their specificity/selectivity for lysines within the histones. Utilizing a label-free, quantitative mass spectrometry based technique, we determined the kinetic parameters of both CBP and p300 at each lysine of H3 and H4, under conditions we would expect to encounter in the cell (either limiting acetyl-CoA or histone). Our results show that while p300 and CBP acetylate many common residues on H3 and H4, they do in fact possess very different specificities, and these specificities are dependent on whether histone or acetyl-CoA is limiting. Steady-state experiments with limiting H3 demonstrate that both CBP and p300 acetylate H3K14, H3K18, H3K23, with p300 having specificities up to 10¹⁰-fold higher than CBP. Utilizing tetramer as a substrate, both enzymes also acetylate H4K5, H4K8, H4K12, and H4K16. With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300. With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP. This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

Show MeSH
Related in: MedlinePlus